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Department of Pharmacology and MRC Centre for Synaptic Plasticity, University of Bristol, Bristol BS8 1TD, UK.

Address correspondence to Neil V. Marrion, Department of Pharmacology and MRC Centre for Synaptic Plasticity, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK. Fax: (44) 1179250168; email: N.V.Marrion{at}bristol.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H⁺ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H⁺ ions. KCNQ2/3 current was inhibited by H⁺ ions with an IC₅₀ of 52 nM (pH 7.3) at -60 mV, rising to 2 µM (pH 5.7) at -10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H⁺ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (I_max) and the voltage dependence of steady-state activation. Reduction of I_max was antagonized by extracellular K⁺ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of I_max was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state (8 pS) to a subconductance state (5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H⁺ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H⁺ ions was conferred by the KCNQ3 subunit.

Key Words: acidosis • conductance • K⁺ channel • M-current • pH

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Dynamic changes in extracellular pH occur during periods of physiological neuronal activity, such as synaptic activation (Chesler, 1990). Changes in extracellular pH also occur during pathological events such as epileptic seizures and cerebral ischemia (Chesler and Kaila, 1992; Tombaugh and Sapolsky, 1993). Extracellular pH can vary over a wide range, falling to below pH 6.5 during ischemia (von Hanwehr et al., 1986) and rising by several tenths of pH units during seizure activity and spreading depression (Chesler and Kaila, 1992; de Curtis et al., 1998; Xiong and Stringer, 2000). Fluctuations of extracellular pH cause marked functional changes to neurons (Chesler and Kaila, 1992), and during synaptic transmission H⁺ ions may act to modulate both pre- and postsynaptic function (Traynelis and Cull-Candy, 1990; DeVries, 2001). During pathological events such as ischemia and seizure, the consequences of pH changes are unclear. Changes in pH may exascerbate neuronal excitation and damage, or may give rise to neuroprotective processes (Tombaugh and Sapolsky, 1993). Effects of H⁺ on neuronal activity have been attributed to the modulation of a variety of different neuronal ion channel types (Tombaugh and Somjen, 1996; Mott et al., 2003).

The KCNQ family of potassium channels contains five members to date, with mutations of KCNQ1–4 showing linkage to human disease (Robbins, 2001). Mutations in the genes encoding KCNQ2 and KCNQ3 channels are thought to give rise to benign familial neonatal convulsions (BFNC), an inherited form of human epilepsy (Biervert et al., 1998; Charlier et al., 1998; Singh et al., 1998). Heteromeric KCNQ2/3 channels have been proposed to underlie the neuronal M-current (Wang et al., 1998), a potassium current that is active in the sub-threshold voltage range for action potential initiation (Brown and Adams, 1980). The M-current contributes to the resting membrane potential in a variety of neurons, dictates general neuronal excitability, controls spike frequency adaptation after stimulation, and facilitates network oscillations within the brain (Brown, 1988; Marrion, 1997; Brown and Yu, 2000; Hu et al., 2002). Blockade or receptor-mediated suppression of M-current is associated with depolarization of the resting membrane potential, increased neuronal excitability, and a propensity for epileptic events (Brown and Adams, 1980; Aiken et al., 1995). Conversely, enhancement of M-current causes a hyperpolarization of the resting membrane potential and reduces neuronal excitability (Tatulian et al., 2001; Otto et al., 2002).

KCNQ2 and KCNQ3 subunits are expressed widely within the nervous system at postsynaptic sites and regions of the central nervous system expressing these subunits are known to be involved in the generation, perpetuation, and termination of seizure activity (Tinel et al., 1998; Yang et al., 1998; Rogawski, 2000; Cooper et al., 2001). In addition, KCNQ2 subunits have been localized to presynaptic regions (Cooper et al., 2001). KCNQ2 and KCNQ3 subunits are therefore likely candidates for modulation by extracellular H⁺, as they are associated with physiological processes and disease states in which pH changes are known to occur and are present in brain regions that exhibit marked fluctuations of extracellular pH.

In this study, extracellular H⁺ ions were found to modulate KCNQ2/3 channels and neuronal M-current in a concentration- and voltage-dependent manner. Regulation of KCNQ2/3 channels by H⁺ ions was subunit dependent, with homomeric KCNQ3 channels exhibiting a high sensitivity and homomeric KCNQ2 subunits exhibiting a relatively low sensitivity. The mechanism of action of H⁺ ions was investigated on a single-channel level. Single KCNQ2/3 channel open-probability (P_o) was reduced by acidosis and gating kinetics were altered, with open times being dramatically reduced and channels recruited into a relatively long-lived ( 50–200 ms) closed state. The sensitivity of KCNQ2/3 current and neuronal M-current to extracellular H⁺ ions suggests that these currents are tonically regulated by relatively small fluctuations in extracellular pH. Such modulation by extracellular pH is likely to have marked effects on neuronal activity and neuronal survival observed during physiological processes such as synaptic transmission as well as pathological events such as ischemia and epilepsy.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Molecular Biology
Rat KCNQ2 (Splice form B, Pan et al., 2001; otherwise, similar to EMBL/GenBank/DDBJ accession no. AF087453) and rat KCNQ3 (EMBL/GenBank/DDBJ accession no. AF091247) constructs were provided by David McKinnon (SUNY, Stony Brook, NY). KCNQ2 and KCNQ3 inserts were subcloned independently into the plasmid vector pcDNA3.1 (Invitrogen). Site-directed mutagenesis was performed using the QuikChange-XL Mutagenesis kit (Stratagene). All sequences were confirmed by dye termination sequencing (Department of Biochemistry, University of Oxford, UK).

Tissue Culture and Transfection
HEK-293 cells (ECACC, UK) and HEK-293T cells (obtained from David Brown, UCL, London, UK and Dr. Galen Flynn, University of Washington, Seattle; also known as tsA-201 cells—an SV-40 virus-transformed HEK-293 cell line known to give rise to enhanced expression of certain plasmid DNA) were grown at 37°C and 5% CO₂ in air. The culture medium was MEM for HEK-293 cells, and DMEM for HEK-293T cells, supplemented with 10% fetal calf serum and 0.2% penicillin/streptomycin. Cells were split twice weekly when confluent and were plated in 35 mm dishes (Falcon Primaria) using a reduced serum content (3%) medium in preparation for transfection. Cells were transiently transfected 24 h after plating using Superfect transfection reagent (QIAGEN). Transfected cells were identified for recording by visualization of cotransfected green fluorescent marker EGFP (CLONTECH Laboratories, Inc.). Coexpression experiments were performed in HEK-293 cells, whereas homomeric channels were expressed in HEK-293T cells (due to low expression levels of these channels in HEK-293 cells). A total of 2 µg of DNA was used for each plate of cells, using equal molarities of KCNQ2 and KCNQ3 for coexpression experiments and a channel:marker ratio of 10:1. For expression of homomeric channels, the channel:marker ratio was 2:1. For coexpression studies, cells were used for recording within 24 h of transfection, whereas homomeric channel expression was studied 2–4 d after transfection.

Superior cervical ganglion neurons were obtained from 16- to 20-d-old Sprague-Dawley rats and cultured as described previously (Beech et al., 1991). Neurons were grown in 35-mm dishes (Falcon Primaria) and were used for recording after 1–2 d in culture.

Electrophysiology
Whole-cell voltage-clamp recordings were made from single, uncoupled cells at room temperature (20–24°C) using an Axopatch 200A amplifier (Axon Instruments, Inc.). Fire-polished electrodes (3–5 M) pulled from borosilicate glass contained (in mM): K-gluconate (110), KCl (10), KOH (20), MgCl₂ (1.5), HEPES (20), EGTA (10), CaCl₂ (4.976; calculated free [Ca²⁺] = 80 nM), K₂ATP (3), pH 7.2 with KOH. The external bathing solution was constantly perfused (8–10 ml/min) and contained (in mM): NaCl (144), KCl (2.5), CaCl₂ (2.52), MgCl₂ (1.2), HEPES/MES/TAPS (10), D-Glucose (22), pH 5.0–8.4 with NaOH. The buffer used was dependent on the pH of the solution: HEPES was used for pH 6.9 and 7.4, MES was used for pH 5.0–6.4, and TAPS was used for pH 8.0 and 8.4. Control experiments showed that buffer type had no effect on current characteristics and that similar effects of pH on current characteristics to those described were seen when HEPES was used (unpublished data). For experiments investigating the effect of K⁺ on pH sensitivity, NaCl concentration was altered to give a total ([Na⁺] + [K⁺]) of 146.5 mM. Otherwise the solutions were the same as above. Initial offset potentials were corrected for before recording. Junction potential shifts associated with solution changes varied by <1 mV and were not adjusted for. Currents were measured with capacitance compensation and series resistance compensation (>90%), filtered at 1 kHz using an 8-pole Bessel filter (Frequency Devices), and sampled at 5 kHz using Pulse (HEKA). Currents were not leak subtracted. Only cells with negligible current rundown after stabilization of whole-cell recording conditions (<1% current amplitude at -30 mV in 30 s) were used for experiments. Small endogenous currents were observed in HEK-293 and HEK-293 T cells at potentials positive to -20 mV (e.g., 9 + 1 pA at -30 mV; 40 ± 4 pA at 0 mV, n = 6 HEK-293T cells). These currents were small in comparison to KCNQ currents studied here (e.g., at -30 mV amplitude was typically >1 nA for KCNQ2 and KCNQ2/3 currents, 344 ± 38 pA for KCNQ3 currents, n = 7). However, in order to minimize the contribution of endogenous current to the relatively small KCNQ3 currents, analysis of KCNQ3 currents was restricted to potentials negative to 0 mV.

Cell-attached patch recordings were made at room temperature in a high K⁺ concentration bathing solution, which resulted in a resting membrane potential of 0 mV. The composition of this solution was (in mM): KCl (150), MgCl₂ (5), CaCl₂ (0.1), HEPES (10), D-Glucose (22), pH 7.4 with NaOH. The composition of the intrapipette solution ("extracellular" solution under cell-attached patch conditions) was: KCl (5.85), NaCl (144), MgCl₂ (5), CaCl₂ (0.1), D-Glucose (22), HEPES/MES/TAPS (10), pH 5.9–8.4 with NaOH. The buffer used was dependent on the pH of the solution: HEPES was used for pH 7.4, MES was used for pH 5.9–6.4, and TAPS was used for pH 8.4. Recordings were made using thick-walled quartz electrodes with resistances of 10–14 M. Single-channel currents were recorded using an Axopatch 200A amplifier, filtered at 1 kHz (or 2kHz in one case, see Fig. 6) using an 8-pole Bessel filter (Frequency Devices) and acquired at 10 kHz using Pulse (HEKA).

Data Analysis
Voltage dependence of activation and deactivation time constants.
Time constants were measured from whole-cell activation and deactivation relaxations. The dependence of these time constants on the applied voltage was assessed by fitting the mean data at different voltages with a single exponential function of the form:

where is the activation time constant or reciprocal deactivation time constant, V is the membrane potential, A is an amplitude coefficient, ₀ is the minimum value of , and x is the slope factor (mV/e-fold).

Activation curves.
The voltage dependence of steady-state channel activation was estimated from measurements of tail currents at -120 mV unless stated otherwise. The data were fit with Boltzmann distributions as shown below:

where I(V) is the normalized tail current amplitude at a test potential V, V_1/2 is the half-activation potential, k is the slope constant related to the apparent equivalent charge involved in channel gating, and I_max is the maximal amplitude of the Boltzmann distribution (or the maximum P_o for single-channel data).

Concentration-response Curves
Curves were fit to mean data using the following equation:

where y is the response, A1 is the minimum limiting value of y, A2 is the maximum limiting value of y, x is the pH, and k is the Hill slope. Hill slopes can indicate the possibility of cooperativity in ligand binding. Hill slopes equal to unity indicate a single binding site or noncooperative binding, whereas Hill slopes >1 can indicate a degree of positive cooperativity in binding. Hill slopes <1 can suggest either multiple binding sites with differing affinity, or an allosteric process affecting ligand binding affinity (e.g., Dahlquist, 1978; Koshland, 1996).

Whole-cell data were analyzed using PulseFit (HEKA) and Origin 6.0 (OriginLab). Single-channel recordings were analyzed using TAC (Bruxton), using the "50% threshold technique" to determine event amplitude and duration. All transitions were visually inspected before being accepted. Open- and closed-state histograms, as well as P_o, were generated using TACFit (Bruxton). Statistical significance of multiple components was determined using the ratio of maximum likelihoods method (Horn and Lange, 1983; Colquhoun and Sigworth, 1995). Open- and closed-state histograms were derived only from patches containing a single channel. Patches containing a single channel were defined by the absence of current superimpositions at depolarizing potentials where P_o was maximal (e.g., +20 mV) over prolonged time periods (>60s). For example, in the case of a patch containing two channels and an overall P_o of 0.8, the likelihood of observing superimposed channel openings in a fixed period would be 0.4 x 0.4 x number of openings. In a 60-s time period containing 1,000 openings, 160 examples of channel opening superimpositions would therefore be expected. Even at the lowest P_o observed (0.15 in pH 5.9), 6 superimpositions would be expected from this order of events. Absence of superimpositions within patches of similar or higher P_o and duration was used as the working definition of a "single-channel patch."

For the amplitude histograms, events lasting <1 ms were excluded to prevent analysis of openings that did not reach full amplitude (Cloues et al., 1997). A square-root function was used for the ordinate in order to reduce apparent variance between the number of events at each amplitude (as in Sigworth and Sine, 1987). All events were included in analysis of closed times and all events except those containing transitions were included in analysis of open times. Channel P_o was first estimated as NP_o, the product of the open probability x number of channels. NP_o was calculated as (dwell-time x level number/total time). N was estimated during maximally depolarizing sweeps and P_o was obtained by dividing NP_o by N.

All values are expressed as mean ± SEM (Origin 6.0). Where error bars are not visible, they lie within symbols. Significance of results was compared using an appropriate t test and considered to be significant if P < 0.05.

Materials
Reagents were obtained as follows: standard chemicals (Sigma-Aldrich), calcium chloride (Fluka), Superfect transfection reagent (QIAGEN), fetal calf serum (Harlan Sera-Lab), DMEM, MEM, penicillin/streptomycin (GIBCO-BRL).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Extracellular H⁺ Ions Modulate KCNQ2/3 Current
Coexpression of KCNQ2 and KCNQ3 subunits in HEK-293 cells gave rise to voltage-dependent potassium currents that were activated at potentials positive to -70 mV under normal physiological ionic conditions (Fig. 1 A). These currents were blocked by extracellular TEA ions with a sensitivity (IC₅₀ = 3.2 mM, Hill slope = 1.3 ± 0.2; unpublished data) that was appropriate for a single population of heteromeric KCNQ2/3 channels with a fixed 2:2 stoichiometry (Hadley et al., 2003). Expressed KCNQ2/3 currents were sensitive to fluctuations of extracellular pH, with acidosis inducing a reduction of whole-cell currents (Fig. 1 A). This reduction was brought about by a depolarizing shift in the activation curve and a decrease in the maximum whole-cell current (I_max) (Fig. 1 B). The effects of acidosis were rapid (immediate upon exchange of bathing solution) and were reversible upon washout (unpublished data). Alkalosis (pH 8.4) caused an approximately -4 mV shift in the activation curve of whole-cell currents relative to those in pH 7.4 (Fig. 1 B; from individual cells, shift was -1.3 ± 0.5 mV, n = 9; paired t test P < 0.05).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Modulation of heteromeric KCNQ2/3 current by extracellular H+ ions. (A) Whole-cell KCNQ2/3 currents from a HEK-293 cell in bathing solutions of differing pH were elicited by depolarizing voltage steps (1.5 s duration) from a holding potential of -70 mV. (B) Whole-cell KCNQ2/3 current activation curves in bathing solutions of pH 7.4 (n = 18), pH 8.4 (n = 9), pH 6.4 (n = 9), and pH 5.9 (n = 7). Tail currents at -120 mV were normalized to the amplitude of the tail current subsequent to a voltage pulse to +20 mV in pH 7.4. Curves are Boltzmann distributions fitted to the mean data, with parameters: V1/2 = -36.4 mV, (pH 7.4), -40.5 mV (pH 8.4); -32.6 mV (pH 6.4) and -24.9 mV (5.9); Imax = 1.00 (7.4), 0.99 (pH 8.4), 0.71 (6.4), and 0.41 (5.9); k = 11.1 mV (pH 7.4), 11.7 mV (pH 8.4), 9.7 mV (pH 6.4), and 10.5 mV (pH 5.9). (C) Modulation of the V1/2 of whole-cell KCNQ2/3 currents by extracellular pH. V1/2 values were determined from fits of Boltzmann distributions to mean data in each pH (n = 5–9). Curve shown is the fit of a concentration-response curve (see MATERIALS AND METHODS) to the mean data, with the following parameters: minimum shift of V1/2 = -2.15 mV; maximum shift of V1/2 = +30.1 mV; EC50 = 6.13; and slope = -0.67 ± 0.16. This slope was significantly different to unity at the 10% confidence level (i.e., unity lies outside the 90% confidence interval of the mean). (D) Modulation of the Imax of whole-cell currents by extracellular pH. Imax values were determined from fits of Boltzmann distributions to the mean data in each pH (n = 5–9). Curve shown is the fit of a concentration-response curve (see MATERIALS AND METHODS) to the mean data, with the following parameters: maximum fractional decrease = 0.60, EC50 = 6.68, and slope = -1.23 ± 0.2.

Steady-state KCNQ2/3 currents showed a sensitivity to extracellular H⁺ that was steeply voltage dependent (Fig. 2, A and B) . This resulted in a large change in current amplitude near the channel activation threshold (e.g., -60 mV). Even small fluctuations in extracellular pH were found to have profound effects on the amplitude of KCNQ2/3 current. The mean pH giving half-maximal block of KCNQ2/3 currents ranged from 7.28 (52 nM H⁺) at -60 mV to 5.69 (2 µM H⁺) at -10 mV (Fig. 2 C). Alkalosis induced by a change from pH 7.4 to pH 8.4 (i.e., 39 to 3 nM H⁺) resulted in a 50 ± 11% increase of steady-state current at -60 mV and a 16 ± 6% increase at -50 mV (n = 9). At potentials more positive than -50 mV, steady-state current was relatively unaffected by such alkalosis (unpublished data).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Sensitivity of heteromeric KCNQ2/3 current to extracellular H+ ions. (A) Whole-cell KCNQ2/3 current activation relaxations in bathing solutions of different pH were evoked by depolarizing voltage pulses (1.5 s duration) from a holding potential of -70 mV. Currents shown are from the same cell. (B) Fractional decrease of whole-cell steady-state KCNQ2/3 current in response to pH changes from pH 7.4 to the pH values indicated. Steady-state currents were measured at the end of depolarizing voltage pulses (1.5 s duration). (C) Voltage dependence of the IC50 values for H+-induced KCNQ2/3 current decrease. Concentration-response curves were constructed for KCNQ2/3 current inhibition at each voltage and the IC50 values determined at each voltage. IC50 values are shown plotted against voltage. Solid line is for display purposes only.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Modulation of KCNQ2/3 current activation and deactivation kinetics by extracellular H+ ions. (A) Whole-cell KCNQ2/3 current activation relaxations in bathing solutions of different pH were evoked by depolarizing voltage pulses (1.5 s duration) from a holding potential of -70 mV. Currents shown are from the same cell. (B) Activation time constants () for KCNQ2/3 current. Time constants were derived by fitting a single exponential to activation relaxations between the times 10 ms (to account for sigmoidal phase of activation) and 1500 ms after the onset of the depolarizing voltage pulse. The solid line shown is the fit of an exponential distribution to the time constants at different voltages. Parameters of this distribution were: A = 26.1 ms (pH 7.4) and 66.7 ms (pH 6.4); 0 = 107.1 ms (pH 7.4) and 143.3 ms (pH 6.4); and x = e-fold/16.3 mV (pH 7.4), and e-fold/19.7 mV (pH 6.4). (C) Whole-cell KCNQ2/3 current deactivation relaxations in bathing solutions of different pH were evoked by hyperpolarizing voltage steps (1.5 s duration) after a depolarizing voltage pulse to -30 mV (1.5 s duration). Currents shown are from the same cell. (D) Reciprocal deactivation time constants (1/) for KCNQ2/3 current. Time constants were derived by fitting a single exponential to deactivation relaxations between the times 6 ms and 1,500 ms after the onset of the hyperpolarizing voltage pulse. Solid line is the fit of an exponential distribution to the time constants at different voltages. Parameters of this distribution were: A = 10-5 ms-1 (pH 7.4) and 3 x 10-5 ms-1 (pH 6.4); 0 = 0.0024 ms-1 (pH 7.4) and 0.0015 ms-1 (pH 6.4); and x = e-fold/12.7 mV (pH 7.4) and e-fold/15.1 mV (pH 6.4).

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Extracellular K+ ions antagonize the H+-induced decrease in KCNQ2/3 Imax. (A) Whole-cell KCNQ2/3 current activation relaxations in bathing solutions of different pH which contained 0 mM extracellular K+ were evoked by depolarizing voltage pulses (1.5 s duration) from a holding potential of -70 mV. Currents shown are from the same cell. Activation curves shown were calculated from tail currents at -100 mV and were normalized to the tail current obtained subsequent to a depolarizing pulse to 0 mV. Curves are Boltzmann distributions fit to the mean data (n = 5), with the following parameters: V1/2 = -43.1 mV (pH 7.4) and -35.2 mV (pH 6.4); Imax = 1.06 (pH 7.4) and 0.50 (pH 6.4); k = 5.9 mV (pH 7.4) and 5.7 mV (pH 6.4). (B) A similar protocol was used to evoke relaxations in bathing solutions of different pH when 15 mM extracellular K+ was present. Curves are Boltzmann distributions to the mean data (n = 4), with the following parameters: V1/2 = -43.5 mV (pH 7.4) and -38.8 mV (pH 6.4); Imax = 0.99 (pH 7.4) and 0.78 (pH 6.4); k = 7.6 mV (pH 7.4) and 6.4 mV (pH 6.4). (C) Dependence of H+-induced KCNQ2/3 current modulation on extracellular K+ concentration. Imax values and V1/2 values were obtained from Boltzmann distributions fit to data from individual cells exposed to pH 7.4 and pH 6.4. Shifts shown are the shifts induced upon changing the pH from 7.4 to 6.4. Asterisk indicates significantly different to the corresponding value in 0 mM K+ (unpaired t test; P < 0.05).

Single KCNQ2/3 channel activity from cell-attached patches under conditions of different extracellular pH is shown in Fig. 5 A. Single KCNQ2/3 channel currents displayed two amplitude levels, both of which displayed sublinear current-voltage relationships at potentials positive to 0 mV (Fig. 5 B). At pH 7.4, a predominant amplitude level (₁) with a slope conductance (₁) of 8.2 ± 0.1 pS (mean of six individual patches; range: 7.8–8.5 pS) was observed, in addition to a smaller amplitude level (₂) with a slope conductance (₂) of 5.1 ± 0.2 pS (mean from the same 6 individual patches; range: 4.1–5.6 pS). Both amplitude levels were present in all patches for every pH analyzed. The smaller amplitude level ₂ was much less frequent than the larger ₁ at pH 7.4 (Fig. 5 B, insets). Under conditions of acidic extracellular pH, the relative occurrence of ₁ and ₂ became approximately equal, while alkalosis had no obvious effect (Fig. 5 B, insets). Measurement of the number of ₁ and ₂ events during a fixed time period (50 s, as shown in the insets to Fig. 5 B) enabled a quantitative assessment of the effect of acidosis on the frequency of ₁ and ₂ events (lasting >1 ms). In pH 7.4, 86% ₁ events and 14% ₂ events were observed out of a total 2630 events. In pH 5.9, 50% ₁ events and 50% ₂ events were observed out of a total 2578 events. These results show that acidosis induced a gain of ₂ events at the expense of an approximately equal number of ₁ events. This suggests that acidosis caused a switch of single-channel activity from the full-conductance state (8 pS) to a sub-conductance state (5 pS). The frequency of opening events was not markedly altered by acidosis (pH 5.9) at 0 mV, even when all events (including those lasting <1 ms) were analyzed: 3,236 events in pH 7.4 and 3,317 events in pH 5.9 over 50 s. This result has implications for the mechanism underlying alteration of closed-state kinetics by H⁺ ions (see below).

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Modulation of single KCNQ2/3 channels by extracellular H+ ions. (A) Cell-attached patch recordings from HEK-293 cells expressing KCNQ2/3 channels. Single KCNQ2/3 channel activity from separate patches is shown at a range of voltages under conditions of different extracellular (intrapipette) pH. The dotted lines show the closed-state level, with channel openings being upward. (B) Conductance of single KCNQ2/3 channels exposed to different extracellular pH. Two amplitude classes (1 and 2) of KCNQ2/3 channels were observed (see insets, showing Gaussian distributions fit to data recorded at 0 mV over a fixed time period of 50 s). Slope conductances of these two amplitude classes (1 and 2, respectively) are shown as solid lines. Slopes are linear fits to the mean data (n = 5–7 patches) between -60 and 0 mV for 1 and between -40 and 0 mV for 2. (C) Mean slope conductance of single KCNQ2/3 channel amplitude classes (derived from individual patches, n = 5–7) under conditions of different extracellular pH. Asterisk indicates significantly different to corresponding value at pH 8.4 (unpaired t test; P < 0.05).

Subconductance States of Single KCNQ2/3 Channels
Multiple conductance values have been described previously for single KCNQ2/3 channels (Schwake et al., 2000; Selyanko et al., 2001). However, it was unclear from these previous studies whether multiple amplitude levels arose from separate channels (for example, by virtue of differing subunit stoichiometry), or whether they represented subconductance states of single KCNQ2/3 channels. Single KCNQ2/3 channels displayed two amplitude levels ₁ and ₂ (Figs. 5 and 6) . Superimpositions of ₁ on ₂ were never observed in patches that did not also display superimpositions of ₁ on ₁ or ₂ on ₂. In addition, transitions between these two amplitude levels were sometimes observed in nominally single-channel patches (see MATERIALS AND METHODS for the working definition of a "single-channel patch"), with no resolvable intervening closures separating the two amplitude levels, even at a higher filtration rate of 2 kHz (Fig. 6, asterisks). Such transitions were rare (n = 15 out of >10³ events) and appeared to occur preferentially from large amplitude to small amplitude (n = 13 out of 15 transitions). The absence of ₁/₂ superimpositions and the occurrence of transitions suggests that the amplitude levels ₁ and ₂ are derived from the same single KCNQ2/3 channel, with ₂ representing a sub-conductance state of the full conductance state ₁.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Subconductance states of single KCNQ2/3 channels. Single-channel recordings from cell-attached patches showing the two amplitude classes (1 and 2) of KCNQ2/3 channel opening. "Transitions" between these two amplitude levels are indicated by asterisks. A section from a patch recorded at a higher filtration rate (2 kHz) is shown, in addition to records filtered at 1 kHz.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Subconductance states of single KCNQ2/3 channels exhibit distinct open-state kinetics. Top left shows the amplitude histogram obtained at 0 mV from a patch containing a single channel. Mean channel amplitudes at 0 mV, derived from Gaussian distributions, were: 1 = 667 fA and 2 = 435 fA. Open time analysis of all events (excluding openings containing transitions) gave a distribution best fit with the sum of two exponentials with time constants of 4.7 and 36.5 ms (top right). Excluding the smaller amplitude class (2) from the analysis (center left) showed that the larger amplitude class (1) displayed open state kinetics described by two exponentials with time constants of 9.1 ms and 42.4 ms (center right). Excluding 1 from the analysis (bottom left) showed that 2 also displayed open-state kinetics described by two exponentials, but with time constants of 1.2 and 27.1 ms (bottom right).

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Modulation of single KCNQ2/3 channel open probability by extracellular H+ ions. (A) Voltage dependence of single KCNQ2/3 channel Po for individual amplitude classes is shown under conditions of extracellular (intra-pipette) pH 7.4 and 5.9 (n = 3 patches containing a single channel). All events were included in analysis, except openings containing transitions. Curves are Boltzmann distributions fit to the mean data for the larger amplitude class (1), with parameters: V1/2 = -35.5 mV (pH 7.4), -46.4 mV (pH 5.9); Maximum Po = 0.88 (pH 7.4), 0.28 (pH 5.9); k = 18.1 mV (pH 7.4) and 20.2 mV (pH 5.9). (B) Voltage and pH dependence of total single KCNQ2/3 channel Po. Solid curves show Boltzmann distributions when all events (both amplitude classes) were pooled (n = 7 patches). Parameters were: V1/2 = -40.0 mV (pH 7.4), -24.1 mV (pH 6.4), -19.1 mV (pH 5.9), -54.7 mV (pH 8.4); Maximum Po = 0.91 (pH 7.4), 0.67 (pH 6.4), 0.38 (pH 5.9), 0.68 (pH 8.4); k = 14.4 mV (pH 7.4), 22.5 mV (pH 6.4), 24.2 mV (pH 5.9) and 15.4 mV (pH 8.4).

View larger version (39K): [in this window] [in a new window]   FIGURE 9. Modulation of single KCNQ2/3 channel open-state kinetics by extracellular H+ ions. (A) Open-state kinetics of large amplitude 1 events exposed to an extracellular (intrapipette) pH of 7.4 or 5.9, recorded at a range of membrane potentials. Values shown are time constants for each kinetically distinct open state, obtained from the fitting of exponential functions to histograms including all events (from three patches containing a single channel). (B) Open-state kinetics of small amplitude 2 events exposed to an extracellular pH of 7.4 or 5.9, recorded at a range of membrane potentials. Values shown are time constants for each kinetically distinct open state, obtained from the fitting of exponential functions to histograms including all events (from the same three single-channel patches used in A). Individual exponential functions and their sum are shown as solid curves. Openings containing transitions were excluded from analysis.

Extracellular H⁺ Ions Modulate Single KCNQ2/3 Channel Closed-state Kinetics
The closed-state kinetics of single KCNQ2/3 channels were found to be modulated by changes in extracellular H⁺ concentration (Fig. 10) . To assess the effect of H⁺ ions on the closed-state kinetics of single KCNQ2/3 channels, it was necessary to group closure events from both amplitude levels. KCNQ2/3 channels displayed three kinetically distinct closed states in pH 7.4 (Fig. 10, left column), an observation that is consistent with previous studies (Selyanko et al., 2001; Tatulian and Brown, 2003). One closed state (C₁) was described by a time constant of 1–2 ms, a second (C₂) with a time constant of 10–20 ms, and a third (C₃) with a time constant of 50–200 ms over the voltage range studied. Closed times showed voltage dependence, becoming shorter at more depolarized potentials. The contribution of C₃ to total channel closures was negligible at potentials positive to -20 mV. Upon exposure to pH 5.9, closed-state kinetics were altered (Fig. 10, right column). Acidosis caused an increase in the magnitude of the time constants describing each of the closed states at all voltage studied. Acidosis also led to a dramatic increase in the relative contribution of the long-lived closed state C₃ to total channel closure events at all voltages studied (P < 0.01, t test; n = 3 patches). In addition, acidosis caused the presence of this closed state to be maintained at depolarized potentials (Fig. 10). These results indicate that at negative membrane potentials (negative to -20 mV), a proportion of long duration closures may correspond to a closed state whose presence is not dependent upon acidosis. This would be similar to the long-lived closed state previously observed for native M-channels, in which channels were proposed to reside at negative membrane potentials (Selyanko and Brown, 1999). Subsequent discussion of the "C₃" state will relate to the long closures thought to be derived from acidosis. The duration of long closures were reduced by depolarization, falling from 180 ms at -40 mV to 80 ms at +40 mV in pH 5.9.

View larger version (36K): [in this window] [in a new window]   FIGURE 10. Modulation of single KCNQ2/3 channel closed-state kinetics by extracellular H+ ions. (A) Closed-state kinetics of single KCNQ2/3 channels exposed to different extracellular (intra-pipette) pH, recorded at a range of membrane potentials. Values shown are time constants for each kinetically distinct closed state. These were obtained from the fitting of exponential functions to histograms including all events (including both amplitude levels). Each set of histograms for each pH is derived from three separate patches containing a single KCNQ2/3 channel. Individual exponential functions and their sum are shown as solid curves.

View larger version (28K): [in this window] [in a new window]   FIGURE 11. Subunit-specific modulation of KCNQ channel gating by extracellular H+ ions. (A) Whole-cell KCNQ2 currents from a HEK-293T cell in bathing solutions of different pH were elicited by depolarizing voltage steps (3.0 s duration) from a holding potential of -70 mV. (B) Whole-cell KCNQ2 current activation curves in bathing solutions of pH 7.4 (n = 7), pH 6.4 (n = 6) and pH 5.9 (n = 4). Curves are Boltzmann distributions with parameters: V1/2 = -35.5 mV (pH 7.4), -34.6 mV (pH 6.4), and -32.8 mV (pH 5.9); Imax = 1.00 (pH 7.4), 0.78 (pH 6.4), and 0.58 (pH 5.9); k = 10.3 mV (pH 7.4), 10.5 mV (pH 6.4), and 10.4 mV (pH 5.9). (C) Whole-cell KCNQ3 currents from a HEK-293T cell in bathing solutions of different pH were elicited by depolarizing voltage steps (3.0 s duration) from a holding potential of -70 mV. (D) Whole-cell KCNQ3 current activation curves in bathing solutions of pH 7.4 (n = 7), pH 6.4 (n = 7), and pH 5.9 (n = 5). Curves are Boltzmann distributions with parameters: V1/2 = -48.5 mV (pH 7.4), -35.4 mV (pH 6.4), and -27.5 mV (pH 5.9); Imax = 0.98 (pH 7.4), 0.81 (pH 6.4), and 0.65 (pH 5.9); k = 8.8 mV (pH 7.4), 11.7 mV (pH 6.4), and 13.9 mV. Data used for activation curves were derived from tail currents at -120 mV, which were normalized to the amplitude of the tail current subsequent to a voltage pulse of +20 mV (for KCNQ2) or 0 mV (for KCNQ3) in pH 7.4.

View larger version (22K): [in this window] [in a new window]   FIGURE 12. Subunit-specific sensitivity of KCNQ channels to extracellular H+ ions. (A) Whole-cell currents elicited by depolarizing voltage steps from a holding potential of -70 mV are shown in different extracellular pH. (B) Fractional decrease of whole-cell steady-state KCNQ2 and KCNQ3 currents in response to a pH change from pH 7.4 to 6.4. Curves are for display purposes only. Steady-state currents were measured at the end of depolarizing voltage pulses (3.0 s duration). (C) Shifts in V1/2 and fractional decreases of Imax of whole-cell currents caused by a change in pH from 7.4 to the values indicated. Asterisks indicate significantly different to the corresponding value for KCNQ2 (unpaired t test; P < 0.01).

View larger version (17K): [in this window] [in a new window]   FIGURE 13. The S5–pore linker region of KCNQ2/KCNQ3 subunits. Residues discussed in the text are shown in bold, including residues H260 of KCNQ2 and K260 of KCNQ3. EMBL/GenBank/DDBJ accession nos. used were: Kv1.4 (P22459), Kv1.5 (P22460), and Kv2.1 (NP_004966).

View larger version (22K): [in this window] [in a new window]   FIGURE 14. Pore determinants of KCNQ2/3 channel sensitivity to H+ ions. (A) Whole-cell KCNQ2/3 (n = 9), KCNQ2/KCNQ3(K260Q) (n = 4) and KCNQ2(H260Q)/KCNQ3 currents elicited by depolarizing voltage steps from a holding potential of -70 mV are shown in different extracellular pH. "Q2" represents KCNQ2 and "Q3" represents KCNQ3. (B) Fractional decrease in response to a pH change from pH 7.4 to pH 6.4 of whole-cell steady-state wild-type KCNQ currents and mutant KCNQ currents. Curves are for display purposes only. Steady-state currents were measured at the end of depolarizing voltage pulses (1.5 s duration). (C) Shifts in V1/2 and fractional decreases of Imax of whole-cell currents caused by a change in pH from 7.4 to 6.4. Asterisks indicate significantly different to the value for the corresponding wild-type channel (unpaired t test; P < 0.01).

View larger version (15K): [in this window] [in a new window]   FIGURE 15. Modulation of neuronal M-current by extracellular H+ ions. (A) Whole-cell M-currents from a rat SCG neuron in bathing solutions of different pH were elicited by hyperpolarizing voltage steps (1.5 s duration) from a holding potential of -30 mV. The dotted line denotes the zero-current level. (B) Fractional inhibition of current amplitude induced by a switch from extracellular pH 7.4 to 6.4. Inhibition of steady-state M-current ("M") and KCNQ2/3 current ("Q2/Q3") at -30 and -50 mV is shown, in addition to the inhibition of M-current deactivation relaxation amplitude (induced by stepping from -30 to -50 mV). No significant difference was seen between the inhibition of M-current (n = 4) and the inhibition of KCNQ2/3 current (n = 9) at any voltage studied (P > 0.05, t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Modulation of KCNQ2/3 Channel Activation Gating by Extracellular H⁺ Ions
Modulation of the voltage dependence of steady-state activation of KCNQ2/3 current showed an IC₅₀ of 740 nM (pH 6.1) and a Hill slope of approximately -0.7. This slope value of <1 may indicate that sensitivity of steady-state activation to H⁺ ions is dictated by multiple binding sites, or by allosteric effects. The latter possibility would be consistent with conformational changes that occur during channel activation. Both the activation and deactivation kinetics of macroscopic KCNQ2/3 channels were slowed by increasing H⁺ concentration (Fig. 3). Slowing of both activation and deactivation kinetics in response to increased H⁺ is inconsistent with simple fixed surface charge-neutralization alone, where slowing of activation and speeding of deactivation would be expected. This suggests that H⁺ ions also modulate specific channel regions involved in channel activation and deactivation. Although speculative, a mechanism involving neutralization of both surface charge and the charge on specific acidic residues