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Marriage::husband Their::woman Women::property Family::status Rights::legal Which::married
A wife is a female partner in a continuing marital relationship. A wife may also be referred to as a spouse. The term continues to be applied to a woman who has separated from her partner and ceases to be applied to such a woman only when her marriage has come to an end following a legally recognized divorce or the death of her spouse. On the death of her partner, a wife is referred to as a widow, but not after she is divorced from her partner.
The rights and obligations of the wife in relation to her partner and her status in the community and in law varies between cultures and has varied over time.
Wife sections
Intro Summary Related terminology Termination of the status of a wife Legal rights of the wife Exchanges of goods or money Changing of name upon marriage Childbearing Differences in cultures Expectation of fidelity and violence related to adultery See also References
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Tradeoffs between Internal and External Governance: Evidence from Exogenous Regulatory Shocks
Financial Management, Forthcoming
51 Pages Posted: 14 Jul 2011 Last revised: 19 Feb 2016
Date Written: May 21, 2014
We use the 2002 NYSE and NASDAQ listing requirements mandating firms to have a majority of independent directors on the board as an exogenous shock to examine the interaction between internal and external governance. Relative to compliant firms, non-compliant firms significantly reduced exposure to three external governance mechanisms: the market for corporate control, shareholder activism, and credit markets, by adding antitakeover provisions, adopting officer and director protection provisions, and reducing debt levels, respectively. The results are stronger in firms with greater exposure to the relevant external governance mechanism. The evidence suggests that firms treat internal and external governance as substitutes.
Keywords: external governance, internal governance, shareholder activism, corporate control
JEL Classification: G34, G38
Suggested Citation: Suggested Citation
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Carla Geurts (born 20 April 1971 in 2000 Olympic Games in Sydney, Australia for the Netherlands. Geurts won three silver medals during her career at the European Swimming Championships. She retired in 2001, after the 2001 World Aquatics Championships in Fukuoka. Since September 2006, Geurts had been a professor of Kinesiology and Physiology at Brock University, in St. Catharines, Ontario. Geurts lives in Hamilton, Ontario, Canada, since marrying swimming coach Andrew Cole in 2003.
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Austria Bike Tour Reviews 241 to 270 of 651
Country: Austria • REMOVE
- 71% 5 star: 404 reviews
- 21% 4 star: 118 reviews
- 5% 3 star: 31 reviews
- 2% 2 star: 11 reviews
- 1% 1 star: 5 reviews
Along the Danube - Four Country Tour
by Denise G. 2 years ago Permalink/Reply Verified ReviewerWe had a mixed experience on this trip, having completed two other bike and barge trips on smaller boats (and smaller waterways). This is a wonderful region for riding - much of the route follows bike paths that parallel the river and provide gorgeous views of river towns. Unlike our other trips, participants ride on their own without a guide, and this worked well for us. Because the boat travels from Passau to Budapest and back again, there is quite a bit of travel time on the boat. At times, we enjoyed watching the scenery, but this also meant that our time on land was quite limited. Food was decent and several options were available at each meal. The dining room was noisy and it does take quite a bit of time to serve everyone, with the staff clearly working hard! During our tour, there were engine problems which resulted in a change in itinerary. The company struggled to manage this and identify reasonable options. In the end, they have promised us a partial refund, which Read more… seems quite fair. We would have preferred that this offer had come sooner in the process, without the numerous conversations that were needed to help the company understand the concerns of passengers.
Boat: Arkona
Along the Danube - Four Country Tour
by Carla - Partner at Tripsite 2 years ago Permalink/Reply Staff
Hi Denise,
Thanks so much for taking the time to leave us your feedback.
It's unfortunate the ship had engine troubles during your tour. These ships are machines and sometimes things can go wrong. I know it is always a very stressful time for everyone involved. Our local partners work hard to make the best of the situation. I'm glad in the end everything worked out fine. Another adventure in the books! I hope you will be back for more....
Thank you again! ~Carla, Partner at Tripsite
Along the Danube - Four Country Tour
by Dan C. 2 years ago Permalink/Reply Verified Reviewer
Best part? The cycling along the Wachau valley
Boat: Carissima
Along the Danube - Passau to Vienna
by Lois K. 2 years ago Permalink/Reply Verified Reviewer
The entire trip was greater than expected! The staff bent over backwards to accommodate. The gorgeous Danube!
Boat: Arkona
Along the Danube - Four Country Tour
by Lori C. 2 years ago Permalink/Reply Verified Reviewer
I adored the trip from start to finish. The scenery, the towns and cities and of course seeing everything by bike. I met wonderful people who share a love of travel and adventure. The only problem with the trip was the tour leader. He was condescending and rude to several guests. The rest of the staff was phenomenal. I would do this trip again tomorrow.
Boat: Normandie
Along the Danube - Four Country Tour
by Gail W. 2 years ago Permalink/Reply Verified Reviewer
Bike Boat 4 countries Germany, Austria, Slovakia and Hungary - most memorable part of the trip was experiencing the Danube River up close; boat was comfortable and so smooth sometimes I had to look out the window to see if we were moving
Boat: Arkona
Along the Danube - Four Country Tour
by Wendy T. 2 years ago Permalink/Reply Verified Reviewer
Most Memorable Part? The beauty & grandeur of the Danube...and the ability to enjoy this while riding a bike!
Boat: Carissima
St. Moritz to Innsbruck
by Melody S. 2 years ago Permalink/Reply Verified Reviewer
We decided to have our honeymoon on the St. Morritz to Innsbruck self-guided bike tour. We are so happy we did as the scenery was beautiful and the people we met along the way made it memorable. I recommend staying an extra night in St. Morritz so you can take the gondola up the mountain (free from the hotel when you stay 2 nights) to hike and enjoy the views. The bikes were sturdy and had the gears necessary for the hills. Bring your own helmet for this tour as they are not available to rent.
Along the Danube - Passau, Bratislava, and Vienna
by Earl W. 2 years ago Verified Reviewer
Best part of the trip was bicycling through the scenic Wachau wine region. The views of the Danube River and vineyards were magical. Views like you see in PBS commercials for Viking cruises. Bratislava was the most pleasant surprise. A beautiful city with an active cafe culture that is not as big and intimidating as Vienna.
If you are unsure of your bicycling skills consider the hop-on hop-off tour option for Vienna instead of the bicycle tour. The crowds and heavy traffic make the bicycle tour slightly treacherous.
The hardest bicycling day ironically was the first day with steep hills up to the Mauthausen concentration camp and the Clam Castle both of which were worth seeing. If you question whether you are in good enough physical condition for this trip rent the electric bikes, they make a huge difference.
Boat: Theodor Körner
Along the Danube - Four Country Tour
by Susan W. 2 years ago Permalink/Reply Verified Reviewer
I would highly recommend this trip. The food was fantastic. The rooms were of a good size and clean. The staff were friendly and helpful. The cycle route were scenic and enjoyable. I would definitely do another.
Boat: Normandie
Along the Danube - Passau to Vienna aboard the Primadonna
by Janet C. 2 years ago Permalink/Reply Verified Reviewer
Riding along the Danube river was fantastic! We loved all the port cities and the scenery was picturesque. Our favorite memory of the trip was being able to explore on our own each day. Guiding ourselves on the daily rides was challenging but very rewarding. Usually we saw our fellow shipmates along the way and had lunch companions for our daily Danube picnics!
Boat: Primadonna
Along the Danube - Four Country Tour
by Paul H. 2 years ago Permalink/Reply Verified Reviewer
What an unforgettable trip! We were able to take our 75 year old dad and he cycled the whole thing easily. The touring days in the cities were full, overwhelming, and inspiring for someone who's never seen the rich history of Europe. Cruising down the river while eating a 4 course dinner was a perfect finish to cycling though forests and orchards all day. It was a perfect combination of cycling, cruising, relaxing and sight seeing. I would recommend it to anyone!
Boat: Normandie
Along the Danube - Four Country Tour
by Paula H. 2 years ago Permalink/Reply Verified Reviewer
This trip provides a chance to explore the rural areas along the Danube by bike and to visit the great cities of Budapest and Vienna. There is a lot packed into this trip.
Boat: Normandie
Along the Danube - Four Country Tour
by Sherry D. 2 years ago Permalink/Reply Verified Reviewer
This was a perfect way to spend a holiday. The food was AMAZING! The staff was personable, kind, and so very efficient with all they do. I was pleasantly surprised with all the paved bike paths and the number of cyclists out traveling on vacation. It was inspirational to interact with them along the way. Thank you, Normandie and staff. We had a fantastic time!
Boat: Normandie
Along the Danube - Four Country Tour
by Sandi B. 2 years ago Permalink/Reply Verified Reviewer
Cycling past the remnants of the Iron Curtain. WOW!
There were so many highlights, but having Axel as our guide certainly added a great deal to our daily rides. Thanks Axel!
Boat: Carissima
Danube Bike Path - Passau to Vienna
by Diane W. 2 years ago Permalink/Reply Verified Reviewer
The beautiful scenery along the river was the most memorable part of the trip. We also loved all the little cafes right by the path for lunch and breaks.
Along the Danube - Four Country Tour
by Ursula T. 2 years ago Permalink/Reply Verified Reviewer
Biking along the Danube and in the evening sitting watching the sunset on the top deck. I would like more time on the bike, but happy we got to see so much!
Boat: Arkona
Along the Danube - Four Country Tour
by Tracy F. 2 years ago Permalink/Reply Verified Reviewer
The entire trip was wonderful! The food was great, accommodations were perfect and the scenery is spectacular.
Boat: Arkona
Around Lake Constance
by Andrea S. 2 years ago Permalink/Reply Verified Reviewer
Most memorable thing from our Lake Konstanz trip?? How in the world do I pick?? The Biergartens? Picnics in medieval towns? Watching World Cup with locals in small family cafés? The acres of apple orchards? Seeing zeppelins flying over crystal blue water? Feeling the thunder of Rhein Falls? Getting lost and finding amazing parks and kind people? It was the BEST vacation we've ever had, and we're already talking about which one to do next year!
Along the Danube - Four Country Tour
by Larry B. 2 years ago Permalink/Reply Verified Reviewer
Biking along river was the greatest. Budapest and Bratislava wonderful. Time in Vienna too rushed. Strongly advise participants to take an extra day or two in Passau before the trip—a lovely small town.
Boat: Normandie
Around Lake Constance
by Tama L. 2 years ago Permalink/Reply Verified Reviewer
My dissatisfactions, two nights in the suburbs of Constance, out of the way, should not be used. Konono staff excellent, but cost us for taxi to get there and back to the train. Route book imprecise. What is the route sign we were supposed to follow? What good is a dotted line on a map when there are multiple routes? Which are more scenic? Less hilly? Passing by cafes? Unpaved in a forest? Exactly how do you get to the Rhein Falls ferry? Book never mentioned the three elevators that take you up and down. We had frustrations every day.
Is it a beautiful trip? Absolutely. The Mozart and Park Villa are beautiful hotels, although not air conditioned.
Around Lake Constance
by Jehiel from Tripsite 2 years ago Permalink/Reply Staff
Hi Tama,
Sorry to hear you were dissatisfied with certain aspects of this tour. As far as the hotel locations, we always try to provide the best hotels possible (location, quality, reputation) but this is based on availability. This region is very popular and hotel development hasn't quite caught up with demand.
In reference to the Route Book....we strongly recommend that clients also request GPS tracks to supplement Route Books. The tracks are more interactive and tend to be more precise. If requested, these tracks can be downloaded beforehand and used with a GPS or your smartphone. See this Blog to learn more.
Im glad you still had an enjoyable time and appreciated the beauty of this region. We hope to help you plan your next trip!
Around Lake Constance
by Peg Hanson 2 years ago Permalink/Reply Verified ReviewerI did the 7 day Around Lake Constance. This was my 4th Tripsite bike tour. I will list the positive things first.
I booked the Category 4 hotel package and was extremely pleased with all the hotels. Location was good for all except the Park Hotel in Uberlingen. It is located at the top of a very steep hill and was very difficult to find even after you saw the sign pointing the way!!
The breakfasts at all the locations were amazing and so beautifully displayed. A wonderful way to start the day!
The bikes were comfortable and almost brand new. Panniers, front bag and trip meter were all very nice.
Now the not so positive...
I had read all the reviews before booking and noticed the negative comments about the bike route directions. Since these comments were from a year and 2 years ago I assumed they had improved this info. NOT TRUE. The book with the routes is almost worthless. Getting from city to city was not always the problem but once in a city the Read more… situation changes. It is even more challenging when you do not speak the language. The locals are more than friendly and always willing to help. However they often assume we know more than we do and even their help does not get us to our destination. A suggestion would be to provide the cyclists with GPS or a HOTSPOT so that we could download the routes on our phones. I realize that if the customer is from Europe they can do that easily but those of us from the USA depend on a wifi connection for this service. I stayed recently at a hotel in Madrid that provided each room with a Hotspot at NO EXTRA charge. If a mid level hotel can afford to do this you would think a cycling touring company could do the same. I realize Tripsite does not have control over some of these situations but if they could encourage this company to try some more advanced methods of guiding their clients I am sure everyone would be happier. I had a wonderful holiday with fabulous weather, beautiful scenery and memorable meals. I will be booking with Tripsite for my next cycling tour. Just wanted to raise this issue to help future travelers be aware.
Around Lake Constance
by Annette 2 years ago Permalink/Reply
I have found I download the app mapsme and put in the hotels before I leave home. I can still access this by satellite and not needing data when getting near. or can probably do the same with the Google maps app and again use offline...also helpful en route.
Pleased the trip went well, we are looking into it for next year.
Along the Danube - Four Country Tour
by Dean Y. 2 years ago Permalink/Reply Verified Reviewer
Great bike routes. Vast majority on nice bike paths.
Boat: Carissima
Along the Danube - Four Country Tour
by Donald V. 2 years ago Permalink/Reply Verified Reviewer
The staff on the boat were wonderful. Very helpful, friendly and hard working. The food on the boat was excellent. They consistently turned out perfectly prepared delicious meals. The bicycles were in excellent condition and performed perfectly. I thoroughly enjoyed this trip.
Boat: Normandie


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*Isoform 1; a splice variant with an additional 25 amino acids is known (isoform 2): this does not form functional channels)
The human a7 gene is partially duplicated in the same chromosomal region
used extensively in investigations of receptor structure and function by electron microscopy (see below).
The muscle-type ACh receptor is a glycoprotein complex (-290kDa), which consists of five subunits arranged around a central membrane-spanning pore. Nicotinic subunits are similar in amino acid sequence and have the same topology (Fig. 11.1): each subunit consists of a large extracellular amino-terminal domain, four predicted membrane-spanning segments (M1-M4), and a long cytoplasmic loop between M3 and M4.
These characteristics are shared with sub-units that form other ion channels/receptors and thus define a receptor superfamily, usually referred to as the nicotinic family. All members in this superfamily function as either cation- or anion-selective channels, thereby mediating fast excitatory or inhibitory synaptic transmission. In mammalian cells, the cation-selective members include nicotinic and 5HT3 receptors, whereas the anion-selective members include GABAa, GABAc, and glycine receptors. Anion-selec-tive channels in this family are also found in invertebrates: these channels are gated by glutamate, 5-HT, histidine, and acetylcholine (1).
The muscle-type receptor has the composition (al)2, ft y, and 8 in embryonic (ordener-vated) muscle, but in the adult the ysubunit is replaced by an e subunit. The adult receptor is found, at high density, only in the end-plate region of the muscle fiber, but before innervation embryonic receptors are distributed over the whole muscle fiber. The electric organ contains only the embryonic y-form of the receptor. All the subunits share a high degree cf homology (typically 31 —41 % pairwise identity to the a subunits, depending on the species).
The properties of and interactions between individual subunits have been explored extensively by a range of biochemical, molecular genetic, and electrophysiological techniques [for recent reviews, see Karlin (2); Corringer et al. (3)]. Their order around the pore is most likely to be a, y, a, ft and 5 going in the clockwise sense and viewed from the direction of the synaptic cleft. Opening of the channel occurs upon binding of ACh to both a subunits (a, and a,) at sites that are at, or close to, the interfaces made with neighboring y and 5 sub-units (4-6). These sites are shaped by three separate regions of the polypeptide chain (3) and include the so-called C-loop (see below).
2.2.1 Molecular Architecture. The tubular crystals from the Torpedo ray form the basis of almost all quantitative three-dimensional studies of the whole receptor [e.g., see Kistler and Stroud (7) ;Miyazawaet al. (8)1. Tubesare built from tightly packed ribbons of receptor dimers and intervening lipid molecules (9). They grow naturally from the isolated postsynaptic membranes, retaining a curvature similar to that at the crests of the junctional folds. Apparently, there is a close structural correspondence between the tubes, which are simply elongated protein-lipid vesicles, and the receptor-rich membrane as it exists in vivo.
Ice-embedded tubes, imaged with the electron microscope, can be made to retain their circular cross section and be analyzed as helical particles (10). At low resolution, using this approach, the receptor appears as an approximately 70 x 160-A (diameter x length) cylinder composed of five similar rod-shape sub-units arranged around the central axis and aligned approximately normal to the membrane plane. The ion-conducting pathway, delineated by the symmetry axis, appears as a narrow (unresolved) pore across the mem-
Figure 11.2. Architecture of whole receptor, emphasizing the external surface and openings to the ion-conducting pathway on the outer (extracellular) and inner (cytoplasmic) sides of the membrane. The positions of the two a subunits, the binding pockets (asterisks),gate of the closed channel (upper arrow), and the constrictingpart of the open channel (lower arrow) are indicated. See color insert.
Figure 11.2. Architecture of whole receptor, emphasizing the external surface and openings to the ion-conducting pathway on the outer (extracellular) and inner (cytoplasmic) sides of the membrane. The positions of the two a subunits, the binding pockets (asterisks),gate of the closed channel (upper arrow), and the constrictingpart of the open channel (lower arrow) are indicated. See color insert.
brane, bounded by two large (-20-A diameter) vestibules. Further development of this approach has led to resolutions of 9 A (ll)and, more recently, 4.6 A (8), being achieved.
Figure 11.2 shows the appearance of the whole receptor at 4.6-A resolution. In the extracellular portion, the subunits form a pentagonal wall around the central axis and make the cylindrical outer vestibule of the channel. The outer vestibule is about 20 x 65 A (width length). About halfway up this portion are the ACh-binding regions in the two a subunits (asterisks). In the cytoplasmic portion, the subunits form an inverted pentagonal cone, which comes together on the central axis at the base of the receptor, so shaping a spherical inner vestibule of the channel. This is about 20
A in diameter. The only aqueous links between the inner vestibule and the cell interior are the narrow (<8-9 A wide) ' "windows" between the subunits lying directly under the membrane surface. The gate of the channel, made by the pore-lining segments, M2, is near the middle of the membrane (upper arrow), and the constriction zone (the narrowest part of the open channel) is at the cytoplasmic membrane surface (lower arrow).
2.2,2 The Vestibules. One likely physiological role of the vestibules is to serve as prese-lectivity filters for ions, making use of charged groups at their mouths and on their inner walls, to concentrate the ions they select for (cations), while screening out the ions they discriminate against (anions). In this way, the ionic environment would be modified close to the narrow membrane-spanning pore, increasing the efficiency of transport of the per-meant ions and enhancing the selectivity arising from their direct interaction with residues and/or backbone groups lining the constriction zone. A more direct means of increasing the cation conductance may be achieved by rings of negative charge located at the mouth of the pore. These rings (at positions —4\ -1\ and 20' of M2, using the numbering system for M2 residues defined in Fig. 11.3) are significant in that they have been shown to influence channel conductance (12).
Consistent with a screening role, the cylindrical shape and about 10-A radius of the outer (extracellular) vestibule provide a route that is narrow enough for charged groups on the inner wall to influence ions at the center, but not too narrow to restrict their diffusion. The design of this portion of the receptor might therefore have some parallels with the fast-acting enzyme acetylcholinesterase, where the whole protein surface plays a role in producing an electrostatic field that guides the positively charged ACh substrate to the active site (13). The inner (cytoplasmic)vestibule is architecturally distinct from the outer vestibule, yet presumably plays a similar functional role in concentrating the cations, given that electrophysiological experiments on the muscle-type receptor have shown that there is no marked preference for cations to go in one direction across the membrane (i.e., rectifica-
Figure 11.3. Helical net plot of the amino acid sequence around the membrane-spanning segment 1VE (Torpedo a subunit). The leucine residue near the middle of the membrane (yellow) is the conserved leucine L251 (at the 9' position), which may be involved in forming the gate of the channel. The dots denote other residues that have been shown to affect the binding affinity of an open channel blocker (17,18) and ion flow through the open pore (19). The numbers shown refer to the numbering scheme for M2 residues used in the text. See color insert.
Figure 11.3. Helical net plot of the amino acid sequence around the membrane-spanning segment 1VE (Torpedo a subunit). The leucine residue near the middle of the membrane (yellow) is the conserved leucine L251 (at the 9' position), which may be involved in forming the gate of the channel. The dots denote other residues that have been shown to affect the binding affinity of an open channel blocker (17,18) and ion flow through the open pore (19). The numbers shown refer to the numbering scheme for M2 residues used in the text. See color insert.
tion). Negatively charged groups framing the windows would have a strong local effect, given that the windows are not significantly wider than the diameter of an ion, including its first hydration shell.
The large proportion of mass (-70%) that is not within the membrane, and shapes these vestibules is also needed for other purposes, such as making the (complex) ACh-binding pockets, and providing sites of attachment for regulatory molecules and other proteins (such as rapsyn) that are concentrated at the synapse.
and therefore must correspond to M2, the stretch of sequence shown by chemical labeling (14,15) and by site-directed mutagenesis/ electrophysiology experiments (12, 16, 17) to be lining the pore. In the shut-channel form cf the receptor, this helix is bent inward, toward the central axis, making the lumen of the pore narrowest near the middle of the membrane. This is the most constricted region of the whole ion pathway and therefore presumably corresponds to the gate of the channel.
A tentative alignment can be made between the three-dimensional densities and the amino acid sequence of M2 (Fig. 11.3) (11). This alignment places the charged groups at the ends of M2 symmetrically on either side of the lipid bilayer and a highly conserved leucine residue (Torpedo aLeu251) at the level of the bend. It seems likely that the leucine side chains, by side-to-side interactions with neighboring M2 segments, are involved in making the gate of the channel. Site-directed mutagenesis experiments, combined with electrophysiological study of function, have highlighted the uniqueness of the conserved leucine residue in relation to the gating mechanism. The profound effects of mutating this leucine to a hydrophilic amino acid on the agonist sensitivity of the receptor and its desen-sitization properties were first reported for the recombinant homomerica7 neuronal nicotinic receptor by Revah et al. (20). In the muscle-type receptor, progressive replacement of leucines by serines (21) or by threonines (22) increases, by roughly uniform increments, the sensitivity of the channel (i.e., decreases the EC,, value for ACh). A similar effect is seen in neuronal nicotinic receptors that contain a3, P4, and j83 subunits (23). However, other experiments [e.g., Wilson and have been interpreted to indicate that the gate is located closer to the cytoplas-mic membrane surface.
2.2.3 Membrane-SpanningPore. The membrane-spanning portion of the receptor has not yet been completely resolved by direct structural methods, although the pore-lining segments are partially visible as a ring of five rod-shape densities, consistent with an a-heli-cal configuration. This helix is the part of the structure closest to the axis of the receptor
2.2.4 The Snail ACh-Binding Protein. Before going on to discuss the agonist binding site, we next discuss the snail acetylcholine-binding protein (AChBP). Glial cells in the snail, Lymnea stagnalis, produce and secrete this protein, which is a homopentamer having structural homology with the large N-termi-nal portion of the extracellular domain of ion
Cys M2-M3
loop loop
Figure 11.4. Three subunits of the Lymnaea AChBP viewed perpendicular to the fivefold axis of symmetry and from the outside of the pentamer. The inner and outer sheets of the j8-sandwich are blue and red, respectively, whereas the putative ligand HEPES is purple. The approximate positions of the a carbons of residues discussed in the text are marked with arrows on the foremost subunit. The approximate positions of the cell membrane and of the M2-M3 loop are shown diagrammatically. The inner j3-sheet (blue) is thought to rotate after agonist binding and to interact with the M2-M3 loop (as indicated by the asterisk). See color insert.
Cys M2-M3
loop loop
Figure 11.4. Three subunits of the Lymnaea AChBP viewed perpendicular to the fivefold axis of symmetry and from the outside of the pentamer. The inner and outer sheets of the j8-sandwich are blue and red, respectively, whereas the putative ligand HEPES is purple. The approximate positions of the a carbons of residues discussed in the text are marked with arrows on the foremost subunit. The approximate positions of the cell membrane and of the M2-M3 loop are shown diagrammatically. The inner j3-sheet (blue) is thought to rotate after agonist binding and to interact with the M2-M3 loop (as indicated by the asterisk). See color insert.
channels in the nicotinic superfamily. The protomer of AChBP is composed of 210 amino Is and has 20-23% sequence identity with muscle-type ACh receptor subunits. It tains most of the residues that were previ-ly suspected to be involved in ACh binding the receptor. Its crystal structure was ed recently to 2.7-A resolution (25), re-vealling the protomer to be organized around sets of j3-strands, forming Greek key-like ;ifs, folded into a curled /3-sandwich. The indwich can be divided into inner- and jr-sheet parts, shown respectively in blue and Ired in Figure 11.4, which are covalently linked together through a disulfide bond. The -loop11 disulfide bond (C128-C142 in Tor> and human al subunits) plays an important! structural role in stabilizing the three-dimensional fold (25) and is absolutely conserved among all members of the ion channel superfamily.
AChBP has been crystallized only with HEPES, rather than ACh, bound, and "owing to low occupancy and limited resolution, the precise orientation of the HEPES molecule cannot be definitely resolved" (25). In overall appearance, it is very similar to the extracellular domain of the receptor (Fig. 11.5). The C-loop is particularly prominent both in AChBP (Fig. 11.4) and in the receptor, where it is shown as the projection labeled C in Fig. 11.5. The C-loop contains several conserved residues that are thought to be part of the acetylcholine binding site: two adjacent cysteines that are characteristic of a subunits, homologs of o:C192, «C193, two tyrosines (c*Y190, <*Y198), and an aspartic acid (aD200). It is orientated more tightly against the neighboring
Figure 11.5. Wooden model of the extracellular part of the ACh receptor, based on the 4.6-A map of the shut channel (8). The membrane surface is at the bottom of the figure. C denotes the C-loop region of the receptor (see also AChBP, Figs, 11.4 and 11.6), which makes part of the ACh-binding site. The width of each wooden slab corresponds to 2 A. [From Unwin et al. (30).]
Figure 11.5. Wooden model of the extracellular part of the ACh receptor, based on the 4.6-A map of the shut channel (8). The membrane surface is at the bottom of the figure. C denotes the C-loop region of the receptor (see also AChBP, Figs, 11.4 and 11.6), which makes part of the ACh-binding site. The width of each wooden slab corresponds to 2 A. [From Unwin et al. (30).]
subunit in AChBP than it is in the (unligan-ded) receptor. These residues on the C-loop, and other nearby aromatic residues (see below) that form part of the binding site in AChBP, are homologs of residues that have been postulated to form the binding site of nic-otinic receptors, on the basis of mutational studies and/or photoaffinity labeling studies. One exception is H145 (see Fig. 11.6a),which aligns with oY151 and which had not been thought to be important. Also, some residues postulated to be part of the binding site by other methods do not appear to be in the AChBP binding site (e.g., aY86).
The two ACh binding sites are located in the extracellular domain, about 30 A from the membrane surface, or 45 A from the gate. Although the actual ligand-binding site has not yet been identified definitively within the three-dimensional structure of the receptor, is expected to bind through cation-a interactions, where the positive charge of its quaternary ammonium moiety interacts with electron-rich aromatic side chains (26). The recently solved structure of AChBP (25), discussed above, shows that the "signature"
aromatic residues lie in a pocket next to tht interface with the anticlockwise-positionec protomer, as seen from the "synaptic cleft.' The pocket identified in AChBP would lie be hind the protruding densities, labeled C in Fig 11.5, near the a/y and a/8 interfaces. The den sities at C can also be identified with the C loop structure in AChBP (see Fig. 11.4), al though they do not curve around so tightly toward the neighboring subunits, making i more open cleft in the (unliganded) receptor.
The key aromatic residues at the binding site are most likely aY93, aW149, aY190, anc aY198. These residues are located in thre< separate loops of the polypeptide chain (3) designated A (Y93), B (W149), and C (Y19( and Y198). All were identified as being neai the agonist binding site by labeling with i small photoactivatable ligand that covalentlj reacts with the receptor upon UV-irradiatior and acts as a competitive antagonist (27), anc the first three are highly conserved in alignec positions of all muscle and neuronal a sub units. Experiments in which a series of unnat ural tryptophan derivatives were substitutec in place of the natural residues have suggestec that the side chain of aW149 is in van dei Waals contact with the quaternary ammo nium group of ACh in the bound state of thi receptor (26). Chemical labeling has alsc shown that the pair of adjacent cysteines (a;C192 and <xC193) is likely to be close to the binding site (28).
Figure 11.6a shows the binding site regior for the AChBP with the ligand (HEPES), tc show the position of the residues mentionec above. The "plus" side of the interface (Fig 11.6a) is analogous with the a subunit of the nicotinic receptor.
The most important residues in neighbor. ing subunits that influence ACh binding art: W57 of the 5 subunit and the homologous W5£ of the y subunit (6, 29). The "minus" side ol the AChBP interface, shown in Fig. 11.6b would be the y/e or the 8 subunit in the receptor, but in the AChBP it is another identica" subunit. The residues shown to be in contaci with the ligand, labeled in Fig. 11.6b, mostlj have no obvious analog in the ACh receptor The one important exception is W53, which corresponds to the tryptophan residues men. tioned above. Consequently, the snail protein
Figure 11.6. The binding site of AChBP. The ligand (HEPES) is in yellow. Blue and red regions denote the inner- and outer-sheet parts of the j3-sandwich (30). The views in a and b are with the fivefold axis vertical, and the "'membrane" at the bottom. (a) The structure shown is analogous to the binding site of the the Torpedo or human ctl subunit, to which the numbering of identified residues refers. Numbers in parentheses refer to AChBP. (b) Surface of the neighboring protomer that faces the binding site of AChBP. In the receptor this would be part of the y, e> or 6 subunit. Numbers in parentheses refer to the AChBP. W53 aligns with W55 in mouse y, e, and 6 nicotinic receptor subunits, but most of the residues have no obvious analog in the y, e, or 6 subunits of the nicotinic receptor. For example, Q55 aligns with mouse 7E57, eG57, and SD57; LI 12 aligns with mouse ?Y117 or ST119, and Y164 is A or G in the nicotinic subunits. See color insert.
model is rather less helpful about the non-a side of the receptor interface.
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