Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

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Biophysical Journal Volume 100 February 2011 885–894 885

Li-Juan Ma, Iris Ohmert, and Vitya Vardanyan* Institut fu¨r Neurale Signalverarbeitung, Zentrum fu¨r Molekulare Neurobiologie, Universita¨t Hamburg, Hamburg, Germany

ABSTRACT Controlled opening and closing of an ion-selective pathway in response to changes of membrane potential is a fundamental feature of voltage-gated ion channels. In recent decades, various details of this process have been revealed with unprecedented precision based on studies of prototypic potassium channels. Though current scientific efforts are focused more on a thorough description of voltage-sensor movement, much less is known about the similarities and differences of the gating mechanisms among potassium channels. Here, we describe the peculiarities of the KCNQ1 gating process in parallel comparison to Shaker. We applied alanine scanning mutagenesis to the S4-S5 linker and pore region and followed the regular- ities of gating perturbations in KCNQ1. We found a fractional constitutive conductance for wild-type KCNQ1. This component increased significantly in mutants with considerably leftward-shifted steady-state activation curves. In contrast to Shaker,no correlation between V1/2 and Z parameters was observed for the voltage-dependent fraction of KCNQ1. Our experimental find- ings are explained by a simple allosteric gating scheme with voltage-driven and voltage-independent transitions. Allosteric features are discussed in the context of extreme gating adaptability of KCNQ1 upon interaction with KCNE b-subunits.

INTRODUCTION KCNQ1 (Kv7.1) is the founding member of Kv7 delayed positive potentials (1,15,19,22). In contrast, KCNE2 and rectifier potassium channels, which belong to the voltage- KCNE3 subunits eliminate the voltage dependence of gated cation channel superfamily. In native tissues, currents mediated through the KCNQ1 pore, converting it KCNQ1 is associated with ancillary subunits (1–4) and to a leak channel (20,23). To understand how such dramatic modulatory proteins (5–7), which shape the biophysical transformations occur, scientific efforts were predominantly properties of the channel to fulfill a distinct function in directed toward the identification of the interaction sites particular cells (4,8). The gating of KCNQ1 has been studied between a- and b-subunits (24–32). It is particularly remark- with increasing intensity in recent decades (9–11), not only able that the capability for extreme modulation is attributable because of the involvement of KCNQ1 mutations in many to the unique properties of the KCNQ1 pore-forming subunit human inherited diseases (12–14), but also due to its unusual itself, since a number of other Kv channels known to interact biophysical properties. One remarkable feature of KCNQ1 is with KCNE subunits in heterologous expression systems do its very slow activation that reaches the steady state within not undergo such radical gating modulations (24,33–36). seconds, compared to the millisecond activation range for In this study, we investigated the unique gating properties most of the Kv channels. KCNQ1 undergoes an uncommon of KCNQ1 by applying the alanine mutagenesis scan to the inactivation, recovery from which causes a characteristic S4-S5 linker and the pore region. This approach has been hook in tail currents (9,15,16). It has been proposed that inac- proven previously to supply essential information about the tivation of KCNQ1 is coupled to the phenomenon of higher voltage-dependent gating process of the Shaker potassium channel conductance by Rbþ than by Kþ (10,16). At first channel (37–39). Availability of analogous data for Shaker þ glance, higher Rb conductance of KCNQ1 is in contrast gave us an exceptional opportunity to evaluate some funda- to the widely accepted notion that Kv channel pores are mental aspects of potassium channel gating in general. þ structurally and energetically optimized for higher K Analysis of steady-state activation for a large number of conductance over other monovalent cations (17,18). mutants revealed that KCNQ1 gating is consistent with the The most extraordinary feature of the KCNQ1 channel is allosteric gating model, which includes voltage-driven and reflected in its capability for extreme gating transformations voltage-independent transitions. These features explain the upon interaction with ancillary proteins (1,4,8,19–21). Asso- extreme flexibility of KCNQ1 channel gating upon the inter- ciation of the KCNE1 b-subunit with KCNQ1 both in native action of this channel with different KCNE subunits. tissues and in heterologous expression systems slows the channel’s activation and deactivation rates, abolishes its MATERIALS AND METHODS inactivation, and shifts its steady-state activation toward Mutagenesis and heterologous expression of potassium channels

Submitted August 5, 2010, and accepted for publication December 15, 2010. Point mutations to human KCNQ1 and Shaker D6–46 cDNA were intro- *Correspondence: [email protected] duced through site-directed mutagenesis by means of overlapping PCR Editor: Kenton J. Swartz. Ó 2011 by the Biophysical Society 0006-3495/11/02/0885/10 $2.00 doi: 10.1016/j.bpj.2010.12.3726 886 Ma et al. and Quick Change kit (Stratagen, La Jolla, CA). Mutations were verified Data analysis by automated DNA sequencing. In vitro transcriptions were made using the mMessage mMachine kit (Ambion, Austin, TX). Quality of synthesized Conductance-voltage relations for wild-type and mutant KCNQ1 channels RNA was checked in gel electrophoresis and quantified with the RNA 6000 were determined from tail currents at hyperpolarized potentials (see Fig. 2, Nano kit (Agilent Technologies, Santa Clara, CA). Xenopus laevis frogs Fig. S2, and Fig. S4). Tail currents of KCNQ1 channels were fitted to a two- (Nasco, Chicago, IL) were kept in the animal facility of the University and, when necessary, a three-exponential function to account for inactiva- Hospital Eppendorf according to the guidelines of the local animal welfare tion (hook-in-tail current), as described previously (9). We used Pulsfit authorities. Xenopus oocytes were removed surgically and defolliculated (HEKA Elektronik, Lambrecht, Germany) or Igor Pro 6.0 (WaveMetrics, 2þ with 2 mg/ml collagenase (Sigma, St. Louis, MO) in Ca -free OR2 Lake Oswego, OR) software for this procedure. Nonspecific leak current solution containing: 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2,5mM for each test potential was estimated by averaging the peak tail currents HEPES, pH 7.5. Stage IV or V oocytes were injected with 2–50 ng from four to seven water-injected oocytes. Subsequently, the average leak cRNA using Nanoject 2000. Oocytes were incubated in OR2 solution sup- was subtracted from the inactivation-corrected tail currents for every poten- plemented with 2 mM CaCl2, 5 mM sodium pyruvate, and 50 mg/ml genta- tial. Errors were calculated according to linear error propagation law. mycin (Sigma) for 1–7 days before electrophysiological recording. The resulting data were normalized to their maximum (G/Gmax). Data points were afterward fitted with a modified Boltzmann function of the (V1/2V)ZF/RT form G/Gmax ¼ (1 Gmin)/(1 þ e ) þ Gmin, where Gmin is Electrophysiology the voltage-independent conductance fraction given by the horizontal asymptote of the function (see Fig. S1). Z, F, R, T, and V1/2 have their usual Whole-cell currents were recorded at room temperature by two-electrode meanings. Kaleidagraph 4.0 (Synergy Software, Reading, PA) and Mathcad voltage clamp (TEVC) using an OC-725C amplifier (Warner Instruments, 13 (Mathsoft Engineering & Education, Cambridge, MA) software were Hamden, CT) linked to Pulse software (HEKA Electronics, Lambrecht/ used for data-fitting and mathematical modeling. Pfalz, Germany) through ITC-16 digitizer for data acquisition and monitoring. Pipettes were pulled from borosilicate glass (WPI, Worcester, MA) using the PB/7 puller (Narishige, Tokyo, Japan). Tips of the recording electrodes were prefilled with 1% agar-3M KCl solution to prevent KCl RESULTS leakage into the oocytes. They were then backfilled with 3M KCl solution. Pipette resistance was 0.1–0.4 MU for current recordings <10 mA. We used Mutation-induced gating perturbations in KCNQ1 pipettes with resistance <0.1 MU for recording currents >10 mA. Record- and comparison to Shaker ings were performed in modified ND96 solution containing (in mM) þ 75 NaCl, 20 KCl, 2 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.5; 20 mM K We explored the properties of KCNQ1 steady-state activa- and 20 mM Naþ in this solution were replaced by 40 mM Rbþ for tion by systematic mutational substitution of amino acids measuring the Shaker D6–46 channel. Membrane voltage was continuously from G245 in the S4-S5 linker to V355 in S6 with alanine monitored during the recordings. Since KCNQ1 channels showed (Fig. 1). Approximately 35% of mutations yielded no a voltage-independent current component, protocols for leak-current subtraction were not used. To subtract a small unspecific current (usually current in the Xenopus oocyte expression system (see ranging from 80 to 200 nA at 100 mV), a parallel water-injected batch Table 1). Although we did not study the reasons for this, it of oocytes served as control. The average amplitude of nonspecific currents is worth mentioning that most of these mutations were local- was almost identical to the current remaining after the block of channels ized between the G297 and Q321 residues of the pore. The with specific KCNQ1 channels blocker XE911 (see Fig. S3 in the Support- majority of Shaker alanine mutants in the analogous region ing Material). Inhibition of KCNQ1 channels was achieved by bath application of XE911 (Tocris Biosciences, Bristol, United Kingdom). XE911 did exhibit current in response to depolarization pulses (37) was dissolved in 0.1 N HCl and kept in aliquots at 20C, as described despite the high sequence homology of Kv channels in this previously (40). region (Fig. 1).

S4 - S5 S5 FIGURE 1 Sequence alignment of representative Kv channels from beginning of S4-S5 linker to the end of S6. Diagram above sequences indi- Shaker SKGLQILGRTLKASMRELGLLIFFLFIGVVLFSSAVYFAEAG------SENSFFK KCNQ1 GGTWRLLGSVVFIHRQELITTLYIGFLGLIFSSYFVYLAEKDAVN--E---SGRVEFG cates secondary structure motifs according to the Kv1.2 SKGLQILGQTLKASMRELGLLIFFLFIGVILFSSAVYFAEAD------ERDSQFP crystal structure of Kv1.2-Kv2.1 (59). Conserved Kv2.1 STGLQSLGFTLRRSYNELGLLILFLAMGIMIFSSLVFFAEKD------EDDTKFK amino acids are in yellow, strongly similar amino Kv3.1 FVGLRVLGHTLRASTNEFLLLIIFLALGVLIFATMIYYAERIGAQPNDPSASEHTHFK acids in blue, and weakly similar amino acids in Kv4.1 SQGLRILGYTLKSCASELGFLLFSLTMAIIIFATVMFYAEKG------TNKTNFT green. The most divergent regions of KCNQ1 are outlined in red (G245–G269 and L338–355). The 245 255 265 275 285 295 overall sequence conservation between Shaker and KCNQ1 is 34%. The asterisk indicates the P- loop S6 position of the gating hinge in eukaryotic Kv chan- * nels, and the black and red bars show the PxP motif Shaker SIPDAFWWAVVTMTTVGYGDMTPVGFWGKIVGSLCVVAGVLTIALPVPVIVSNFNYFY and the hydrophobic seal, respectively. Numbers KCNQ1 SYADALWWGVVTVTTIGYGDKVPQTWVGKTIASCFSVFAISFFALPAGILGSGFALKV correspond to the human KCNQ1 sequence Kv1.2 SIPDAFWWAVVSMTTVGYGDMVPTTIGGKIVGSLCAIAGVLTIALPVPVIVSNFNYFY (GI 32479527). The National Center for Biotech- Kv2.1 SIPASFWWATITMTTVGYGDIYPKTLLGKIVGGLCCIAGVLVIALPIPIIVNNFSEFY nology Information sequence identiﬁcation Kv3.1 NIPIGFWWAVVTMTTLGYGDMYPQTWSGMLVGALCALAGVLTIAMPVPVIVNNFGMYY numbers for other proteins are: Shaker,GI Kv4.1 SIPAAFWYTIVTMTTLGYGDMVPSTIAGKIFGSICSLSGVLVIALPVPVIVSNFSRIY 288442; rat Kv1.2, GI 24418849; human Kv2.1, 305 315 325 335 345 355 GI 4826784; human Kv3.1, GI 163792201; and human Kv4.1, GI 27436981.

Biophysical Journal 100(4) 885–894 KCNQ1 Gating 887

TABLE 1 Steady-state gating parameters for KCNQ1 channels Table 1. Continued

V1/2 (mV) Z Gmin n V1/2 (mV) Z Gmin n WT 25.3 5 0.5 2.95 5 0.06 0.046 5 0.006 35 G306A NC G245A 24.3 5 0.6 2.69 5 0.04 0.029 5 0.005 5 V307A NC G246A 40.6 5 0.2 2.88 5 0.04 0.076 5 0.002 5 V308A NC T247A 25.5 5 0.6 3.12 5 0.17 0.029 5 0.004 4 T309A NC W248A 2.70 5 2.1 1.85 5 0.08 0.033 5 0.002 9 V310A 34.9 5 1.9 1.50 5 0.04 0.104 5 0.008 4 R249A 22.8 5 0.5 3.18 5 0.05 0.033 5 0.003 4 T311A NC L250A NC T312A NC L251A 11.7 5 0.8 2.88 5 0.06 0.021 5 0.004 6 I313A NC G252A 29.8 5 0.3 3.02 5 0.07 0.057 5 0.005 6 G314A NC S253A 35.2 5 0.4 2.46 5 0.15 0.088 5 0.008 4 Y315A NC V254A NA G316A NC V255A 18.7 5 0.7 2.01 5 0.05 0.047 5 0.002 5 D317A NC F256A 10.9 5 1.0 2.36 5 0.03 0.033 5 0.002 4 K318A NC I257A NC V319A NC H258A 35.7 5 1.9 3.35 5 0.27 0.101 5 0.009 5 P320A NC R259A 28.9 5 1.5 1.54 5 0.04 0.048 5 0.004 5 Q321A 22.3 5 0.8 3.27 5 0.13 0.045 5 0.003 4 Q260A 2.92 5 0.8 1.95 5 0.05 0.051 5 0.002 6 T322A NC E261A 27.6 5 0.7 2.49 5 0.09 0.038 5 0.006 7 W323A 26.3 5 1.1 2.60 5 0.05 0.039 5 0.002 5 L262A 18.4 5 2.0 1.77 5 0.04 0.053 5 0.006 4 V324A 27.5 5 1.8 2.78 5 0.13 0.049 5 0.001 7 I263A 17.7 5 0.8 3.37 5 0.26 0.039 5 0.001 10 G325A NC T264A NA K326A NC T265A 29.2 5 0.4 1.91 5 0.03 0.053 5 0.005 5 T327A 27.8 5 0.5 2.87 5 0.06 0.039 5 0.002 5 L266A 20.9 5 0.3 2.56 5 0.18 0.052 5 0.001 7 I328A NC Y267A 13.5 5 0.8 1.17 5 0.06 0.062 5 0.004 5 A329 I268A 18.7 5 2.1 1.39 5 0.07 0.128 5 0.024 7 S330A 29.1 5 0.6 2.81 5 0.07 0.040 5 0.002 4 G269A NA C331A 31.5 5 0.7 2.88 5 0.09 0.042 5 0.003 4 F270A 27.0 5 0.3 2.61 5 0.04 0.038 5 0.002 5 F332A 0.64 5 0.9 3.07 5 0.07 0.034 5 0.002 5 L271A 7.90 5 0.9 1.42 5 0.06 0.038 5 0.001 4 S333A 21.9 5 0.8 3.41 5 0.20 0.042 5 0.001 4 G272A 26.4 5 0.6 3.03 5 0.14 0.051 5 0.006 4 V334A NC L273A NC F335A 4.80 5 1.0 1.23 5 0.01 0.031 5 0.003 4 I274A 35.3 5 1.7 2.90 5 0.10 0.068 5 0.002 7 A336 F275A 38.3 5 1.6 1.86 5 0.05 0.076 5 0.007 5 I337A 20.7 5 0.2 1.95 5 0.04 0.056 5 0.009 6 S276A 18.8 5 2.1 3.23 5 0.23 0.038 5 0.002 5 S338A 3.80 5 1.5 1.49 5 0.05 0.036 5 0.001 6 S277A 28.2 5 0.3 2.59 5 0.07 0.049 5 0.002 4 F339A 23.9 5 1.4 2.24 5 0.08 0.057 5 0.004 5 Y278A 8.50 5 0.9 3.02 5 0.11 0.151 5 0.003 4 F340A 52.0 5 0.4 2.20 5 0.06 0.127 5 0.013 4 F279A 32.3 5 0.9 2.93 5 0.08 0.054 5 0.004 4 A341 V280A NC L342A 51.7 5 0.7 2.28 5 0.12 0.159 5 0.011 5 Y281A NC P343A NC L282A 20.9 5 0.8 1.64 5 0.11 0.112 5 0.007 5 A344 A283 G345A 8.30 5 1.4 1.47 5 0.06 0.112 5 0.002 5 E284A NC I346A NC K285A 34.1 5 1.7 1.75 5 0.08 0.060 5 0.001 5 L347A 36.0 5 4.9 1.33 5 0.14 0.021 5 0.007 4 D286A NC G348A 35.6 5 0.6 1.78 5 0.10 0.111 5 0.006 4 A287 S349A 2.80 5 1.2 2.24 5 0.08 0.026 5 0.005 4 V288A 23.9 5 2.6 1.88 5 0.06 0.052 5 0.002 4 G350A 4.50 5 0.9 1.36 5 0.03 0.027 5 0.007 4 N289A 24.3 5 0.6 1.94 5 0.03 0.051 5 0.002 5 F351A 21.8 5 0.6 3.20 5 0.12 0.036 5 0.001 4 E290A 22.2 5 1.4 3.00 5 0.29 0.055 5 0.003 5 A352 S291A 24.3 5 1.4 4.13 5 0.31 0.054 5 0.011 4 L353A 42.5 5 2.1 0.86 5 0.06 0.213 5 0.012 4 G292A NC K354A 8.90 5 0.7 1.93 5 0.08 0.043 5 0.007 4 R293A 21.1 5 0.7 2.31 5 0.01 0.051 5 0.001 5 V355A 35.5 5 0.5 1.71 5 0.02 0.157 5 0.012 5 V294A 25.8 5 0.8 2.27 5 0.06 0.054 5 0.003 5 G246AI274A 60.9 5 1.1 1.53 5 0.03 0.213 5 0.002 6 E295A 22.0 5 1.6 3.33 5 0.10 0.043 5 0.002 5 G246AF275A 77.7 5 1.5 1.92 5 0.04 0.280 5 0.031 7 F296A NC I274AF275A 36.6 5 2.2 1.27 5 0.04 0.116 5 0.007 9 G297A 20.3 5 3.4 2.73 5 0.23 0.042 5 0.002 4 I274AF340A 51.6 5 0.7 1.55 5 0.01 0.360 5 0.014 4 S298A NC F275AF340A 96.9 5 3.3 1.39 5 0.09 0.589 5 0.014 7 Y299A NC F275AL342A 61.9 5 1.6 1.49 5 0.05 0.240 5 0.016 6 A300 F275AG348A 41.4 5 1.9 1.62 5 0.04 0.210 5 0.009 6 D301A NC F340AL342A 40.8 5 1.1 2.79 5 0.09 0.313 5 0.016 4 A302 F340AG348A 73.0 5 3.2 1.79 5 0.05 0.285 5 0.026 10 L303A NC F340AV355A 48.9 5 1.1 2.16 5 0.03 0.188 5 0.013 6 W304A NC L342AG348A 58.2 5 1.1 1.81 5 0.03 0.333 5 0.016 7 W305A NC (Continued on next page)

Biophysical Journal 100(4) 885–894 888 Ma et al.

activation shifts in KCNQ1 was markedly different from Table 1. Continued that seen in Shaker (Fig. 2 D). In particular, more positive V1/2 (mV) Z Gmin n activation shifts were induced by KCNQ1 mutations than L342AL355A 54.7 5 0.5 2.23 5 0.08 0.300 5 0.021 7 by analogous Shaker alanine mutations. The abundance of 5 5 5 G348AV355A 51.2 2.5 1.59 0.06 0.256 0.020 7 mutants causing positive V1/2 shifts may point to the altered G246AS276A 38.8 5 0.8 2.58 5 0.08 0.046 5 0.012 4 closed-open equilibrium of the KCNQ1 channel toward the G246AS333A 45.5 5 1.8 2.59 5 0.03 0.061 5 0.002 4 5 5 5 open states, in contrast to Shaker channels. In this respect, G252AF340A 54.2 1.2 2.40 0.08 0.137 0.008 7 D F275AS276A 32.4 5 0.8 1.69 5 0.03 0.079 5 0.005 4 it is remarkable that wild-type KCNQ1 and Shaker 6–46 F275AS333A 38.5 5 1.4 1.98 5 0.07 0.101 5 0.011 6 channels display very similar V1/2 and Z values (Fig. 2 C). S276AF340A 48.6 5 0.8 2.13 5 0.04 0.139 5 0.008 6 The only difference is the availability of a small constitu- S333AF340A 51.1 5 1.8 1.91 5 0.05 0.125 5 0.013 4 tively open component in KCNQ1 (see below). Third, it ap- 5 5 5 S333AF342A 55.9 0.8 1.88 0.06 0.165 0.010 6 peared that the majority of mutants with significant negative The procedure for calculating standard errors (mean 5 SE) is described in activation shifts also exhibited a larger constitutively open Materials and Methods. NC, no current; NA, not analyzed. component than the wild-type (Table 1, Fig. 2, A and B). Taken together, our data indicate that the gating mechanism The steady-state conductance-voltage relationship for the of KCNQ1 and the structure of its S6 segment differ signifi- wild-type and 63 single-point mutants was determined from cantly from Shaker. tail currents at negative potentials after their correction for fast inactivation, as described earlier (9). Exemplary current The constitutively open component of KCNQ1 traces and corresponding voltage protocols are shown in and its correlation with steady-state activation Fig. S2. The steady-state activation of Kv channels is usually parameterized by the half-maximal voltage of acti- Under our experimental conditions, Xenopus oocytes vation, V1/2, and the equivalent charge, Z. These parameters expressing KCNQ1 channels displayed a considerable are determined by fitting the two-state Boltzmann function steady-state inward current at hyperpolarized holding to steady-state activation data. Since we detected a small potentials (100 mV and 120 mV). No similar current constitutively open fraction for KCNQ1 (see below), an components were present either in oocytes expressing extra parameter, Gmin, was introduced to measure the extent KCNQ2 channel or in water-injected oocytes from the of this fraction represented as an offset in the Boltzmann same batch. We noticed also that the amplitude of function (Fig. S1). V254A and T264A channels were not mentioned inward currents increased with the augmentation analyzed due to problems in fitting the respective tail of channel expression. We hypothesized that this current currents (see Fig. S2). Fitting the activation curve of could originate from KCNQ1 channels due to their incom- G269A resulted in high fit errors. Therefore, we also plete closure at hyperpolarized potentials. To examine this excluded G269A from analysis. possibility, we applied the specific blocker XE911 to the Of 63 alanine mutants, 41 demonstrated voltage-gating KCNQ1 channels (Fig. 3 A and Fig. S3). In agreement perturbations not exceeding 10 mV V1/2 shifts (Table 1). with previous reports from Cohen and co-workers (40), Alanine substitution in these cases obviously had no signifi- bath application of 300 mM XE911 inhibited the voltage- cant influence on steady-state activation of the KCNQ1 dependent KCNQ1 current (Fig. S3). In addition, it also channel. In contrast, ~35% of all functional mutants (22 of inhibited the voltage-independent current, demonstrating 63) had a marked impact on steady-state activation, reflected the presence of a KCNQ1-specific constitutively open in steady-state activation shifts of >10 mV.Shifts occurred in component. Since 100% block could not be achieved by both the positive and negative potentials over a wide range of application of saturating XE991 concentrations, we exam- voltages (Fig. 2). Mutations that caused activation shifts ined the possibility of whether the averaged leak current, above the defined threshold (DV1/2 ¼ 510 mV) were local- recorded from the same batch of water-injected oocytes, ized in the S4-S5 linker, and in the S5 and S6 segments might serve as an unspecific current measure. Therefore, (Fig. 2). Comparison of our results to Shaker alanine muta- we calculated and compared the constitutively open fraction genesis data (37) revealed some important differences. First, of conductance (Gmin) before and after the partial inhibition no correlation was detected between V1/2 and Z values for the of channels. As Fig. 3 B demonstrates, for wild-type KCNQ1 channel (Fig. 2 C). This was in contrast to Shaker KCNQ1, very similar values were observed after partial mutants, for which the negative activation shifts were accom- block by 100 mM XE911. Similar results were obtained panied by an increase in Z (37,41,42). The strong correlation also for I274A, F340A, F275A/S276, and G246A/I274A between V1/2 and Z in Shaker was explained by mutational mutant channels. This gave us confidence that a small influence on the late opening transition step of the channel unspecific current could be estimated by recording of (37), in agreement with earlier predictions based on a sequen- parallel batches of water-injected oocytes. tial gating scheme (37,43,44). Second, in the S6 segment, A marked increase in Gmin was observed in 15 muta- starting from the F332 position, the pattern of steady-state tions, whereas for some others, especially for those with

Biophysical Journal 100(4) 885–894 KCNQ1 Gating 889

A wt I274A E295A

40 mV 40 mV 40 mV

- 100 mV - 100 mV - 100 mV -120 mV -120 mV -120 mV

1µA 1µA 1µA 1s 1s 1s F332A F340A F351A

FIGURE 2 Impact of alanine mutations on 50 mV 20 mV 60 mV steady-state activation of KCNQ1. (A) Current - 100 mV -80 mV µ traces for six representative KCNQ1 channels. 1 A -100 mV µ -80 mV µ 1 A -60 mV 1 A 1s 0.5 s 1s Currents were elicited by depolarizing test pulses with 10 mV increments. (B) Steady-state activation curves of the channels presented in A. Smooth

B C 16 curves are the ﬁts of the two-state Boltzmann func- 1.0 WT 14 tion with offset to the data. Error bars represent the F351A mean 5 SE. (C) Correlation plot for V1/2 and Z F332A 12 0.8 E295A gating parameters for the wild-type and mutant 10 F340A KCNQ1 channels along with some Shaker D6–46 max 0.6 I274A Z 8 mutants measured under identical experimental G/G 0.4 6 conditions. Red open circles, KCNQ1 single 4 mutants; red solid circles, KCNQ1 double 0.2 D 2 mutants; blue open circles, Shaker 6–46 channels (see also Table 2). Smooth curve representing the 0.0 -100 -50 0 50 -50 050 V1/2-Z relationship for Shaker channels is adopted Voltage (mV) V1/2 (mV) from Yifrach and MacKinnon (37). Positions of the wild-type KCNQ1 and Shaker D6–46 channels are D Shaker indicated by red and blue dashed lines, respectively. (D) Comparative diagram of the mutational 379 389 399 409 419 425 435 445 455 465 475 485 effect on steady-state activation of KCNQ1 and D S4 - S5 S5 P- loop S6 Shaker 6–46 channels at analogous positions. Errors bars are omitted for simplicity. 60 50 40

(mV) 30 20 1/2 10 Δ V 0 -10 -20 -30

245 255 265 275 285 295 305 315 325 335 345 355 KCNQ1

remarkable positive V1/2 shifts (F332A, S338A, L347A, fraction and voltage dependence of KCNQ1 activation and F351A), we detected a slight decrease. Upon examina- (Fig. 3 D). tion of data, we noticed an indication that the mutants with negative DV values exhibited an increase in G 1/2 min Allosteric model for KCNQ1 channel gating (see Table 1 and Fig. 3, C and D). We investigated this issue further by measuring a number of double mutants The sequential schemes proposed previously to describe with even larger negative activation shifts (Fig. S4). The KCNQ1 gating (9,15,16) could not explain the correlation mean steady-state activation curves for some representa- between V1/2 and Gmin observed in our study. The presence tive mutants are illustrated in Fig. 3 C. The summary of of a constitutively open conductance in KCNQ1 channels results reﬂecting the analysis of 85 channels revealed suggests voltage-independent transition steps in the gating a strong correlation between the constitutively open pathway of KCNQ1. Hence, we considered the allosteric

Biophysical Journal 100(4) 885–894 890 Ma et al.

A B

0.10 FIGURE 3 Constitutive conductance of KCNQ1 1μA channels and its correlation with steady-state acti- 1s 0.08 vation. (A) Overlay of current traces recorded n i

m 0.06 before (black) and after (red) the inhibition of m G wild-type KCNQ1 channel with 100 M XE991. 40 mV 0.04 Only current traces corresponding to a 40 mV 0.02 test potential are shown. A representative trace -100 mV recorded from water-injected oocytes with the -120 mV 0 identical protocol is shown in gray. The light WT + XE911 (100μM) gray area corresponds to overlay of seven different traces. (B) Calculated Gmin values for the wild-type KCNQ1 channel before (black) and after (red) current inhibition with 100 mM XE991 (n ¼ 12), with error bars representing the mean 5 SE. (C) C D Conductance-voltage relationship for some mutants with high Gmin values. Data analysis and 1.0 0.7 ﬁtting of steady-state data of KCNQ2 were per- 0.6 formed in the same way as for KCNQ1 channels. 0.8 Error bars are represented as mean 5 SE. (D)

xam 0.5 Gmin-V1/2 relationship as a result of analysis of 85 KCNQ1 channels. Red open circles correspond n 0.6 im G 0.4 to KCNQ1 single mutants and red solid circles to G G/ KCNQ2 KCNQ1 double mutants. The dashed curve is the 0.3 0.4 I268A mathematical approximation of the G -V rela- WT min 1/2 G246A 0.2 tionship according to Eq. 3 (see Allosteric model L342A ¼ 0.2 of KCNQ1 gating for details). Parameters Kv I274A/F340A 0.1 3 F275A/F340A 0.41, Ka ¼ 0.05, and b ¼ 1.59 10 were obtained L342A/G348A 0.0 0.0 by ﬁtting the conductance-voltage data of the wild- type KCNQ1 to Eq. 2.Z¼ 2.37 corresponds to the -100 -100 -150 -50 0 -50 0 50 average Z value of all KCNQ1 channels measured. Voltage (mV) V1/2 (mV)

gating model, which includes potential-independent vertical observed relationship between V1/2 and Gmin. The theoret- transitions, as illustrated in Scheme I below. ical deﬁnition for such a correlation corresponding to Scheme I was very complex. Therefore, we simpliﬁed the 4 K(v) 3 2 1 2 K(v) 3 K(v) 4 K(v) C C1 C2 C3 C4 diagram to a four-state allosteric scheme (Scheme II): 2 3 4 Ka Kab Ka b Ka b Ka b Z F V R T Kv e C C1 O O1 O2 O3 O4 1K(v)b 2K(v)b 3K(v)b 4 K(v)b 4 3 2 Ka K a b (SCHEME I)

O Z F O For simplicity, we have omitted the inactivation steps V 1 R T described earlier in detail (9,15,16,45). The open probability K v e b of the channel with four independently moving voltage (SCHEME II) sensors and spontaneous closed-open transitions (Scheme A similar scheme has been used already to describe the I) is given by (46,47) constitutive conductance of some Shaker mutants (50) and 1 Shaker-KcsA chimeras (51). For Scheme II, Eq. 1 takes Po ¼ 4 (1) the form 1 þ 1 1þKðvÞ 1þ ð Þ 1 Ka bK v ¼ (2) G V ZF V 1 1 þ K eRT where Ka and K(v) are equilibrium constants for the sponta- 1 þ v ZF neous and voltage-dependent transitions, respectively, and Ka V 1 þ bKveRT b is a parameter describing the degree of coupling between Ka and K(v). Scheme I is analogous to the models developed where the Kvexp(ZFV/RT) component describes the for mSlo calcium-activated potassium channel (48) and voltage-dependent equilibrium constant K(v); Z, F, and HCN channels (49). We were interested in whether the allo- R are known physical constants, and T is the absolute steric-gating Scheme I could explain the experimentally temperature. Although the four-state diagram is a very plain

Biophysical Journal 100(4) 885–894 KCNQ1 Gating 891 approximation of allosteric gating, it enabled us to derive (Fig. 4). The mean steady-state activation parameters for the mathematical relationship between the constitutively these channels are shown in Table 2. open component, Gmin, and the midpoint voltage of potential-sensitive fraction, V (see Appendix). Equation 3 1/2 DISCUSSION describes the theoretical link between Gmin and V1/2 that corresponds to Scheme II: The major finding of our study is the close correlation between voltage-dependent activation and fractional consti- ZF 1 þ V1=2 tutive conductance in the KCNQ1 channel. This type of ¼ KveRT : (3) Gmin ZF correlation was not found in large-scale mutagenesis screens ð 1Þ V1=2 b KveRT of Shaker channels (37) except in the case of hydrophilic substitutions of residue P475, the second proline of the The dashed line in Fig. 3 D represents an approximation conserved PVP motif of Kv channels (50). Similar to of the experimentally observed Gmin-V1/2 relationship with Eq. 3. The Kv and b parameters were obtained from fitting Eq. 2 to steady-state activation data of the wild-type channel. A It describes well the Gmin-V1/2 relationship obtained experi- L366A E395A mentally. At positive potentials, the theoretical curve slightly deviates from experimental data points, although a tendency for Gmin reduction is evident. Further, we examined the possibility that mutation-induced variations in b and Kv parame- -25mV -41mV 2μA ters could account for the observed discrepancy at positive 2μA -100mV 20ms -80mV 20ms potentials. Therefore, the steady-state activation data of ΔV=5mV ΔV=3mV mutants with gating perturbations exceeding 10 mV activation shifts (jDV1/2j R 10mV) were fitted with Eq. 2 and T442A V476A average values for b and Kv were calculated (Table S1). Consequently, we simulated the Gmin-V1/2 relationship with minimum and maximum values of the parameter b (Fig. S5 -20mV 2μA μ -55mV 2 A A). Consistent with our model, most of the experimental -100mV 20ms 20ms ΔV=5mV -100mV data points were located between two theoretical curves. ΔV=3mV However, no pronounced improvement of overlay at positive L366A/V476A V1/2 values was observed even with mean Kv and b values that E395A/V476A correspond to channels with marked positive activation shifts (Fig. S5 B). As stated earlier in this section, the model from which Eq. 3 was derived is only a rough approximation of 2μA 2μA -75mV -67mV the allosteric gating. Therefore, we did not expect a perfect 20ms 20ms overlap between experimental data and theoretical curves. -120mV -100mV ΔV=3mV ΔV=3mV At this time, we do not have data explaining the significant deviation of I268A, Y278A, L282A, and G345A B 1.0 mutants form the theoretical model. One possible explanation could be that these mutations induced conformational 0.8 changes influencing the S6 gate to a much higher extent max 0.6 G

than the electromechanical coupling machinery. In this /

respect, it is impressive that all KCNQ1 channels with G 0.4 Shaker Δ6-46 significant negative activation shifts (DV1/2 % 10 mV) L366A E395A demonstrated an increase in Gmin. 0.2 T442A V476A In contrast to KCNQ1, the vast majority of Shaker E395AV476A 0.0 L366AV476A mutants with significant negative V1/2 shifts did not exhibit any detectable constitutively open component (37,41,50). -100 -75 -50 -25 0 Voltage (mV) The only exceptions were mutants with a hydrophilic substitution at the P475 position (39,50), which we discuss below. FIGURE 4 Shaker D6–46 mutants with large negative activation shift To check the availability of constitutively open fraction for display marginal constitutive conductance. (A) Representative current Shaker channels under our experimental conditions, we traces for Shaker D6–46 mutants measured under the same experimental conditions as for wild-type KCNQ1. Unspecific currents are not subtracted. tested six mutants with known large negative activation (B) GV curves for channels shown in A after the subtraction of average shifts (37). Our data show that only a marginal constitutively unspecific current and subsequent Boltzmann fit of the steady-state activa- open component was present in the Shaker mutants tested tion data. Error bars represent mean 5 SE.

Biophysical Journal 100(4) 885–894 892 Ma et al.

TABLE 2 Gating parameters for Shaker D6–46 channels not be estimated. At this time, our model cannot explain the measured under the same experimental conditions as for reason for large constitutive conductance of the above- KCNQ1 channels mentioned channels. It is possible that these mutations V1/2 ZGmin n induce global structural rearrangements, reflected, for Shaker D646 (Rbþ) 30.0 5 0.62 3.18 5 0.19 0.001 5 0.005 7 example, in the tail-current overshoot of V254A, the L366A 56.4 5 0.72 5.25 5 0.16 0.005 5 0.003 5 unusual deactivation kinetics of T264A, and the very fast 5 5 5 E395A 58.9 0.41 17.2 0.81 0.007 0.003 14 microscopic current decay of G269A (Fig. S2). It is inter- T442A 53.6 5 0.93 5.86 5 0.14 0.025 5 0.006 7 V476A 73.9 5 0.55 16.4 5 1.02 0.014 5 0.005 8 esting to note that alanine substitution at the G345 position, L366A/V476A 91.7 5 0.43 25.0 5 4.47 0.024 5 0.005 9 the Shaker P475 equivalent residue, showed pronounced E395A/V476A 82.2 5 1.17 17.9 5 1.92 0.029 5 0.014 5 deviation from the observed V1/2-Gmin correlation (Table 1). Unspecific currents were subtracted using parallel batches of water-injected This indicates that the G345 position corresponding to the oocytes. Tail-current amplitudes were measured at the position immediately PXP motif of Kv channels also may have an important after the decrease of capacitive currents, determined by recording the water- role in KCNQ1 channel gating. injected oocytes with identical protocol. Normalized tail currents were fit to All alanine substitutions of KCNQ1 residues from S298 a two-state Boltzmann function with offset. Shaker D6–46 was measured þ to P320, with the only exception V310A, yielded no currents under 40 mM extracellular Rb conditions. Exemplary current traces are shown in Fig. 4. Values are represented as mean 5 SE. in the Xenopus oocytes. In the analogous region of Shaker, 13 of 19 alanine mutants did show functional expression (Fig. 2 D). These results were somewhat unexpected in KCNQ1 mutants, in respective Shaker channels, the nega- view of the high sequence conservation of Kv channels in tive steady-state activation shift was accompanied by frac- this region (Fig. 1). In addition, in S5 (F256–Y278) and tional constitutive conductance. Based on detailed analysis in distal S6 (F332–V355), where the most remarkable acti- of the P475 Shaker mutants, Swartz and colleagues vation shifts were observed in KCNQ1, analogous Shaker concluded that in addition to causing negative activation mutations induce activation shifts in the opposite direction shifts, hydrophilic mutations at P475 deteriorate the electro- (Fig. 2 D). Given these substantial differences, we refrained mechanical coupling between the voltage sensor and the from interpretation of mutation-induced structural changes pore (50). Fitting Eq. 2 to the steady-state activation data using the available structural models (55), because these for KCNQ1 channels revealed that coupling constant models are based either on the crystal structure of Shaker- b varies from 0.43 103 to 4.93 103 among KCNQ1 related channels (56) or even distantly related prokaryotic mutants, which is roughly five orders of magnitude smaller potassium channels (57). than in the wild-type Shaker channel (52). Consequently, we On the other hand, we observed some similarities among suggest that a weaker electromechanical coupling is the key Shaker and KCNQ1 channels. For example, alanine muta- factor determining the allosteric gating features of KCNQ1. tions of residues in the extracellular loop connecting S5 The structural properties of the S4-S5 linker and/or the to the P-helix (F279–G297 in KCNQ1) and in the proximal charge paucity of the S4 segment in KCNQ1 could account S6 (Q321–C331 in KCNQ1) did not cause noticeable for such a weak coupling. Indeed, it has been reported that gating perturbations in either Kv channel. The higher flex- the charge neutralization of the Kv7.4 S4 segment, which ibility of the extracellularly located S5-P-Helix loop is mimics the KCNQ1 charge balance, generates channels a likely explanation for its mutational insensitivity. In with partial constitutive conductance (53). However, contrast, the gating insensitivity of residues located on focused experimental investigations will be required to proximal S6 was unexpected, since intensive side-chain properly address these questions in the future. contacts have been revealed in crystal structures of Kv Our data indicate that constitutively open conductance in channels in this region (56,58). One possible explanation KCNQ1 originates from spontaneous transitions between is that the packing of side chains in this region is so stable closed and open states of the channel. We consider the alter- that single point mutations had no significant influence on native explanation that the channel’s pore is leaky as less the overall structure. plausible, since 1), a leaky pore cannot describe the correla- The proposed allosteric model explains the high adapt- tion between V1/2 and Gmin; 2), no leaky gate has been ability of KCNQ1 upon interaction with accessory subunits. observed in single-channel recordings of wild-type The model predicts that the factors that severely shift the KCNQ1 (54); and 3), Shaker P475 hydrophilic mutants, closed-open equilibrium of KCNQ1 toward negative poten- which exhibit a very similar gating phenotype, did not tials would mimic the effect of KCNE2 or KCNE3 subunits. show a leaky pore in single-channel recordings (50). Positive shifts, in contrast, would resemble currents medi- Four mutants (I268A, Y278A, L282A, and G345A) ex- ated through KCNQ1/KCNE1 complexes. hibited a larger constitutive conductance than expected The proximal C-terminal stretch after the S6 helix has been from their V1/2 values. The closed-open equilibrium of identified as a calmodulin interaction site for the KCNQ1 þ V254A, T264A, and G269A channels is also shifted toward channel (6,7). Assuming that the binding of Ca2 to positive potentials, even though V1/2 and Z parameters could calmodulin may change the structure of distal S6, the

Biophysical Journal 100(4) 885–894 KCNQ1 Gating 893

þ reported Ca2 -dependent regulation of KCNQ1 (6) can be ZF 1 þ V1=2 ¼ KVeRT (A5) explained by allosteric modulation of the channel gating Gmin ZF V1=2 from the S6 segment. In this respect, it is less plausible that ðb 1ÞKVeRT a small constitutive component of KCNQ1/KCNE1 conductance would significantly contribute to the excitability. 2þ However, the Ca -dependent shifts of steady-state activa- SUPPORTING MATERIAL tion, due to the allosteric gating mode, may have a significant physiological relevance. On the other hand, the size of the One table and four figures are available at http://www.biophysj.org/ biophysj/supplemental/S0006-3495(11)00003-8. constitutive conduction component in the KCNQ1/KCNE2 or KCNQ1/KCNE3 complexes may be regulated at certain We thank Prof. Olaf Pongs for his valuable support. Dr. C. Siebrands and physiological conditions. The allosteric gating features, Dr. N. Schmitt helped us to characterize some KCNQ1 mutants at the early from this point of view, open what we believe to be a new stages of this work. We also thank Dr. J. R. Schwarz and Dr. B. Attali for perspective for reevaluation of KCNQ1/KCNE complex helpful comments on the manuscript. regulation both in heterologous expression systems and in Olaf Pongs is indebted to the Deutsche Forschungsgemeinschaft (Po137/ native tissues. 36-1,2) and to the German-Israeli DIP program (Po137/41-1; At 119/1-1) for their invaluable support.

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