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An Epithelial Ca2+-Sensor Protein is an Alternative to Calmodulin to Compose Functional KCNQ1 Channels

Background/Aims: KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+-sensor protein, calmodulin (CaM), to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. Methods: We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL) and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. Results: When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. Conclusion: Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels.


Introduction
KCNQ1 is a pore-forming subunit of voltage-gated K + channels. Inherited mutations in KCNQ1 alter the duration of cardiac action potentials, leading to life-threatening arrhythmias in the worst cases [1,2]. KCNQ1 channels are also expressed in multiple types of epithelia and In-gel fluorescence detection The E. coli cell lysate (100 µl) prepared as described above was incubated at different temperatures for 10 min, and then centrifuged at 50,000 x g for 20 min. A small aliquot of the supernatant (1.25 µL) was diluted with lysis buffer, mixed with standard Laemmli sample buffer, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fluorescence imaging was performed using a Typhoon 9410 scanner (GE Healthcare), where the GFP were excited by a 488-nm laser, and the images were subjected to densitometry analysis using ImageJ 1.40g software [46].

Immunoprecipitation analysis
HEK cells were transfected with plasmids carrying KCNQ1, CaM, or Calml3 using X-tremeGENE 9 DNA transfection reagent (Roche, Mannheim, Germany). Two days after transfection, the cells were rinsed and collected into ice-cold phosphate buffered saline. The cells were sonicated in 500 µL of membrane preparation buffer (20 mM HEPES, 150 mM NaCl, 12.5 mM KCl, 0.5 mM EDTA, 1 mM PMSF supplemented with complete protease inhibitor cocktail). Membrane fractions were collected by ultracentrifugation and stored at -80°C. The membrane proteins were solubilized with 2% (v/v) Triton X-100 in the membrane preparation buffer. After pre-absorbed non-specific binding with non-immune IgG and protein G Sepharose (GE Healthcare), either rabbit anti-KCNQ1 (APC-022; Alomone) or mouse anti-c-Myc antibody (Clontech) was mixed with the extract to isolate the protein complexes. The immune-complexes were separated by SDS-PAGE. Before transferring the electrophoresed proteins to polyvinylidene difluoride membranes, the gels were cut off below 60 kDa for KCNQ1 detection and above 25 kDa for Calml3 detection.

Electrophysiological recording and data analysis
The treatment of frogs (Xenopus laevis) was carried out in accordance with the Guidelines for the Use of Laboratory Animals of Osaka University Medical School. The oocytes were surgically removed from frogs anaesthetized with 0.35% tricaine methanesulfonate (Sigma-Aldrich, MO), and were defolliculated in 1 mg/ mL type I collagenase solution (Life Technologies). All clones were transcribed in vitro with mMESSAGE mMACHINE Transcription kits (Life Technologies). The cRNA of KCNQ1 (0.5 ng) was injected into oocytes in various combinations with the cRNA of either CaM or Calml3 cRNA (10 ng), and the cRNA of Ci-VSP (1-5 ng). Membrane currents were recorded using a conventional two-microelectrode voltage-clamp technique with a GeneClamp 500 amplifier (Molecular Devices, CA) 3-5 days after injection of cRNA into the oocytes. All experiments were conducted at ambient temperature (20-24°C). The glass electrodes had a resistance of 0.4-1.2 MΩ when filled with 3 M KCl. The bath solution for recording KCNQ1 contained 2 mM KCl, 96 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES, and 150 µM niflumic acid (pH 7.35 with KOH). For recording Kir2.3, the concentrations of KCl and NaCl were changed to 40 mM and 58 mM, respectively. Voltage clamp protocols are described in each figure legend. The leak subtraction was carried out by subtracting the linear ohmic function calculated from the difference in current amplitudes at -100 mV and -90 mV. Tested oocytes were prepared from at least five different frogs. Data acquisition and fitting was carried out using the Clampfit software (Molecular Devices) and SigmaPlot (Systat Software, Chicago, IL). Data are presented as the mean ± S.E.M. (n = number of observations).

Development of fluorescence-based detection techniques for KCNQ1-CaM complex
We next expressed a KCNQ1cL construct tagged with GFP on its N-terminus (Fig. 1A). The cell lysate was separated on a size exclusion chromatography (SEC) column, which is connected in series to a fluorescence monitor to detect GFP fluorescence in the eluate (FSEC) (Fig. 1C) [47]. In the absence of CaM, there are two fluorescence peaks: the first was collected at the void volume, which presumably reflects the contaminated aggregates, and the second was collected at an elution volume close to the molecular weight of GFP, suggesting degradation products. However, in the presence of CaM, a fluorescence peak was prominently observed in fractions corresponding to a molecular weight of approximately  Thermal stability is one of the biochemical characteristics of a protein complex. To assess the thermal stability of the KCNQ1cL-CaM complex, aliquots from the same E. coli cell lysate described above were incubated at different temperatures for 10 min, clarified by centrifugation, and then subjected to FSEC ( Fig. 2A). The height of the fluorescence peak corresponding to the hetero-octamer was lowered by increasing incubation temperatures. On the other hand, the fluorescence peak recovered at the void volume disappeared after low heat treatment (45°C), but the fluorescence peak corresponding to the GFP was stable at temperatures up to 65°C.
GFP is resistant to low concentrations of SDS, and the fluorescence can be detected in SDS-PAGE gels, even though it is fused to other proteins [48]. Given the thermal stability of the GFP-KCNQ1cL-CaM complex in FSEC and the recovery of the majority of the fulllength product in the hetero-octamer fractions, we were interested in studying its thermal stability using this in-gel fluorescence-detection technique. The lysates of cells expressing both KCNQ1cL and CaM were incubated at different temperatures for 10 min, clarified  Assembly of selective Ca 2+ -sensors with KCNQ1 Using biochemical techniques, we tried to assess the ability of Ca 2+ -sensor proteins to facilitate the folding of KCNQ1 cytoplasmic domain. Marine Ca 2+ -sensor proteins (Calml3, Calml4a, Calml4b, NCS-1, NCALD, Hpca, VILIP-1, KChIP2a, and Calp) were expressed in cells with GFP-KCNQ1cL. Among CaM homologues, Calml3 is highly similar and Calml4a is mildly similar to CaM (93% and 53% amino acid similarity, respectively), while other neuronal Ca 2+ -sensor (NCS) family members share relatively low amino acid similarity (8-12%) (Fig.  3A). The protein expression of two CaM homologues (Calml3 and Calml4a) and three NCS family members (NCS-1, NCALD and NCS-1) was confirmed by Coomassie staining of SDS-PAGE gels loaded cell lysates (Fig. 3B). Expression of the homologues did not appear to perturb the overall protein expression level of GFP-KCNQ1cL (Fig. 3C). After clarifying each lysate by centrifugation, the supernatant was analyzed by FSEC (Fig. 3D). The remarkable fluorescence peak at the position of the hetero-octamer was only observed in cell lysates coexpressing GFP-KCNQ1cL and either Calml3 or CaM. The number of amino acids in Calml3 is equal to the number in CaM (Fig. 3F). Calml3 shares a dumbbell-like crystal structure with CaM, but the structures of the two proteins show displacement of the central helix connecting the two globular domains [50,51]. Furthermore, Calml3 has an 8-fold lower affinity for Ca 2+ , but undergoes large structural changes upon metal binding [52]. These observations suggest that the two Ca 2+ -sensors Fig. 6. Ca 2+ -sensor proteins did not perturb Ci-VSP activity. A. Representative currents from oocytes expressing a canonical inward rectifier K + channel, Kir2.3, in combination with Ci-VSP, CaM, and Calml3. Membrane potential was stepped to +20 mV and then to -120 mV from a holding potential of -20 mV. The bath solution contains 40 mM K + and the equilibrium potential of K + was estimated to be -20 mV. The duration of the depolarizing potential pulses was varied from 0 sec to 1.4 sec by 100 msec increment. The Kir channel blocker, Ba 2+ (1 mM), was added to the bath solution to measure the leak current level. Arrowheads point the level at zero current. B. Voltage-dependent kinetics of Kir2.3 run-down. The normalized Ba 2+ -sensitive current amplitude at the end of hyperpolarizing pulses was plotted against the duration of the depolarizing pulses.C. The averages of time constants for fitting the plots from different cells. The time constants were obtained by fitting to a single exponential function. The numbers in the parenthesis indicate the number of cells recorded.
may distinctly impact KCNQ1cL. The thermal denaturation profile of the KCNQ1cL-Calml3 complex measured by the in-gel analysis indicates that the midpoint of denaturation was 49.4 ± 0.2°C (n = 4) (Fig. 3E). The slight difference in the thermal stability between the two assemblies of KCNQ1cL and Ca 2+ -sensor implies that, although Calml3 forms a complex with KCNQ1cL in a comparable manner to CaM, the two Ca 2+ -sensors confer unique property on KCNQ1 channels.

Interaction of KCNQ1 and Calml3
We next tested whether Calml3 interacts with the full length KCNQ1 (Fig. 4). KCNQ1 was expressed in HEK cells with or without either GFP-tagged CaM or c-Myc-tagged Calml3. After solubilization with Triton X-100, KCNQ1 was isolated with a specific antibody and then analyzed by immunoblotting or in-gel GFP fluorescence analysis. As CaM formed the complex with KCNQ1 (Fig. 4A), Calml3 was found in the immunoprecipitates with KCNQ1 (Fig. 4B). Furthermore, KCNQ1 was also immunoprecipated with Calm13 when using an anti-c-Myc antibody. Therefore, Calml3 appears to bind to KCNQ1. To address the mode of Calml3 binding, we examined the effect of CaM over-expression on the interaction of KCNQ1 and Calml3 (Fig. 4C). CaM over-expression reduced the amount of Calml3 by 45.2 ± 8.5% (n=8) recovered in the immunoprecipitate with KCNQ1, while Calml3 over-expression lowered the amount by 29.2 ± 9.3 % (n=5) (Fig. 4D). These results indicate that Calml3 binds to KCNQ1 competitively with CaM, thereby suggesting that Calml3 is an alternative to CaM.

Effects of Calml3 on KCNQ1 channel activity
Since Calml3 and CaM are known to differently regulate the activities of target enzymes [37,53], we next tested their effects on KCNQ1 activity. KCNQ1 cRNA was injected into Xenopus oocytes with or without either CaM or Calml3 cRNA, and K + currents were measured using the two electrode voltage clamp technique. Fig. 5A shows typical current traces of KCNQ1 alone and of KCNQ1 co-expressed with CaM or Calml3 obtained with depolarizing voltage steps from -100 mV at 10-mV increments in a solution containing 2 mM K + . KCNQ1 was rapidly activated by depolarizing step pulses. The current amplitude at the end of the test pulses was augmented in a voltage-dependent manner (Fig. 5B). Co-expression of both Ca 2+ -sensors did not modulate the current expression level or the voltage dependence of KCNQ1. Therefore, it is likely that the KCNQ1-Calml3 complex is functionally comparable to the KCNQ1-CaM complex. PIP 2 plays pivotal roles in KCNQ1 activation [54,55]. Amino acid residues responsible for PIP 2 -dependent activation of KCNQ1 channels have been mapped to the proximal half of the C-terminus, sandwiched between putative CaM-binding sites [56][57][58][59]. Based on this result, we hypothesized that bound Ca 2+ -sensor proteins affect the affinity of KCNQ1 for PIP 2 . A voltage-sensitive phosphatase (Ci-VSP) hydrolyzes PIP 2 in the inner leaflet of cell membranes in a voltage-dependent manner [45]. It is highly active at depolarization, but less active at hyperpolarization. Thus, Ci-VSP could deplete PIP 2 by increasing the membrane potential or the duration of membrane potential depolarization. Over-expression of Ci-VSP/WT did not modulate the current amplitude at low membrane potentials (Fig. 5C). However, the current amplitude of KCNQ1 was reduced at membrane potentials above +20 mV, peaked at +40 mV and decreased by 47 ± 4% at +100 mV (n = 16), when compared to the current amplitude recorded at +10 mV (Figs. 5C and D). Therefore, in the presence of Ci-VSP/WT, the currentvoltage characteristics of KCNQ1 was an apparent bell-shaped curve. The Ca 2+ -sensor proteins did not modify the trend of the voltage-dependent effect of Ci-VSP. However, in the presence of Calml3, but not CaM, the bell-shaped voltage-dependent curve of KCNQ1 became tall and its peak was shifted to right by approximately 10 mV. Furthermore, the current amplitude only decreased by 21 ± 5% at +100 mV (n = 27). On the other hand, voltage-dependent modulation of the KCNQ1 current did not occur in the presence of phosphatase-dead Ci-VSP mutant (C363S) even though the gating currents were detected when membrane potentials changed. These data indicate that the expression of Calml3 lessens the KCNQ1 response to the voltage-dependent depletion of PIP 2 by Ci-VSP.

Cellular Physiology and Biochemistry
There remained the possibility that the effect of Ca 2+ -sensors on the affinity of the KCNQ1 channel for PIP 2 was based on the modulation of phosphatase activity of Ci-VSP. To address this possibility, we expressed the sensor proteins to measure the voltagedependent modification of a canonical inward rectifier K + channel, Kir2.3, which requires PIP 2 for activation, by Ci-VSP (Fig. 6A). Expression of Ci-VSP reduced the current amplitude of Kir2.3 by one third without influencing the current voltage relationship (Fig. 6A). The membrane potential was stepped to +20 mV from the holding potential of -20 mV and then hyperpolarized to -120 mV to measure inward current amplitude passing through Kir2.3. The length of the depolarizing membrane potential was varied by increasing the duration in steps of 100 msec. Without Ci-VSP, the depolarizing membrane potential did not affect the current profile of Kir2.3. However, in the presence of Ci-VSP, the current amplitude was significantly reduced by increasing the length of the depolarizing membrane potential (Fig. 6B). The time constant was 0.56 ± 0.05 sec (n = 9) (Fig. 6C). Co-expression of CaM and Calml3 did not perturb the reduction of the overall current amplitude of Kir2.3 by Ci-VSP. Furthermore, the voltage-dependency of Ci-VSP in the presence of CaM and Calml3 (0.55 ± 0.04 sec, n = 11) and 0.56 ± 0.03 sec, n = 7, respectively) was consistent with that in the absence of Ca 2+ -sensors. Therefore, neither CaM nor Calml3 appears to modify the phosphatase activity of Ci-VSP, thereby suggesting that direct association of Ca 2+ -sensor proteins with KCNQ1 influences PIP 2 -sensitivity of the channels.

Discussion
In this study, we developed an evaluation system to analyze the association between KCNQ1 and Ca 2+ -sensor proteins. The setup comprises two steps. The first step is the coexpression of the GFP-fused KCNQ1 cytoplasmic domain with CaM in E. coli, which lacks endogenous CaM-like Ca 2+ -sensor proteins (Fig. 1). The second step is the measurement of the GFP fluorescence in the tagged KCNQ1-CaM complex using FSEC and in-gel detection systems ( Figs. 1 and 2). Since these approaches allowed us to identify Calml3 as an alternative to CaM for the formation of functional KCNQ1 channels (Fig. 3), these biochemical methods appear to accurately detect the interaction between KCNQ1 and Ca 2+ -sensor proteins.
Calml3 and CaM competitively bind to the KCNQ1 channel (Fig. 4), and the electrophysiological property of KCNQ1 in complex with Calml3 was equivalent to that in complex with CaM (Fig. 5). These results suggest that the roles of Calml3 in the folding of KCNQ1 and the channel formation are comparable to those of CaM. However, the two complexes showed slight differences in thermal stability (Fig. 3) and in channel affinity for PIP 2 (Fig. 5). These results are in agreement with the observations that Calml3 and CaM have distinct potencies for controlling target enzymes [37,53]. Structural features of the two Ca 2+ -sensors, such as the domain orientation and the electrostatic surface potential, may underlie the difference in their ability to control the targets [51].
To detect the effects of Calml3 on the KCNQ1-PIP 2 interaction, we needed to inject its cRNA at a maximal dose (Fig. 5). This interfered the evaluation of functional competition between Calml3 and CaM by injecting different ratios of the two Ca 2+ -sensor cRNAs into oocytes. CaM is reported to be abundantly present in most of cells (approximately 25 µM) [32][33][34][35]. Therefore, the requirement of high expression of Calml3 could be mainly accounted for by the presence of endogenous competitor CaM. In the case of the L-type voltage-gated Ca 2+ channel, competitive interaction between CaBP1 and CaM was found to modulate Ca 2+dependent inactivation [60]. While a functional unit of Ca 2+ channel harbors a single Ca 2+sensor protein at its IQ domain, the tetrameric KCNQ1 channel associates with four Ca 2+sensors. This implies that the number of molecules binding to KCNQ1 is also involved in the control of sensitivity to PIP 2 . On the other hand, it becomes apparent that the binding of cytoplasmic auxiliary proteins influences the gates at the selectivity filter of voltage-gated K + channels [12,61]. Therefore, although the absence of a quantitative functional analysis hampers any rigorous assessment of how Ca 2+ -sensors differently modulate the affinity of KCNQ1 for PIP 2 , it might link to the general roles of cytoplasmic proteins in direct and  [65][66][67], voltage-gated Na + channels [68,69], and transient receptor potential canonical (TRPC) channels [70,71]. These Ca 2+ -sensor proteins share less than 15% amino acid similarity with CaM, suggesting a broad specificity in binding to these ion channels. In contrast, KCNQ1 bound only to the Ca 2+ -sensors CaM and Calml3, which share strong amino acid similarity [38]. Thus, it seems likely that Ca 2+ -sensor binding to KCNQ1 is considerably selective.
The C-terminus of KCNQ1 contains two putative CaM binding sites. There is a cysteine residue between these sites, at position 445, that is targeted for S-nitrosylation [72]. CaM appears to switch its mode of binding in a Ca 2+ -dependent manner [30,31,73], which influences the redox-dependent modulation of the channel. Furthermore, the binding of phosphatidylinositols and CaM regulate the TRPC6 in an integrative manner [74]. Therefore, Ca 2+ -sensors may dynamically regulate KCNQ1 channel activity and contribute to the molecular mechanisms underlying the divergence in the modulation of channel activity.
Calml3 is abundant in multiple types of epithelial cells, including those found in tissues of the breast [39], kidney, intestine, and skin [41]. On the other hand, KCNQ1 channels prominently function in the repolarization of cardiac action potential and contribute to the functions of epithelial cells; for example, gastric acid secretion from parietal cells [3][4][5][6], thyroid hormone biosynthesis [7,9], and cerebrospinal fluid secretion [8]. Their overlapping distribution in epithelial tissues strongly suggests that KCNQ1 and Calml3 assemble to form functional channels. However, in epithelial cells, CaM is also expressed abundantly. This led us to speculate that the functional units of epithelial KCNQ1 channels harbor both Ca 2+sensor proteins. Although we tried to isolate these complexes from native tissues by the immunoprecipitation, the commercially available antibodies that were specific to Calml3 and CaM did not work in this study. This is a clear limitation of this study designed to determine how Ca 2+ -sensor proteins participate in the formation of functional KCNQ1 channels in the epithelium. Nevertheless, since a variety of cell signals modulate PIP 2 metabolism, the Calml3-induced increase in PIP 2 affinity may maintain KCNQ1 activity to support the intimate functions of the epithelium.