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Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis

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Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biophysical Journal Volume 100 February 2011 885–894 885 Allosteric Features of KCNQ1 Gating Revealed by Alanine Scanning Mutagenesis 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 (V1/2ÀV)ZF/RT form G/Gmax ¼ (1 À 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 À100 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 appli- cation 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 À20C, as described despite the high sequence homology of Kv channels in this previously (40).
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