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When you arrive in Amsterdam after a long flight or train ride the last thing you want to do is hunt for your hotel. In this case you walk out the train station, make a right and you have arrived. This is a great place to choose if you're just in town for a few days and need access to the train. Internet is fast, facility is clean. Noise level can be a little high at times. Rooms are a little cramped for two people but overall this place gets a major thumbs up.
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Fright-master Steve Niles concludes the all-new, critically acclaimed 30 Days of Night series! The war between vampires reaches a fever pitch as Eben Olemaunâs movement collides head-on with the old guard, with Agent Alice Blood directly in the crossfire. It all ends here!
Harley Quinn's Greatest Moments from "Batman: The Animated Series"
TV, Comic Books

subset: commoncrawl


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Hotel Star
Hotel Star
Hotel Guest Rating: 7.4/10
Avenida Lazaro Cardenas, 1313
Manzanillo, CL 28218
Hotel Freebies Available*
• Free Internet Available show dates
Hotel Star
Featuring a access beach with Palapa parasols, Hotel Star has beautiful oceanfront setting in Manzanillo's Las Brisas district. It offers rooms and bungalows, an outdoor swimming pool and a restaurant.All air-conditioned accommodation at Hotel Star has simple décor, cable TV and free Wi-Fi. Bungalows have a terrace, a fridge and a microwave.Shops, bars and restaurants can be found within 15 minutes' walk of the hotel. The centre of Manzanillo is just over 2 km away.There are 2 golf courses within 15 minutes' drive of the Star. Tecomán and Armería are within 60 km of the hotel. ...read more
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- Outdoor pool
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Hotel Star Amenities
- 24-hour front desk
- Air conditioning
- Bar
- Breakfast in the room
- Family rooms
- Free WiFi
- Luggage storage
- Outdoor pool
- Packed lunches
- Private parking
- Restaurant (à la carte)
- Room service
- Safety deposit box
- Swimming pool
- Tour desk
from $21


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Put a stop to robberiesFebruary 20,2014
Put a stop to robberies
I was at our Kinney Drugs store in Berlin and noticed there was a sheriff sitting in the waiting area of the pharmacy. My understanding is he was hired to keep customers safe while getting their meds; it is so sad that our local community is getting out of control. I think that we need more police officers in all pharmacies and stores, we should hire more police officers and make this happen until these losers see that we are not going to take this anymore. Vermont is really getting out of hand, and we are lacking protection. My child was a victim of one of the stores that was held up, and let me tell you that this has traumatized my child in a way no one will ever know. My child is having nightmares, and every time someone has a face mask on my child freezes in her footsteps. What is our governor doing about this? What are our legislators doing for us?
Francine Belville
BarreMORE IN LettersI believe that Matt Dunne has the energy, experience and creativity to lead Vermont, as our... Full StoryI am writing in support of Sue Minter for governor. Full StoryIm voting for Phil Scott for Governor on Aug. 9. You should too. Phil Scott listens to Vermonters. Full Story
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Join us on our podcast each weekday for an interesting story, well told, from Charisma News. Listen at charismapodcastnetwork.com.
Over the years, Facebook posts, forwarded emails and rumors have led people to believe the Salvation Army does not serve members of the LGBT community. Click below to watch Maj. Jeff Strickler, commander of the Twin Cities Salvation Army, explain that these accusations aren't true.
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- olgakantor3275
- USA, Žena, 25-30
- Nomád nováček
- 2 Recenze
Great location! Very close to all of the big sites. Staff were very helpful, offered easy airport transfer service! View from the rooftop at breakfast was gorgeous! Highly recommend.
- Anonymní
- USA, Muž, 25-30
- Nomád nováček
- 5 Recenze
I recommend this hostel for the terrace view of the Blue Mosque and Sea of Marmara alone. It was very clean, the staff was very friendly, and it's a short walk to the Blue Mosque, Haghia Sophia, and Hippodrome. One minor issue is that it didn't seem too social of a place. We only stayed for one night, but if we were in Istanbul for longer I would have wanted a hostel where it was easier to meet other travelers.
Mobilní aplikace Hostelworld
Rezervujte si pokoj na cestách s novou mobilní aplikací Hostelworld.


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Hmm, interesting to know this thing... anyways, I was ok with teri meri...
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The certified public accounting firm of Adams, Brown, Beran & Ball, Chartered announces the promotion of Turner Polzin, CPA to manager. Polzin is a graduate of Hoisington High School.
Matt Jensen had a plan to land a job as a coding developer in the computer industry. He would first earn a bachelor's degree in information systems, which would immerse him in business management, coding language and computer security.
During her Academy Awards acceptance speech, actress Patricia Arquette delivered a message about equal pay for women.
Participating in your employer's Going Out of Business sale is a solid indicator that a job change is inevitable. However, if your employer's books are solidly in the black, how do you know the right time, if ever, to change jobs?
The ability to communicate, whether written or spoken, was recently noted by the Pew Research Center as the skill American adults think is most necessary to be successful.
Western Kansas Human Resource Management Association (WKHRMA) will hold their March meeting on Wednesday, March 11, at Thirsty's Banquet Room, 2704 Vine Street in Hays. Registration is between 11:15 and 11:30 a.m. with a short business meeting starting at 11:30 a.m. The program for the March meeting will be on "The Risks of Engaging Social Media in the Employment Process" presented by Mike Fowler, Validity Screening Solutions.
Michael F. Goss, former managing director and head of global investor relations for Bain Capital, will be the spring 2015 speaker for the Kansas State University College of Business Administration's Distinguished Lecture Series.
Lego has long been a favorite toy, but the company's recent growth has spurred it to new heights.
Central National Bank announced that Steven Swigart has been named home mortgage lender for the Great Bend Lending Services Office. He joins Central National Bank with seven years of banking experience.
As millennials begin to overtake the baby boomers and Gen X in size it will become increasingly important for businesses to target them as customers.
The dual awards of "Top Group Agency" and "Top Group Agent" were recently presented to Cheyenne Travel/Please Go Away™ Vacations as an organization and Paula Axman individually, by Collette Vacations. District Sales Manager Jordan Marx traveled to Great Bend and presented the awards in recognition of outstanding 2014 promotion and personal service of Collette's products in the arranging and conducting of one-of-a-kind value added travel experiences throughout North America and the rest of the world.
Codi Womochil joins Sunflower Electric Power Corporation
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The Golden Belt Humane Society recently had to purchase a new air conditioning unit. This unexpected expense was a big hit to one of Animal ...
Eakin announces Brock Eakin as employee of the first quarter for 2016.
Someday, women will be paid the same as men for doing the same job. That day, judging from recent news, may not come any time ...
Like every other generation, millennials are so broad a demographic that anything about them, while probably true of one segment, is untrue of another. Still ...
After a months-long investigation into the well-publicized risks of self-balancing scooters, nicknamed hoverboards, the Consumer Product Safety Commission has issued a product recall for more ...
Since Perks Coffee Shoppe features the award-winning PT's Coffee, the staff wanted to learn more about the product and went directly to the source.
St. Rose Health Center staff members and volunteers will be on hand from 6:30 to 9 a.m. Saturday, July 23 for the next ...
Even though Heather Quillin is the new children's services coordinator at Sunflower Diversified Services, she will continue in her role as a speech-language pathologist ...
Tina Mingenback has added a new credential to her formal education to better serve residents at Cherry Village Benevolence.
Few people are able to take paid family leave, but many of both genders would like to. And it's something for which Americans are ...
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A plane frame is constructed by using a pin connection between segments ABC and CDE. The frame has pin supports at A and E and joint loads at B and D (see figure).
(a) Find reactions at supports A and E.
(b) Find the resultant force in the pin at C.


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If you have troubles since the update to WordPress 4.5 please read this post before opening a new thread.
Tagged: login redirect broadscope
I am having a hard time to get users after login to be redirected to a specific page on my site.
Please I need some help with this issue in order to make the visiting experience clearer to my users.
No matter what I do, users gets redirected "home"
Thanks for any info any one may share.
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The sun always prevails after the rain. A relaxing moment from my window. This shot was taken with my 4k…
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Kanye West – All Of The Lights (Instrumental Remake) Had to recreated this classic throwback! Like, Comment & share!
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Did a remake of Ambitions as a Ridah. One time for Pac.
(15 second clip) Making of "Flight school" off of The Clear Sky EP. iTunes
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Ultra 2013: Richie Hawtin Delivers Sensory Overload, No Bottle Anthems
This year's main stage at Ultra Music Festival has seemed particularly heavy on so-called "confetti house," the lineup rife with marquee names from VIP-heavy megaclubs.
But the (relatively) more underground sounds of dance music -- or at least those not so concerned with encouraging bottle sales -- lived on at a few other tents. The Carl Cox and Friends stages at Ultra 2013, two this past Friday and one on Saturday, continue to serve as a refuge for deeper, techier sounds at the festival.
See also:
-Swedish House Mafia Kicks Off Ultra 2013, "F#$%s Miami in the Pussy"
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-Ultra 2013: Snoop Dogg Goes Rasta to Gangsta to Raver, Debuts "No Guns Allowed"
-Kaskade and Tiesto Kick Off UMF's Weekend Two, Reach Epic Pop Peaks
-Avicii Does Country Music and Eric Prydz Proclaims "Love You" UMF!
-Ultra 2013: Slash Does Surprise Guitar Solo, "Pretty Awesome Gig"
-Ultra 2013: Deadmau5 Proves He's a Real Person, Rejects the Easy Headliner Gimmicks
As such, one of the rare treats came early Saturday evening with an appearance by techno god Richie Hawtin. Was a mass-appeal festival with shortened DJ slots the best place to hear him play? Not really -- the best Hawtin sets unfurl over the course of a club night and into the wee hours. Still, by Ultra terms, his allotted two hours seemed like a luxurious sprawl, with the full kind of narrative arc that rewards dancers who stick it out to the finish.
Hawtin's been mocked for helping create the trend for superminimal techno, but played at ear-crushing volumes, the sound yesterday was pleasantly full and dubby, locking into a robot groove from the start. Subtle variations in the tracks, carefully crescendoing song selection, and of course seamless mixing kept things moving without the flash gimmicks of many DJs on the main stage.
And yes, the kids still loved it -- thousands of revelers of a wide spread of ages packed the tent from the beginning, lost in careful concentration on the four-four. There wasn't a single easy vocal hook here, yet it was hard to turn away as Hawtin's set continued on its twists and turns until it reached a frenzied peak right after sundown.
Seemingly all of a sudden, things got very psychedelic. Bass pounded, the mid-range clanks and squeals ground along, and the hectagonal screens on the tent's ceiling lowered in an explosion of psychedelic vocals. It was totally possible to get a kind of euphoric high on the sensory overload, no bottle anthems required.
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Migrating from Word 2003 to Word 2007
Upgrading from one software version to another can sometimes be a difficult transition. In Migrating from Word 2003 to Word 2007, author David Rivers explains the benefits of switching to Word 2007 from Word 2003. The course covers key differences Word 2003 users need to understand, including the new Ribbon interface and the new Office Open XML file format. Exercise files accompany the course.
- Comparing the Word 2003 and 2007 interfaces
- Working in a mixed Word environment
- Dealing with file compatibility issues
- Changing the default file format
- Using keyboard shortcuts
- Understanding Compatibility Mode
- Creating macros
- Subject:
- Business
- Software:
- Word
- Author:
- David Rivers
(upbeat music) - [Voiceover] Hi and welcome to Migrating form Microsoft Word 2003 to Word 2007. I'm David Rivers, Microsoft has incorporated some major changes and improvements in Word 2007 so if you're considering migrating from Word 2003 this title will help to make your transition to Word 2007 a smooth one. We'll begin with a tour of the new and improved user interface, namely the ribbon, where you'll find easy and logical access to the various features and functions in Word 2007.
Then we'll investigate the various options for staying compatible with older versions of Microsoft Word, such as the compatibility checker and compatibility mode which will be very helpful if you're working in a mixed environment. The way you perform many of the common commands in Microsoft Word 2003 may be performed differently in Word 2007. We'll investigate the office button for common file related commands and we'll work with the quick access tool bar to help create a more efficient work environment. We'll also work with the mini tool bars for even faster access to related functionality when selecting content in Word 2007.
Lastly, we'll examine ways you can customize the user interface to personalize your work environment, let's get started.
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These authors contributed equally to this work.
A Role of the Sulfonylurea Receptor 1 in Endocytic Trafficking of ATP-Sensitive Potassium Channels
Version of Record online: 3 JUL 2011
DOI: 10.1111/j.1600-0854.2011.01227.x
© 2011 John Wiley & Sons A/S
Volume 12, Issue 9, pages 1242–1256, September 2011
Additional Information
How to Cite
Bruederle, C. E., Gay, J. and Shyng, S.-L. (2011), A Role of the Sulfonylurea Receptor 1 in Endocytic Trafficking of ATP-Sensitive Potassium Channels. Traffic, 12: 1242–1256. doi: 10.1111/j.1600-0854.2011.01227.x
Publication History
- Issue online: 11 AUG 2011
- Version of Record online: 3 JUL 2011
- Accepted manuscript online: 7 JUN 2011 04:48AM EST
- Received 31 January 2011, revised and accepted for publication 4 June 2011, uncorrected manuscript published online 7 June 2011, published online 3 July 2011
- bungarotoxin;
- endocytosis;
- imaging;
- KATP channel;
- Kir6.2;
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
The ATP-sensitive potassium (KATP) channel consisting of sulfonylurea receptor 1 (SUR1) and inward-rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2, while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live-cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin-binding peptide labeled with a fluorescent dye. We show that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for KATP channels and support a role of SUR1 in this trafficking process.
ATP-sensitive potassium (KATP) channels formed by sulfonylurea receptors (SURs) and inwardly rectifying potassium channels (Kir6.x) have an established role in coupling cellular ATP and ADP levels to membrane excitability to regulate physiological processes. In pancreatic β cells, KATP channels consisting of SUR1 and Kir6.2 regulate insulin secretion to maintain glucose homeostasis. Elevated blood glucose leads to increased ATP production and a higher intracellular (ATP/ADP) ratio which closes KATP channels, causing membrane depolarization, opening of voltage-gated calcium channels, calcium influx and insulin secretion. In contrast, low blood glucose causes KATP channel opening due to a decreased intracellular (ATP/ADP) ratio leaving the β-cell in a hyperpolarized resting state to prevent insulin release (1).
Defective channel function is linked to a number of human diseases including several forms of diabetes (2,3). KATP channel function depends on channel gating properties and the number of channels expressed at the cell surface. Mutations in either subunit, Kir6.2 or SUR1, can affect KATP channel surface expression or gating properties to cause disease. Gain-of-function mutations are associated with permanent neonatal diabetes mellitus (4,5). On the other hand, loss-of-function mutations underlie congenital hyperinsulinism (6–8). The number of KATP channels expressed at the cell surface is dependent on correct trafficking of channels from the endoplasmic reticulum (ER) to the plasma membrane. SUR1 and Kir6.2 each contain an RKR tripeptide ER-retention signal preventing the subunits from exiting the ER when they are expressed independently. Only assembled channels can leave the ER because the octameric structural organization of the SUR1/Kir6.2 complex masks the ER-retention signals (9,10). In addition to regulation in the secretory pathway, the number of KATP channels in the plasma membrane can also be influenced by changes in internalization, recycling or degradation of surface channels. An increase in channel density at the plasma membrane is expected to increase the threshold of glucose concentrations necessary to depolarize β-cell membrane and stimulate insulin secretion. Conversely, a decrease in cell surface channel density renders membrane potential more easily depolarized at a given stimulatory glucose concentration (6,11–13). Endocytosis of KATP channels has been suggested to be clathrin-mediated and solely dependent on Kir6.2. Initially, a dileucine motif in Kir6.2 was proposed to be responsible for KATP channel internalization in response to protein kinase C (PKC) stimulation in COS-7 cells and neurons (14). The role of the dileucine motif was later challenged by Mankouri et al. who reported that channel endocytosis is instead mediated by a tyrosine-based motif 330YSKF333 in the C-terminal cytoplasmic domain of Kir6.2. They further reported that a neonatal diabetes Kir6.2 mutation Y330C that reduces channel sensitivity to ATP inhibition also prevented channel endocytosis. As Y330C disrupts the tyrosine-based motif, it was concluded that the mutation causes neonatal diabetes in part by preventing channel endocytosis and increasing the resident lifetime of the ATP-insensitive mutant Kir6.2. In these studies, trafficking and distribution of KATP channels were assessed primarily with conventional approaches using antibody labeling of fixed cells or biochemical assays (15).
In this study, we used a small, high-affinity α-bungarotoxin (BTX)-binding peptide tag (BTX tag) (16) placed on the extracellular domain of SUR1 or Kir6.2 for tetramethyl rhodamine isothiocyanate (TRITC)-BTX labeling to monitor internalization of KATP channels in living cells. This new approach allowed us to visualize endocytosis of membrane-bound KATP channels in real time. Live-cell imaging of the Kir mutants Y330C and F333I coexpressed with SUR1 showed that these mutant channels do not prevent internalization as previously reported. We also show that SUR1 alone undergoes endocytosis when the ER-retention signal RKR is inactivated by mutation to AAA (SUR1RKRAAA). Taken together, these data suggest that the aforementioned Kir6.2 tyrosine motif is not solely responsible for KATP channel internalization and that the SUR1 protein contains signaling information that is sufficient for its own endocytosis, and thus contributes to regulation of endocytosis of the channel complex.
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
Monitoring endocytosis of KATP channels with live-cell imaging
In previous internalization studies, KATP channels were primarily monitored with either biochemical methods or antibody-based imaging of fixed cells (17–20). These methods only provide snapshots of channel protein distribution at certain time points of the study, however they lack the power to visually follow internalization in real time that live-cell imaging methods offer. To follow internalization of KATP channels in living cells, we tagged the N-terminus of SUR1 with a short peptide sequence (BTX tag-SUR1) that binds fluorophore-labeled BTX (TRITC-BTX) with high affinity (21). The N-terminus of SUR1 is located on the extracellular side of the plasma membrane allowing access of TRITC-BTX, which itself is membrane impermeable, to its targeting sequence without permeabilization of cells (16,22). Coexpression of BTX tag-SUR1 with Kir6.2 generated channels with gating characteristics similar to untagged channels as assessed by inside-out patch clamp recording. Preincubation of cells expressing the channel with TRITC-BTX also had no effect on channel gating or channel density, indicating that ligand binding does not affect channel function or expression (Figure S1).
For live-cell imaging, COSm6 cells transiently transfected with BTX tag-SUR1 and wild-type (WT)-Kir6.2 were loaded onto the microscope stage at 37°C. TRITC-BTX was applied to the medium to label channels at the plasma membrane, and image acquisition began immediately after focusing on the cells. This approach minimizes the time between ligand application and image acquisition such that channels expressed at the cell surface were captured before significant intracellular fluorescence, due to endocytosis, could develop. Cells transfected with BTX tag-SUR1 and WT-Kir6.2 showed fluorescent surface signal at the start of acquisition (Figure 1A, first panel). At later time-points, an increase in punctate intracellular fluorescent signal was detected. We followed trafficking of labeled channels from the plasma membrane over a 30 min time period. Fluorescence intensity profiles were measured with a line scan algorithm as described in Materials and Methods. Fluorescent signals associated with the cell surface are depicted by two initial peaks in the line scan graph at the start of acquisition (Figure 1A, acquisition start). Internalized channels are detected as peaks distributing toward the middle of the graph over time (Figure 1A, panels 10–30 min and Movie S1). Imaging of untransfected cells labeled with TRITC-BTX served as a control. In these cells, no punctate fluorescence signal was observed (data not shown). Furthermore, we examined endocytosis using live-cell imaging of channels formed by SUR1- and BTX-tagged Kir6.2 (Movie S3). Internalization was similar when the BTX tag was fused to either SUR1 or Kir6.2, although the overall fluorescent signal was stronger when the tag was placed on the SUR1 subunit (cf. Movie S1 and Movie S3). We therefore used BTX tag-SUR1 for all subsequent studies.
Similar studies were carried out in rat insulinoma cells (INS-1) cells, which closely resemble pancreatic β-cells, to validate results obtained in the heterologous expression system. For these experiments, INS-1 cells were infected with recombinant adenoviruses containing BTX tag-SUR1 and Kir6.2 cDNAs. Confocal microscopy showed surface-labeled channels trafficked from the plasma membrane toward intracellular compartments over time (Figure 1B and Movie S2). These results confirmed the results obtained in COSm6 cells. For subsequent experiments analyzing mutant KATP channels, we used COSm6 cells as they provide a clean experimental platform devoid of endogenous KATP channels, which might compensate potential effects of mutant constructs.
Analysis of Kir6.2 mutations previously reported to impair KATP channel endocytosis
Earlier studies suggested that the neonatal diabetes Kir6.2 mutations Y330C and F333I impair internalization of KATP channels by disrupting the tyrosine-based endocytosis motif 330YSKF333(15). To see if similar observations could be made with live-cell imaging, COSm6 cells were cotransfected with BTX tag-SUR1 and Kir6.2 harboring the F333I or Y330C mutation. Live-cell imaging was performed as described earlier. Surprisingly, we observed internalization of both Kir6.2-Y330C and F333I mutant KATP channels (Figure 2A, Movie S4 and data not shown). Moreover, we observed significantly reduced fluorescence signal at the cell surface when the dye was added and acquisition was started indicating reduced surface channel expression. The decreased surface expression is opposite to what one would expect if the mutations prevent channel endocytosis and increase channel residence time in the plasma membrane. Quantitative analysis by line scan showed an increase in fluorescent signal levels corresponding to an increased amount of intracellular fluorescent puncta over time (Figure 2A, graphs acquisition start to 30 min).
To substantiate our finding, we utilized a second, more conventional method. COSm6 cells coexpressing BTX tag-SUR1 and WT, F333I-, Y330C- or Y330A-Kir6.2 were labeled at 4°C with TRITC-BTX and chased at 37°C before fixation to assess endocytosis. The chase medium contained a fluid-phase marker, fluorescein isothiocyanate (FITC)-BSA. After 30 min of incubation at 37°C, a punctate BTX staining pattern was observed inside cells expressing WT or mutant channels (Figure 2B). Colocalization with the endocytic vesicle marker FITC-BSA confirmed that puncta were truly of intracellular nature.
The results described earlier are in opposition to previously published results by others. This prompted us to confirm our data with yet additional biochemical approaches. Our imaging experiments showed reduced surface staining of Y330C and F333I mutant channels; therefore, we first compared their expression levels with WT channels using western blot and the chemiluminescence assay described in Materials and Methods. By western blot, the Y330C and F333I Kir6.2 mutants showed reduced steady-state protein levels compared to WT Kir6.2 coexpressed with either FLAG- or BTX tag-SUR1 (Figure 2C, middle blot). Moreover, there was a significant reduction in the mature, complex-glycosylated SUR1 upper band in cells coexpressing the mutant Kir6.2 compared with cells coexpressing WT Kir6.2 (Figure 2C, top blot). Blotting with tubulin served as a loading control (Figure 2C, bottom blot). Reduced mature SUR1 band is indicative of a decrease in the abundance of channels that have trafficked past the medial Golgi, where complex glycosylation of SUR1 occurs. Indeed, quantification of surface channels by chemiluminescence assays revealed ∼50% reduction in surface expression of SUR1/Kir6.2-F333I and Y330C mutants compared with WT channels (Figure 2D).
To monitor channel endocytosis biochemically, we employed a surface biotinylation pulse-chase protocol as detailed in Materials and Methods (Figure 2E). Biotinylated WT and mutant channels were chased at 37°C to examine endocytosis (30 min time-point) or kept on ice (0 min time-point). To assay internalized channels only, samples were treated with a membrane-impermeable reducing agent to strip off residual surface biotin label (Figure 2E, stripped, far right). Samples not treated with the reducing agent served as controls to account for the total amount of signal present at 0 and 30 min (Figure 2E, unstripped, left). Densitometry of the western blots was performed to estimate the percent of the initial signal (0 time, unstripped) that was internalized after 30 min of chase for WT and the Y330C mutant. We observed no significant difference between the two (WT: 31.8 ± 4.7% and Y330C: 36.6 ± 8.7%; Figure 2F). Taken together, the imaging and biochemical data led us to conclude that the integrity of the proposed 330YSKF333 endocytosis motif is not essential for KATP channel internalization.
SUR1 is involved in KATP channel internalization
As the 330YSKF333 motif in Kir6.2 is not essential for channel endocytosis, we asked if the SUR1 subunit alone was capable of undergoing endocytosis. To address this question, we inactivated the ER-retention signal RKR by mutating it to AAA (SUR1RKRAAA), which allows SUR1 to traffic to the cell surface in the absence of Kir6.2 (10). As expected, live-cell imaging showed abundant surface expression of BTX tag-SUR1RKRAAA at the start of acquisition in COSm6 transfected with the construct (Figure 3A). Interestingly, BTX tag-SUR1RKRAAA alone showed internalization comparable to WT BTX tag-SUR1/Kir6.2 channels when imaged over a 30 min interval. Movie screen shots at acquisition start and 10-, 20- and 30 min time-points are shown in Figure 3A (internalized channels are indicated by white arrowheads, arrow and inset; compare also Movie S5 of a different cell in Supporting Information). Pulse-chase experiments with the FITC-BSA fluid-phase marker identified intracellular localization of the puncta validating our live-cell imaging results (Figure 3B). Further analysis revealed that there was no statistically significant difference of the average number of internalized BTX-labeled puncta between the SUR1RKRAAA and WT BTX tag-SUR1/Kir6.2 groups. The average number of puncta colocalized with the fluid-phase marker FITC-BSA was similar as well indicating that there was no difference in the number of intracellular puncta (Figure 3C; BTX-labeled: RKR = 16.5 ± 1.7 and WT = 16 ± 2.4; colocalized FITC-BSA: RKR = 9.5 ± 1.2 and WT = 12.2 ± 1.3, N = 2, n > 20 per condition).
Endocytosis of SUR1RKRAAA was further examined by surface biotinylation pulse-chase experiments. The SUR1RKRAAA protein manifests as both the core-glycosylated and complex-glycosylated forms just as WT SUR1; however, the complex-glycosylated SUR1RKRAAA migrates slower on SDS–PAGE than the complex-glycosylated WT SUR1, consistent with previous reports. Expression levels of WT SUR1/Kir6.2 and SUR1RKRAAA were similar whether the constructs were fused to a FLAG or BTX tag (Figure 4A). Supporting our results obtained from the imaging experiments, we observed internalization of SUR1RKRAAA expressed alone with a time–course similar to WT SUR1 coexpressed with Kir6.2 using chemiluminescence pulse-chase assays (Figure 4B) and in surface biotinylation pulse-chase experiments (Figure 4C). Internalization of surface-biotinylated channels was observed upon 30 min of chase for both WT and SUR1RKRAAA(Figure 4C, cf. stripped 0 and 30 min). Densitometry measurements and normalization to the unstripped controls at 0 min revealed that the percentage of endocytosed surface channels after 30 min was comparable between WT and SUR1RKRAAA (Figure 4D, WT: 39.7 ± 4.8% and SUR1RKRAAA: 39.9 ± 12.9%). The similar time–course of internalization was further observed in surface biotinylation experiments with smaller increment of chase time between 0 and 30 min (Figure 4E). Note that in western blots from surface biotinylation experiments only one band of SUR1RKRAAA is detected representing the complex-glycosylated form that traffics to the cell surface (Figure 4C,E).
The experiments described earlier were all carried out in COSm6 cells. We next tested if SUR1RKRAAA endocytosis might be dependent on tissue origin as a previous study reported that SUR1RKRAAA was unable to undergo endocytosis in the human tsA-201 cell line. TsA-201 cells expressing either WT BTX tag-SUR1/Kir6.2 or BTX tag-SUR1RKRAAA were labeled at 4°C and chased for 30 min with FITC-conjugated transferrin included in the media. Both WT SUR1/Kir6.2 and SUR1RKRAAA displayed robust internalization in tsA-201 cells (Figure 5A, left, top: WT, bottom: SUR1RKRAAA) colocalizing with transferrin (merged pictures and insets). Internalization levels were comparable to those observed in COSm6 cells which served as controls (Figure 5A, right). Quantitative analysis of transferrin colocalization revealed that the percentage of BTX dye-stained puncta also positive for staining with FITC-transferrin was not significantly different between WT SUR1/Kir6.2 and SUR1RKRAAA or between tsA-201 and COSm6 cells (Figure 5B). These findings indicate that SUR1RKRAAA endocytosis is independent of tissue or species origin (cf. tsA-201 cells from human versus COSm6 cells from monkey origin). Moreover, SUR1RKRAAA and WT SUR1/Kir6.2 channels follow the same route of endocytosis as suggested by colocalization with transferrin, a marker for recycling endosomes and clathrin-mediated endocytosis (CME). To further investigate SUR1RKRAAA endocytosis, we performed immunofluorescent staining with the early endosomal marker EEA1. BTX tag-SUR1RKRAAA colocalized with EEA1 to the same degree as WT BTX tag-SUR1/Kir6.2 channels (Figure 5C), another indication that both are internalized via similar mechanisms.
SUR1RKRAAA endocytosis is dynamin-dependent and clathrin-mediated
Previous studies have shown that WT KATP channel endocytosis is dependent on the large GTPase dynamin (14). To verify if SUR1RKRAAA internalization is dynamin-dependent as well, we performed live-cell imaging of cells transfected with BTX tag-SUR1RKRAAA in the presence of the dynamin inhibitor dynasore (23)(24). Incubation of cells with 80 µm dynasore 10 min prior to and during live-cell imaging led to a significant decrease in BTX tag-SUR1RKRAAA internalization compared to untreated control (Figure 6A, Movies S5 and S6). Similar observations were made in cells transfected with WT BTX tag-SUR1/Kir6.2 (images not shown). Quantification of intracellular puncta revealed that SUR1RKRAAA internalization was blocked by 84.25 ± 0.48%, similar to the extent of block observed in WT channels (86.34 ± 0.88%) (Figure 6C). Note a small number of fluorescent BTX tag-SUR1RKRAAA puncta were internalized in dynasore-treated cells, consistent with a previous report showing that dynasore blocks endocytosis by approximately 80–90% (25). Thus, like WT channels, SUR1RKRAAA undergoes endocytosis in a dynamin-dependent manner. As dynamin is involved in CME but also caveolae-mediated endocytosis (26,27), we conducted further experiments to test whether endocytosis of WT KATP channels and SUR1RKRAAA is mediated by the clathrin- or caveolae-dependent pathway. We used the drug nystatin in order to block caveolae-mediated endocytic pathways (28). Treatment of cells with 25 µg/mL nystatin did not block or reduce internalization of either SUR1RKRAAA or WT SUR1/Kir6.2 compared to untreated controls (Figure 6B). Quantitative analysis showed that nystatin treatment had no significant effect on endocytosis of either WT SUR1/Kir6.2 or SUR1RKRAAA (Figure 6C, left: WT = 98.5 ± 0.65% and SUR1RKRAAA = 99.6 ± 1.4% internalization of untreated control), indicating that their endocytosis is unlikely to be caveolae-dependent.
The above results establish that WT and mutant channel endocytosis is dynamin-dependent but not caveolae-mediated. Next, we tested whether internalization is mediated by a clathrin-dependent pathway. Cells expressing WT SUR1/Kir6.2 or SUR1RKRAAA were treated with chlorpromazine or potassium depletion to inhibit CME (29)(30). Internalization of both SUR1RKRAAA and WT SUR1/Kir6.2 was almost completely abolished under these conditions (Figure 7A, middle, chlorpromazine: WT = 8.06% and SUR1RKRAAA = 4.1% of untreated control; bottom, K+ depletion: WT = 1.9% and SUR1RKRAAA = 3.6% of untreated control). Transferrin was included in the chase media during the assay as an internal control for CME, and transferrin endocytosis was also almost completely blocked by chlorpromazine and K+ depletion (compare Figure 7A, pictures labeled FITC-transferrin; top panel is untreated control). These findings strongly support the notion that endocytosis of SUR1RKRAAA, just like internalization of WT KATP channels, is clathrin-mediated.
Taken together, our results show that (i) SUR1, at least the SUR1RKRAAA variant, is capable of undergoing endocytosis independently of Kir6.2 and (ii) internalization of SUR1RKRAAA follows the same route as WT KATP channels and (iii) is dynamin-dependent and clathrin-mediated. These data strongly support a role of SUR1 in mediating endocytic trafficking of KATP channels.
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
Regulation of membrane protein endocytosis and recycling is a common and important mechanism to control protein function and plays a critical role in various cellular events such as signaling, synaptic transmission and hormone secretion. In this study, we utilized a live- cell imaging approach to monitor KATP endocytosis in real time. To our knowledge, this is the first study following the dynamic endocytic trafficking of KATP channels in living cells. The use of a high-affinity BTX-binding peptide tag to study protein trafficking by live imaging has been previously documented for a number of ion channels and G-protein-coupled receptors (22,31–33). It offers several advantages over conventional immunohistochemical approaches using antibody labeling in fixed cells or live-cell imaging using fluorescent protein tags such as green fluorescent protein (GFP). Antibodies are large proteins that may limit spatial resolution and may also lead to clustering of surface proteins with possible confounding effects on trafficking (34,35). GFP tagging does not distinguish between intracellular or surface protein pools. Because of the large size of GFP it can affect protein function or trafficking as it has been documented for KATP channels (36). Using patch-clamp recordings, we showed that the extracellular N-terminal BTX tag itself or binding of fluorophore-conjugated BTX to the tagged SUR1 protein does not alter channel gating properties or channel density (Figure S1). We have previously shown that a FLAG tag placed at the N-terminus of SUR1 also does not affect channel function (6). This is likely because (i) in both cases the tags are small peptides minimizing effects on protein folding and (ii) KATP channel gating is mostly regulated by intracellular ligands.
Comparison with previous KATP channel endocytosis studies
Two internalization signals, both in Kir6.2, have been implicated in mediating KATP channel endocytosis in previous studies. The first is a dileucine motif present in the C-terminus of Kir6.2 (355LL356) that has been proposed to mediate PKC-induced, CME of SUR2A/Kir6.2 and SUR1/Kir6.2 KATP channels to downregulate channel function in heterologous expression systems and in cardiac ventricular myocytes (14). The second is a tyrosine-based motif reported by Mankouri et al., also in the C-terminus of Kir6.2 (330YSKF333). In their study, it was concluded that KATP channels undergo constitutive endocytosis mediated by the tyrosine-based motif in Kir6.2 and that SUR1 does not play a signaling role, as surface-labeled myc-tagged SUR1RKRAAA expressed in the absence of Kir6.2 was not internalized over a 30 min time–course. The authors further showed that introducing neonatal diabetes-associated mutations Y330C or F333I in Kir6.2 prevented endocytosis of KATP channels leading to their proposal that reduced endocytosis is a contributing mechanism to increased channel activity and disease (15). In contrast, the studies we present here—using live-cell imaging complemented by immunocytochemistry and surface biotinylation approaches—clearly show that Y330C, Y330A and F333I Kir6.2 mutants do undergo endocytosis similar to WT channels (Figure 2). Also in contrast to the notion that Y330C and F333I cause neonatal diabetes in part by increasing their surface density, our results showed significantly reduced surface expression of these mutants, suggesting they cause increased channel activity and neonatal diabetes solely via effects on gating (37). It is interesting to note that a similar tyrosine motif 332YSRF335 is present in a closely related channel Kir2.3 and has been reported not to be responsible for mediating endocytosis of the channel (38). Moreover, two residues in the proposed Kir6.2 tyrosine motif, Y330 and F333, are predicted to lie closely to the Kir6.2 subunit–subunit interface or SUR1–Kir6.2 interface and may be involved in ATP binding or ATP-induced gating (37,39). Mutations at either of these residues (Y330C and F333I) cause permanent neonatal diabetes by rendering the channel less sensitive to ATP inhibition (37). These studies raise the question of whether the proposed tyrosine motif residues would be available for interaction with adaptor proteins to signal endocytosis.
A role of SUR1 in KATP channel endocytosis
In our study, we found that SUR1RKRAAA itself is endocytosed in the absence of Kir6.2, with a time–course and intracellular distribution similar to WT SUR1/Kir6.2 channels. As these results again contradict with previous studies (15), we repeated some of our experiments in the human tsA-201 cell line used in the previous study to exclude the possibility that the internalization of SUR1RKRAAA is cell-type or species-dependent (Figure 5). Our results showed clearly endocytosis of SUR1RKRAAA also occurs in tsA-201 cells. One possible explanation for the observed discrepancy between the two studies could be the number of cells analyzed. We analyzed two independent experiments with more than 20 cells per condition and a total of more than 200 individual internalized puncta counted (Figures 3C, 5B, and 6C). When screening by confocal microscopy, we found that a small percentage of cells did not show internalization of either mutant or WT channels, possibly because these cells were in a specific stage of the cell cycle (not shown) (40,41). Smaller sample size in the earlier study could lead to the conclusion that endocytosis is impaired. Another explanation for impaired endocytosis of SUR1RKRAAA reported in the previous study might be the use of an anti-myc antibody to investigate protein internalization. Antibodies, although widely used to monitor internalization behavior, can pose problems due to specificity and/or bulkiness of the antibody molecules, for example with significant impact on membrane trafficking (35,42). Therefore, antibodies should be extensively tested in the system used. Ideally, the SUR1RKRAAA endocytosis assay would have been complemented by a secondary method to draw a definitive conclusion about internalization behavior of SUR1RKRAAA.
Our study, using three different approaches, shows that endocytosis of SUR1RKRAAA is comparable if not more efficient than that of WT SUR1/Kir6.2 channels. Moreover, colocalization with various endocytic compartment markers showed similar patterns between internalized SUR1RKRAAA and WT SUR1/Kir6.2. That SUR1RKRAAA alone is internalized independently of Kir6.2 with similar intracellular distribution as WT KATP channels suggest SUR1 itself likely carries endocytosis signals and may contribute to endocytic trafficking regulation of the channel. Moreover, we found that endocytosis of SUR1RKRAAA can be abolished specifically by dynasore, an inhibitor of the GTPase dynamin, and by inhibitors of CME but not caveolae-mediated endocytosis, strongly supporting the concept that SUR1RKRAAA is internalized by a dynamin-dependent, clathrin-mediated process.
Typical CME motifs are dileucine- or tyrosine-based YXXΦ or NPXY (where X represents any amino acid and Φ represents bulky hydrophobic amino acids). Other non-canonical signals such as di-isoleucine in Kir2.3 have also been reported (38). In some cases, multiple signals may be involved to regulate endocytic protein trafficking and sorting as in the cystic fibrosis transmembrane conductance regulator (CFTR), another ATP-binding cassette (ABC) transporter (43). The SUR1 sequence contains many such motifs (>10) and future systematic analysis of these signals will identify those that are relevant to channel endocytosis. In addition to endocytosis, KATP channels have also been reported to undergo recycling (44). Consistent with this, we have observed colocalization of internalized channels with the recycling endosome marker FITC-transferrin. Which signals regulate further sorting of endocytosed channels is also an important question to address in future studies.
In summary, we have developed SUR1 and Kir6.2 tagged with an extracellular α-BTX-binding peptide to follow KATP channel endocytosis by live-cell imaging. These tools could be adapted to studying physiological trafficking regulation of plasma membrane KATP channels in isolated islets or even in vivo. Our findings that mutations in the proposed Kir6.2 330YSKF333 motif do not prevent channel endocytosis and that SUR1RKRAAA alone undergoes endocytosis challenge the previous view that channel endocytosis is solely mediated by the Kir6.2 subunit. They raise new questions on the signaling role of SUR1 in KATP channel trafficking regulation from the plasma membrane, and open up the possibility that mutations in SUR1 may alter endocytic trafficking of KATP channels to cause insulin secretion disease.
Materials and Methods
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
Molecular biology
Rat Kir6.2 cDNA is in pCDNAI/Amp vector and N-terminal FLAG epitope (DYKDDDDK)-tagged SUR1 (fSUR1) in pECE as previously described. A minimal α-BTX-binding (WRYYESSLEPYPD) peptide tag was inserted at the N-terminus of SUR1 (BTX tag-SUR1) in pECE using the polymerase chain reaction (PCR) and the following primers, forward: 5′-GCTTGTCGACGCCGCCATGTGGCGGTACTACGAGAGCAGCCTGGAGC CCTACCCCACATGCCCTTGGCC-3′ and reverse: 5′-GGCCAAGGGCATGTC GGGGTAGGGCTCAGGCTGCTCTCGTAGTACCGCCACATGGCGGCGTCGA CAAGC-3′. In addition, a 9-amino acid linker (YAYMEKGDL) followed by a minimal BTX epitope tag (WRYYESSLEPYPD) was introduced into the turret region of rat Kir6.2 pCDNAI/Amp (BTX+9 Kir6.2) between amino acids L100 and A101 using the PCR and the following primers, forward: 5′-GCATATATGGAAAAAGGAGACCTGTGGCGGTACTACGAGAGCAGCCTGG AGCCCTACCCCGACGCCCCCGGAGAGGGCACCAATGT-3′ and reverse: 5′-ACATTGGTGCCCTCTCCGGGGGCGTCGGGGTAGGGCTCCAGGCTGCT CTCGTAGTACCGCCACAGGTCTCCTTTTTCCATATATGC-3′. Site-directed mutagenesis was performed using the QuikChange mutagenesis kit (Stratagene). Mutant clones from two or more independent PCRs were analyzed and fully sequenced to avoid false results caused by undesired mutations (6,16).
COSm6 cells were cotransfected with cDNAs for BTX tag-SUR1 and Kir6.2 and the GFP (to facilitate identification of transfected cells) using Fugene 6 (Roche), and plated onto coverslips 24 h after transfection. Inside-out patch-clamp recordings were made 36–72 h post-transfection at room temperature using micropipettes (resistance ∼1.5 MΩ) pulled from non-heparinized Kimble glass (Fisher Scientific) on a horizontal puller (Sutter Instrument). Inside-out patches were voltage-clamped with an Axopatch 1D amplifier (Axon Inc.). The bath and pipette solution (internal high potassium solution; K-INT) contained 140 mm KCl, 10 mm K-HEPES, 1 mm K-EGTA, pH 7.3. Currents were measured at membrane potential of −50 mV (pipette voltage = +50 mV) and inward currents shown as upward deflections.
Western blotting
COSm6 cells plated in 35-mm dishes were transfected with 0.6 µg SUR1 and 0.4 µg Kir6.2 using 3 µL Fugene 6 and lysed 48 h post-transfection in 20 mm HEPES, 150 mm NaCl, 4 mm ethylenediaminetetraacetic acid (EDTA), 1 mm EGTA, 1% Ipegal, 0.1% SDS, 0.04% deoxycholate, pH 7.2 with complete protease inhibitor (Roche) for 20 min at 4°C. Proteins in the cell lysate were separated by 7.5, 10 or 4–12% gradient SDS–PAGE (BioRad; Invitrogen), transferred to nitrocellulose membranes (GE Amersham), blocked in 6% non-fat milk/TBS (Tris-buffered saline) and analyzed by incubation with appropriate primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Amersham), and visualized by enhanced chemiluminescence (Super Signal West Femto; Pierce) using the FluorChem®E Digital Darkroom (Cell Biosciences).
Chemiluminescence assays
COSm6 cells were transfected with channel subunit cDNAs as described earlier and assayed 48 h post-transfection. Cells were fixed with 2% paraformaldehyde/PBS for 20 min at 4°C and preblocked in PBS/0.5% BSA for 1 h. Cells were then incubated with anti-FLAG antibody (10 µg/mL; Sigma) for 1 h, washed 3× 20 min each in PBS/0.5% BSA and incubated in HRP-conjugated sheep anti-mouse antibody (GE Amersham) for 1 h. After four washes 30 min each in PBS/0.5% BSA, cells were incubated for 10 seconds in ELISA Femto luminol solution (Pierce), and chemiluminescence was quantified using a TD-20/20 luminometer (Turner Designs). Results of each experiment are the average of two dishes. For pulse-chase internalization assays, cells were first labeled with anti-FLAG antibody (10 µg/mL; Sigma) for 1 h at 4°C. Cells were chased at 37°C for various times to allow internalization and then fixed and processed as above to obtain chemiluminescence measurements.
Biotinylation assays
COSm6 cells were plated in 10-cm dishes and transfected with 3 µg SUR1 and 2 µg Kir6.2 with 15 µL Fugene 6, and subjected to surface biotinylation 36–48 h post-transfection. Surface biotinylation was performed at 4°C by incubation with 1.5 mg/mL Sulfo-NHS-SS-Biotin (Pierce) in PBS for 20 min. Residual free biotin was quenched with two washes of 50 mm glycine/PBS 5 min each at 4°C. Cells were returned to 37°C and chased for allotted times. Endocytosis was terminated by the addition of ice-cold 4°C 10 mmd-glucose/PBS and cells were kept on ice until all time-points were reached. Biotinylated proteins remaining at the plasma membrane were kept intact (referred to as unstripped samples) or stripped of the biotin label with two washes of 50 mm glutathione buffer (75 mm NaOH, 75 mm NaCl, 1 mm EDTA, 0.1% BSA, pH 9.0) 20 min each at 4°C (referred to as stripped samples). Residual glutathione was quenched with 5 mg/mL iodoacetamide/PBS (Sigma) for 5 min at 4°C. Cells were lysed in 1 mL of extraction buffer (50 mm Tris–HCl, 2 mm EDTA, 2 mm EGTA, 100 mm NaCl, 1% Triton-X-100, pH 7.4 with complete protease inhibitor) for 30 min at 4°C; 1 mg of total lysate was incubated with 100 µL of ∼50% slurry NeutrAvidin Agarose (Pierce) overnight at 4°C. Biotinylated proteins were eluted with 2× protein loading buffer for 15 min at room temperature and analyzed by western blotting using anti-FLAG, anti-SUR1 or anti-Kir6.2 antibodies (19). Immunoglobulin (IgG)-purified anti-Kir6.2 was from rabbit serum raised against the C-terminus of Kir6.2 (aa 170-390) fused with glutathione S-transferase (GST) (45). IgG-purified anti-SUR1 was from rabbit serum raised against the C-terminal peptide of hamster SUR1 (KDSVFASFVRADK) (27). Densitometry was performed on 16-bit Tif image files using AlphaView® software (Cell Biosciences). Density data were determined using the band analysis feature and calculated as the background-corrected average sum of pixel intensities within a defined region.
COSm6, INS-1 and tsA-201 cells were cultured following standard protocols. All imaging experiments described below were performed on a Zeiss LSM710 3-channel spectral confocal microscope and imaging was performed with 63× 1.4 numerical aperture (NA) objective (Carl Zeiss). All imaging data are from at least two independent experiments with at least 10 cells/experiment unless otherwise indicated (total n > 20 per condition).
Live-cell imaging
COS cells were plated onto 35-mm dishes and transfected with WT BTX tag-SUR1 and WT or mutant Kir6.2 or BTX tag-SUR1RKRAAA alone. After additional 24 h, cells were split at the appropriate density onto 18-mm, #1.5 glass coverslips (Warner Instruments). Live-cell imaging was performed 48 h post-transfection. Coverslips were placed in the imaging chamber (Carl Zeiss), and the cell culture media were exchanged to Hanks' balanced salt solution (HBSS) supplemented with 10 mm HEPES. The chamber was placed onto the microscope with a stage top incubator warmed to 37°C (Carl Zeiss). TRITC-BTX (Molecular Probes) was added directly into the imaging solution at a 1:200 dilution and image acquisition was started immediately. TRITC-BTX was present in the medium throughout the imaging experiment. Images were taken every 15 seconds for 30 min. Untransfected cells were imaged as control to account for potential unspecific background staining. INS-1 cells were infected with WT BTX tag-SUR1 and WT Kir6.2 recombinant adenoviruses as described previously (46). Live-cell imaging was carried out as described for COSm6 cells. For experiments with dynasore, cells were serum starved for 2 h and incubated with 80 µm dynasore for 10 min prior to imaging. Dynasore was added to the imaging solution as well.
BTX pulse-chase labeling
COSm6 cells were transfected and plated as described for live-cell imaging. TsA-201 cells were plated the same way but transfected using the Ca2PO4 method. For pulse-chase labeling, cells were incubated with TRITC-BTX (Molecular Probes) at 1:200 diluted in DMEM at 4°C. After 1 h the dye was replaced with pure DMEM and cells were further incubated at 37°C, fixed at the desired time-points and viewed under the microscope. Untransfected cells treated the same way served as control to exclude non-specific binding of the dye. For colocalization studies with the fluid-phase marker, cells were treated as described above but Alexa Fluor 488-conjugated BSA (FITC-BSA; Molecular Probes) was included in the chase media at 1 mg/mL. For experiments where colocalization with transferrin was examined or transferrin was used as CME marker, 10 µg/mL FITC-transferrin (Invitrogen) was included in the chase media. For colocalization studies with the early endosomal marker EEA1, cells were treated the same way, fixed after 30-min chase and stained with mouse anti-EEA1 at 1:1000 (BD Transduction Laboratories) and the appropriate secondary antibody. Cells stained with secondary antibody only and untransfected cells served as controls (data not shown).
Inhibition of different endocytic pathways
Treatment of cells with dynasore is described under live-cell imaging. Nystatin (25 µg/mL) treatment of cells was carried out as described previously (27) and BTX pulse-chase labeling was performed in the presence of the drug. For treatment with chlorpromazine, cells were preincubated with 10 µg/mL chlorpromazine in serum-free media for 45 min and the BTX pulse-chase assay was carried out in the presence of the drug. Potassium depletion was carried out as described previously (30) and the BTX pulse chase assay was performed in potassium-free buffer.
Image analysis
All image analysis was performed using Metamorph (Molecular Devices). For the live-cell imaging experiments shown in Figures 1–3, a line- scan analysis was used to track the migration of fluorescence signals from the cell surface toward the interior of the cell over time. The area of analysis is indicated by the box framed by the two solid lines and the two dotted lines in the image such that most of the area of the cell was covered. The maximum intensity of TRITC-BTX fluorescence was determined by a scan along the dotted line for a set pixel width and the scan repeated across the distance defined by the white lines. The maximum fluorescence intensity detected in each scan was then plotted against the pixel distance across the cell (represented by the white lines). Note this analysis does not quantify the total fluorescence signal associated with the plasma membrane or the space between the membranes but simply detects strong intracellular fluorescence signals associated with the brightest endosomes that develop over time.
For pulse-chase experiments with FITC-BSA, colocalization was determined and puncta were counted according to Glynn and McAllister (47). Transferrin colocalization analysis was performed using the colocalization routine of the program. Pictures from the two different channels (488 and 561 nm) were thresholded for light objects. Colocalized objects had to be positive for staining in both channels. Appropriate secondary antibody only stained controls were employed.
Internalization rate of channels was determined by measuring the average intracellular fluorescence intensity of cells after 30 min of pulse chase. Pictures of individual cells were thresholded and a region including the cytosol but excluding the remaining surface stain was drawn for each cell. The average fluorescence intensity of the cytosol was calculated by dividing the integrated fluorescence of the measured region by its size. Values for internalization are expressed as percentage of internalization observed in control cells.
Data averages are mean ± SEM unless otherwise stated. Statistical tests for normally and non-normally distributed data (t-test and Man–Whitney rank sum test) were employed. When statistical tests were applied, a p value < 0.05 was considered significant.
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
We thank the staff, especially Dr Stefanie Kaech-Petrie, of the Advanced Light Microscopy Core at The Jungers Center (OHSU) for their expert help with image acquisition and image analysis. We thank Dr Emily B. Pratt and Dr Qing Zhou for help with supporting information. The INS-1E cell line clone 832/13 was kindly provided by Dr Christopher B. Newgard. Rat Kir6.2 was from Dr Carol A. Vandenberg. This work was supported by National Institutes of Health Grant DK57699 (to S.-L. S.), the March of Dimes Research Grant Foundation Grant 1-2001-707 (to S.-L. S.) and a Collins Medical Trust Foundation Grant (to C. E. B.).
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
- 1ATP-sensitive potassium channelopathies: focus on insulin secretion. J Clin Invest 2005;115:2047–2058.
- 2The Walter B. Cannon Physiology in Perspective Lecture, 2007. ATP-sensitive K+ channels and disease: from molecule to malady. Am J Physiol Endocrinol Metab 2007;293:E880–E889.
- 3The role of the KATP channel in glucose homeostasis in health and disease: more than meets the islet. J Physiol 2010 Sept 1;588(Pt 17):3201–9., ,
- 4Kir6.2 mutations associated with neonatal diabetes reduce expression of ATP-sensitive K+ channels: implications in disease mechanism and sulfonylurea therapy. Diabetes 2006;55:1738–1746., , , , , ,
- 5Focus on Kir6.2: a key component of the ATP-sensitive potassium channel. J Mol Cell Cardiol 2005;38:927–936., , , ,
- 6Defective trafficking and function of KATP channels caused by a sulfonylurea receptor 1 mutation associated with persistent hyperinsulinemic hypoglycemia of infancy. Proc Natl Acad Sci U S A 2001;98:2882–2887., , ,
- 7Loss of functional KATP channels in pancreatic beta-cells causes persistent hyperinsulinemic hypoglycemia of infancy. Nat Med 1996;2:1344–1347., , , , , , , ,
- 8Of mice and men: K(ATP) channels and insulin secretion. Recent Prog Horm Res 2001;56:47–68., ,
- 9Identification of a familial hyperinsulinism-causing mutation in the sulfonylurea receptor 1 that prevents normal trafficking and function of KATP channels. J Biol Chem 2002;277:17139–17146., , , , ,
- 10A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 1999;22:537–548., , ,
- 11
- 12Hyperinsulinism in infancy: from basic science to clinical disease. Physiol Rev 2004;84:239–275., , , ,
- 13Sulfonylureas correct trafficking defects of disease-causing ATP-sensitive potassium channels by binding to the channel complex. J Biol Chem 2006;281:33403–33413., ,
- 14ATP-sensitive potassium channel traffic regulation by adenosine and protein kinase C. Neuron 2003;38:417–432., , ,
- 15Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels. EMBO J 2006;25:4142–4151., , , ,
- 16Peptide tags for labeling membrane proteins in live cells with multiple fluorophores. Biotechniques 2005;38:945–952., , ,
- 17The cytoplasmic C-terminus of the sulfonylurea receptor is important for KATP channel function but is not key for complex assembly or trafficking. Eur J Biochem 2002;269:5303–5313., ,
- 18Glucose deprivation regulates KATP channel trafficking via AMP-activated protein kinase in pancreatic beta-cells. Diabetes 2009;58:2813–2819., , , , , , ,
- 19
- 20
- 21The binding site of acetylcholine receptor as visualized in the X-ray structure of a complex between alpha-bungarotoxin and a mimotope peptide. Neuron 2001;32:265–275., , , , , , , , , , ,
- 22K(V)4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization. Channels (Austin) 2010 MarApr;4(2):115–23., , , , ,
- 23Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 2006;10:839–850., , , , ,
- 24Dynasore puts a new spin on dynamin: a surprising dual role during vesicle formation. Trends Cell Biol 2006;16:607–609.,
- 25Synthesis of potent chemical inhibitors of dynamin GTPase. Bioorg Med Chem Lett 2010;20:4858–4864., , , , ,
- 26Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 2007;8:603–612.,
- 27Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI. J Lipid Res 2007;48:2560–2570., , , , ,
- 28Selective caveolin-1-dependent endocytosis of glycosphingolipids. Mol Biol Cell 2003;14:3254–3265., , , , ,
- 29Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN. Am J Physiol Lung Cell Mol Physiol 2006;290:L946–L955., , , , ,
- 30Depletion of intracellular potassium arrests coated pit formation and receptor-mediated endocytosis in fibroblasts. Cell 1983;33:273–285., , ,
- 31Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc Natl Acad Sci U S A 2004;101:17114–17119.,
- 32Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin. J Biol Chem 1999;274:26113–26119., , , , ,
- 33A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors. J Biol Chem 2004;279:51460–51465.,
- 34Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures. J Neurochem 2007;102:479–492., , , , , , , , , , , , , , et al.
- 35Antibodies as valuable neuroscience research tools versus reagents of mass distraction. J Neurosci 2006;26:8017–8020.,
- 36Independent trafficking of KATP channel subunits to the plasma membrane. J Biol Chem 1998;273: 3369–3374.,
- 37Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6. 2-SUR1 interactions. EMBO J 2005;24:2318–2330., , , ,
- 38AP-2-dependent internalization of potassium channel Kir2.3 is driven by a novel di-hydrophobic signal. J Biol Chem 2008;283:5973–5984., ,
- 39Functional analysis of a structural model of the ATP-binding site of the K(ATP) channel Kir6.2 subunit. EMBO J 2005;24:229–239., , , ,
- 40Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J Cell Biol 1980;85:660–671.,
- 41Endocytosis resumes during late mitosis and is required for cytokinesis. J Biol Chem 2005;280:41628–41635., , ,
- 42Redistribution and stabilization of cell surface glutamate receptors during synapse formation. J Neurosci 1997;17:7351–7358., ,
- 43Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator. Biochem J 2001;354:561–572., ,
- 44Constitutive endocytic recycling and protein kinase C-mediated lysosomal degradation control K(ATP) channel surface density. J Biol Chem 2010;285:5963–5973., , , , ,
- 45Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels. Am J Physiol Cell Physiol 2005;289:C1351–C1359., , ,
- 46Membrane phosphoinositides control insulin secretion through their effects on ATP-sensitive K+ channel activity. Diabetes 2005;54:2852–2858., , , ,
- 47Immunocytochemistry and quantification of protein colocalization in cultured neurons. Nat Protoc 2006;1:1287–1296.,
- 48Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits. J Gen Physiol 2003;122:225–237., , ,
Supporting Information
- Top of page
- Abstract
- Results
- Discussion
- Materials and Methods
- Acknowledgments
- References
- Supporting Information
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1: BTX tag or BTX incubation does not affect channel function. A) Representative inside-out patch-clamp recordings from COS cells cotransfected with BTX tag-SUR1 and WT Kir6.2 not pretreated (left) or pretreated for 30 min with TRITC-BTX at a concentration used in the imaging experiments (1:200) (right). In both cases, currents were readily inhibited by 1 mM ATP, similar to those seen in cells expressing non-tagged channels (48) (not shown). Recordings were made at −50 mV in symmetrical K-INT solution as described in Materials and Methods and inward currents are shown as upward deflections. B) Average current amplitude observed in patches from control and BTX-treated cells. The bar represents the mean ± SEM from 12 (control) or 10 (BTX-treated) cells. There is no statistically significant difference between the two groups by Student's t-test (p > 0.05).
Figure S2: WT BTX tag-Kir6.2 coexpressed with WT SUR1 shows characteristics similar to WT BTX tag-SUR1+WT Kir6.2. COSm6 cells were labeled with TRITC-BTX at 4°C and chased for 30 min at 37°C with the marker FITC-BSA included in the chase media. Cells transfected with WT BTX tag-Kir6.2/SUR1 displayed punctate TRITC-BTX staining (left panel, red, scale bar 10 µm) which colocalized with the fluid-phase marker FITC-BSA (middle panel, green), and thus confirming the intracellular nature of the channel staining (see merged picture and inset, right panel). Channel behavior was similar to WT BTX tag-SUR1/Kir6.2 channels.
All movies were compiled with METAMORPH software and play at 5 frames/second.
Movie S1: Movie corresponds to Figure 1A. A COS cell expressing BTX tag-SUR1 and WT Kir6.2 shows endocytosis of channels over a 30 min time interval. Images were taken every 15 seconds for 30 min (scale bar 5 µm).
Movie S2: Movie of INS-1 cells expressing BTX tag-SUR1 and WT Kir6.2 corresponding to Figure 1B. Images were taken every 15 seconds for 30 min (scale bar 5 µm). In both cases (Movies S1 and S2), internalization of KATP can be visually followed over time as fluorescent puncta budding from the plasma membrane and migrating to the cytosol.
Movie S3: Movie of a COS cell expressing SUR1 WT and BTX tag-Kir6.2. Images were taken every 15 seconds for 15 min. The BTX tag can be fused to either KATP channel subunit with no difference in internalization behavior (compare to Movie S1A, scale bar 10 µm).
Movie S4: 30 min movie of COS cells expressing BTX tag-SUR1 and Kir6.2 Y330C corresponding to Figure 2A. Internalization of BTX tag-SUR1/Kir6.2 Y330C is similar to wild-type channels (scale bar 5 µm).
Movie S5: Movie of COS cells expressing BTX tag-SUR1RKRAAA only. SUR1 internalizes independently from Kir6.2 when the ER-retention signal is mutated (30 min movie, scale bar 10 µm). This movie corresponds to Figure 6A, top as control for the dynasore-treated cell.
Movie S6: Movie of COS cells expressing BTX tag-SUR1RKRAAA with the drug dynasore in the imaging media. SUR1RKRAAA internalization is greatly reduced when dynasore, an inhibitor of dynamin, was present (30-min movie, scale bar 10 µm).
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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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Perform minimal DBA tasks such as making working copies of databases, setup users and permissions on such DBs. Database Architect, IT....
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|Reviews for Whoever said math teachers aren't torturous lied!
| GurlBye chapter 1 . 12/7/2014
I thought the story was really good! Funny too. Especially the ending about how Shawn remembered the pineapple smoothie!
| TabloidJunkieZStar chapter 1 . 12/18/2013
Oh poor Shawny! Good thing he was okay in the end :)
| ilikepsych chapter 1 . 5/20/2013
Well...I liked the story. The Shawn whumps were fun and detailed, as well as the "hopped up on drugs" act at the end. I got a little disinterested in the middle, but that could be because it is past midnight, when i am reading this. I loved the last sentence. SO CUTE! Nice imagery of Shawn putting his fingers to his head:"Psychic Style".
| cindythechef chapter 1 . 3/28/2013
This was really good :) Love me some shawn whump :)
| readernurse chapter 1 . 7/13/2012
Awesome job! I loved the ending...:D
| Dr.RiverSong1003 chapter 1 . 4/3/2012
I love it!
Sent from my iPhone via Pocket Fiction
| InazumaElle chapter 1 . 3/20/2012
Awesome! But about the title, math teachers, let's see: talk about the MOST boring stuff and expect us to listen, not fall asleep and even UNDERSTAND it, make us make stuff that's COMPLETELY gibberish, and more! So, I don't know if anyone would call a math teacher not torturous... ;)
| Automail-gHost chapter 1 . 10/13/2011
HA! I couldn't stop smiling! Great story! :D
It was awesome! I must say, I LOVE iiiitttt! .
It was really good, I hope you continue to write, I would love to read more of your stories! Bravo! Bravo! ENCORE! -
| Indilwenlily chapter 1 . 12/30/2010
HAHAHAHAHAHAHAHHAHAHA I had to stop reading for about 10 minutes when Shawn said "This is why I'm not allowed to drink coffee!" and then went on to tell LASSITER that drugs are bad for you. hahahahahahahaa I also had to reread it over and over to get over how amazingly funny it was!
hahaha great job! I loooooooooove angsty stories, but this kind of angst, torture stuff, PLUS Shawn on painmeds was just to good! XDDDDDD
| Alpine-Dragon-Queen chapter 1 . 10/16/2010
Good story! Very painful... (gosh, now math teachers scare me even more ; ; ) Poor Lassie, fake-loopy Shawn has to not be good his mental state...! XP
| purple909 chapter 1 . 9/19/2010
Oh, this is gold! Many spelling errors but its the story that counts! Plot is beautiful! Characters are close-to-spot-on, which is pretty good, mind you! Plus, it's your first for Psych so... its pure awesomeness!
| Elliot Green chapter 1 . 7/29/2010
I liked the bit at the end, that was cute. Pretty good, although there were a few typos here and there.
| MonikaLisa chapter 1 . 6/16/2010
Very good story! Thank you and keep writing :)
| Nuka-Winch chapter 1 . 6/7/2010
oh chick, I totally loved this fic!
It was amazing. I love the hurt and you totally satisfied me! And it was funny too. Drugged!Shawn is funnier than normal Shawn. As hard I believe.
Sorry, my english is too bad :(. I hope read more hurt!Shawn fics soon.
| BiRDiEs95 chapter 1 . 3/24/2010
That was great! Loved it!


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Low Back Pain
Causes of lower back pain
Short-term (acute) lower back pain generally lasts a few days to a few weeks. Most acute back pain is the result of trauma to the lower back, or the culmination of conditions such as poor posture, obesity, poor physical condition, smoking, stress, or a poor sleeping position. Conditions such as arthritis or disc disease, osteoporosis or other bone diseases, viral infections, irritation to joints and discs, or congenital abnormalities in the spine can also cause pain. Occasionally, back pain "radiates" from the kidneys, abdomen, or other parts of the body.
- muscle aches or stabbing pain in lower back
- limited flexibility or range of motion
- difficulty standing straight
Being physically active significantly improves low back pain. Treatment involves pain relievers, reducing inflammation, restoring proper function and strength to the back, and preventing recurrence of the injury. Most patients fully recover.
General treatment measures
Heat: Applying heat with heating pads or heated blankets can provide short-term relief of acute back pain. Warm baths can also help muscles relax. Avoid sleeping on a heating pad, which can burn and lead to tissue damage.
Bed rest: At night or while resting, lie on one side, with a pillow between the knees to ease pain and reduce stress on the lower back. Bed rest alone can worsen back pain and lead to complications such as depression, decreased muscle tone, and blood clots in the legs. Resume normal activities as soon as possible.
Spinal manipulation: Trained specialists (such as chiropractors or osteopaths) use spinal manipulation to reduce pain and restore mobility.
Massage: Gentle working of the soft tissues can be performed by a massage therapist to help with pain and restore soft tissue mobility.
Nonprescription: Acetaminophen, naproxen, and ibuprofen are pain relievers that can be taken orally to ease pain and reduce stiffness, swelling, or inflammation. Follow the recommended dosage on the label directions when using acetaminophen. The recommended dose for ibuprofen is three 200 mg tablets every four to six hours, taken with food. Do not exceed 2400 mg daily.
Prescription: Prescription medications may be prescribed to manage severe pain. These medications should be used only for a short period of time and under a clinician's supervision. Side effects can include drowsiness, slower reaction time, and impaired judgment. Chronic use of these drugs may be harmful, adding to depression and possibly increasing pain, and has the potential for addiction.
Stretching and strengthening the back
Exercise that helps strengthen the back and abdominal muscles may be the most effective way to speed recovery. In addition to the exercises below, back-healthy activities such as swimming, walking, or movement therapy can help improve coordination, posture, and balance. Any mild discomfort felt at the start of these exercises should disappear as muscles become stronger. If the pain is more than mild and lasts longer than 15 minutes, stop and consult a clinician.
Stretching exercises
Pelvic tilt: Lie on the back with both knees bent and feet flat on the floor. Press the small of the back against the floor, without using the legs. Hold for five seconds. Repeat ten times.
Piriformis stretch: Lie on the back with both knees bent. Cross one leg over the other. Pull the opposite knee to the chest until a stretch is felt in the buttock/hip area. Hold for 20 seconds and relax. Repeat five times on each side.
Knee to chest: Lie on the back with legs straight out in front. Bring one knee up to the chest and grasp the back of the thigh. Pull the knee toward the chest, stretching the buttock muscle. Hold the position for 15 to 30 seconds and return to the starting position. Repeat three times on each side.
Prone lying: Lie on the stomach with the arms placed along the body and head turned to one side. A small pillow under the hips and an ice pack wrapped in a towel may provide additional pain relief. Maintain this position for three to five minutes.
Prone press-up:Slowly work up to the prone press-up. Lie on the stomach with the hands on the floor near the shoulders. Slowly push the upper body off the floor by straightening the arms, keeping the hips on the floor. Hold for five seconds and repeat ten times.
Standing hamstring stretch: Place the heel on a stool about 15 inches high. Keeping the knee straight, lean forward and bend at the hips until a mild stretch is felt in the back of the thigh. Do not roll the shoulders or bend at the waist. Hold the stretch for 15 to 30 seconds. Repeat three times with each leg.
Hamstring stretch on wall: Lie on the back with the buttocks close to a doorway, and extend the legs forward along the floor. Raise one leg and rest it against the wall next to a door frame. The other leg should extend through the doorway. Feel the stretch in the back of the thigh. Hold for 15 to 30 seconds. Repeat three times on each side.
Hip flexor muscle stretch: Kneel with the left foot in front. Slowly shift weight forward, keeping the back straight. Lifting the stomach muscles and pushing the left buttock toward the floor, hold for 30 seconds. Relax and return to the starting position. Repeat with the opposite leg.
Cat and camel:Start on the hands and knees. Let the stomach sag, allowing the back to curve down. Hold this position for five seconds. Then arch the back and hold for five seconds. Do three sets of ten.
Child's pose: Kneel and rest the head on the floor. Feel the stretch in the lower back. Hold for 20 seconds. Do this stretch after any exercise that arches the back.
Strengthening exercises
Quadruped: Start on the hands and knees, keeping the lower back straight. Lift one arm straight up next to the ear. Then extend the opposite leg out to the back. Try not to twist the body. Hold the arm and leg parallel to the floor for five seconds. Return to the starting position and repeat ten times. Then repeat with the opposite leg and arm.
Bridging: Lie on back with both knees bent and the feet flat on the floor. Raise the hips and the lower back off the floor, keeping the lower back straight. Hold the position for five seconds. Relax and repeat ten times.
Partial sit-up: Lie on the back with both knees bent and the feet flat on the floor. Slowly curl the head and shoulders off the floor. Hold briefly for one to two seconds, and then relax. Do 10 to 20 times.
Tips for a healthier back
- Regularly stretch and exercise to keep the back healthy, and remember to always stretch before any strenuous physical activity.
- Stay active. Speed walking, swimming, or biking for 30 minutes a day can increase muscle strength and flexibility. Yoga is another way to gently stretch muscles and ease pain.
- Wear comfortable shoes without a high heel.
- Do not slouch when standing or sitting. When standing, keep weight balanced on the feet. The back supports weight most easily when it is less curved.
- When studying or using a computer, sit in a chair with good back support and proper position and height for the task. Keep the shoulders back. Switch positions often and periodically walk around or gently stretch muscles to relieve tension. A pillow or rolled-up towel placed behind the small of the back can provide some back support. If sitting for a long time, rest the feet on a low stool or stack of books.
- When lifting objects, lift with the knees, pull in the stomach muscles, and keep the head down and in line with a straight back. Keep the object close to the body. Do not twist when lifting and do not lift anything that may be too heavy.
- Use a backpack, slung over both shoulders, to carry books rather than a shoulder bag. Minimize weight to avoid strain.
- Sleep on the side to keep the spine straight. Always sleep on a medium-firm mattress.
- Eat a balanced diet to maintain a healthy weight. Extra weight at the waistline can strain the lower back. A diet with sufficient calcium, phosphorus, and vitamin D promotes new bone growth.
- Don't smoke. Smoking reduces blood flow to the lower spine and causes the spinal discs to degenerate.
When to contact a clinician
Seek care for the following:
- frequent, painful, or bloody urination
- severe pain that spreads to the legs or buttocks
- leg pain that goes below the knee
- a history of back or spine problems
- pain and inflammation not noticeably reduced after 72 hours of self-care
Some symptoms may indicate a more serious medical problem. If any of the following symptoms are experienced, contact a clinician immediately:
- progressive weakness or numbness in the legs
- pain accompanied by fever
- loss of bowel or bladder control
- inability to empty bladder fully


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65_RedRoses is a 2009 documentary film about Eva Markvoort, a young woman from New Westminster, British Columbia, who suffered from cystic fibrosis. The film follows Markvoort as she lives her life undaunted by her disease, waiting for a lung transplant while blogging about her experiences. Wikipedia
Eva Markvoort: The only people who can really understand how I'm feeling right now are the friends I've made online. Eva Markvoort: The internet has become my lifeline to the world.