Chloride dependence of hyperpolarization-activated chloride channel gates

Abstract

1. ClC proteins are a class of voltage-dependent Cl- channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl- channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values. 2. We studied the dependence on internal Cl- ([Cl-]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl- ([Cl-]o) activated ClC-2 currents. Replacing [Cl-]o by the less permeant Br- reduced channel activity and accelerated deactivation. 5. Gating of the ClC-2 mutant K566Q in normal [Cl-]o resembled that of WT ClC-2 in low [Cl-]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl- by Br- or I- led to a decrease in Po. 6. The [Cl-]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl-. 7. The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl- favours channel opening. Lysine 556 may be important for the relevant binding site.

Figure 1. Schematic diagram summarizing the gating phenotypes of various ClC channels and mutants

For each channel type (or gate for ClC-0), idealized voltage-clamp responses to voltage steps to -120 and +40 mV from a holding potential of -30 mV are shown. Note the various time scales. In two-electrode voltage-clamp recordings the N-terminal deletion mutant ΔNClC-2 of ClC-2 does not exhibit voltage- or time-dependent gating (as shown in the figure), in contrast to patch-clamp recordings (see Fig. 5).

Figure 5. Effect of [Cl⁻]_i on the gating of N-terminally deleted ClC-2

Activation of ΔNClC-2 was studied using a similar pulse protocol as for ClC-0 (A). Currents are from the same patch. Similar results were obtained in five independent experiments.

Figure 2. Effect of [Cl⁻]_i on the slow gating of WT ClC-0

Steady-state slow gate open probability as a function of voltage was assessed with the pulse protocol shown in A. Progressive hyperpolarization cumulatively activates the slow gate. The degree of slow gate activation was then assessed at the fixed tail pulse of +60 mV. At this voltage, the fast gate opens maximally within less than 10 ms, even at low [Cl⁻]_i. In B, C and D, recordings from the same inside-out patch that was superfused are shown, with the indicated Cl⁻ concentrations. In E, the (averaged) current at the +60 mV tail pulse is plotted as a function of the conditioning voltage. Similar results were obtained in six independent experiments.

Figure 3. Effect of [Cl⁻]_i on the slow gating of the ClC-0 mutant P522G

The pulse-protocol (A) is similar to that used for WT ClC-0 (Fig. 2), although with shorter pulse durations. B-F, current traces obtained from one patch perfused with the indicated solutions in chronological order. G, the (averaged) current at the +60 mV tail pulse is plotted as a function of the conditioning voltage. Note the incomplete recovery after the final wash. The dotted lines represent Boltzmann fits with the parameters obtained from several experiments given in the text. Similar results were obtained in seven independent experiments.

Figure 4. Effect of [Cl⁻]_i on the gating of the ClC-1 mutant D136G

Activation of D136G was studied using a similar pulse protocol as for ClC-0 (A). Currents are from the same patch. Similar results were obtained in four independent experiments.

Figure 6. Modulation of WT ClC-2 gating by extracellular halides

A, dependence of ClC-2 on extracellular Cl⁻: voltage-clamp traces from a typical oocyte expressing rat ClC-2 after 1 min perfusion with ND96 (left), ND96 with 103.6 mM Cl⁻ substituted with glutamate (middle), and again in ND96 (right). Voltage was clamped for 9 s each from the resting membrane potential to values between +20 mV and -140 mV in steps of -40 mV, followed by a 3 s test pulse to -60 mV (see inset). B, activation of rat ClC-2 by a reduction in [Cl⁻]_o: tail currents at -60 mV were measured after activation for 9 s at the given prepulses in solutions with [Cl⁻]_o substituted by increasing amounts of glutamate (○, 103.6 mM (ND96); ▪, 5 mM; ▵, 1 mM). Averaged results from experiments as shown in A from five oocytes are shown. Currents were normalized to the tail current after a prepulse of -140 mV and 1 mM [Cl⁻]_o. Data were corrected for liquid junction potentials. C, dependence of deactivation kinetics on extracellular halides: superimposed deactivation currents at +40 mV after 9 s activation at -140 mV dependent on the substitution of extracellular Cl⁻ with bromide. The extracellular solution contained 100 mM NaX⁻ (X = Cl⁻+ Br⁻), 1 mM MgCl₂, 1 mM CaCl₂ and 5 mM Na-Hepes; pH 7.4. The total halide concentration is 104 mM. Deactivation accelerates with Cl⁻ substituted with increasing concentrations of bromide. D, deactivation time constants at +40 mV in relation to extracellular Cl⁻ substituted with bromide. Currents recorded from four oocytes as in panel C were fitted to the bi-exponential equation . Both time constants decrease with increasing concentrations of bromide.

Figure 7. Modulation of ClC-2 mutant K566Q by extracellular halides

A, dependence of K566Q on extracellular Cl⁻: voltage-clamp traces from a typical oocyte expressing mutant K566Q measured as in Fig. 6A. B, averaged tail currents of K566Q mutant at different values of [Cl⁻]_o. Experiments were done as in Fig. 6A and averaged results from four oocytes are shown. Data have been corrected for liquid junction potentials. Activation by hyperpolarization appears to be shifted to more positive potentials at low Cl⁻ concentrations. C, shift in voltage dependence of the K566Q mutant by extracellular halides. Voltage protocol (left): each test pulse (from +40 to -140 mV in steps of -20 mV for 9 s) was preceded by a long 35 s period at the resting membrane potential and concluded by a +40 mV pulse for 3 s. Voltage-clamp traces from a typical oocyte after 1 min perfusion with extracellular solution containing Cl⁻ (left), Cl⁻ substituted by bromide (middle) and Cl⁻ substituted by iodide (right) are shown. Solutions are as in Fig. 6C. Whereas in Cl⁻ a large fraction of channels are already open at the resting membrane potential, substitution by bromide and iodide shifts the voltage dependence of activation to ClC-2 wild-type levels. D, comparison of instantaneous currents after the prepulse at the resting membrane potential in 104 mM Cl⁻ and 4 mM Cl⁻/100 mM bromide-containing solution measured as in C. Currents are normalized to values at -140 mV in 104 mM Cl⁻ (n = 3). E and F, dependence of deactivation kinetics of the K566Q mutant on extracellular halides. Currents and time constants are determined as in Fig. 6C and D.