Strong cooperativity between subunits in voltage-gated proton channels

Abstract

Voltage-activated proton (Hv) channels are essential components in the innate immune response. Hv channels are dimeric proteins with one proton permeation pathway per subunit. It is unknown how Hv channels are activated by voltage and whether there is any cooperation between subunits during voltage activation. Using cysteine accessibility measurements and voltage-clamp fluorometry, we show data consistent with the possibility that the fourth transmembrane segment S4 functions as the voltage sensor in Ciona intestinalis Hv channels. Unexpectedly, in a dimeric Hv channel, the S4 in both subunits must move to activate the two proton permeation pathways. In contrast, if Hv subunits are prevented from dimerizing, the movement of a single S4 is sufficient to activate the proton permeation pathway in a subunit. These results indicate strong cooperativity between subunits in dimeric Hv channels.

Figure 1

State-dependent modification of S4 residues by external MTSET. (a) Alignment of S4 region (dashed box) for Ci-H_V, mouse Hv1 (mH_V), Shaker K, and the first domain from Nav1.2 channels. (*) mark residues mutated to cysteine and tested with MTS reagents. Arrows marks residue S242 in Ci-H_V used for fluorescence experiments. (b-d) Currents from oocyte expressing I248C channels in response to voltage steps from −60 mV to +60 mV (in 20 mV increments, pH_o = 7.5), (b) before MTSET, (c) after a 40-seconds application of 100 μM MTSET at −60 mV (and washed for 20 seconds), and (d) after a 20-seconds application of 100 μM MTSET at +60 mV (and washed for 20 seconds). V_holding= −60 mV. (e) Currents in response to a +60-mV voltage step before and after each 10-second MTSET application on closed I248C channels. Closed-state modification was tested by applying 100 μM MTSET at −60 mV for 10 sec, followed by wash-out for 20 seconds. MTSET application was repeated 4 times. (f) Currents in response to a +60-mV voltage step during open-state MTSET application. Open-state modification was tested by applying 100 μM MTSET continuously while stepping to +60 mV for 2 seconds every 10 seconds. MTSET significantly increased the rate of activation. (g) The rate of MTSET modification at −60 mV (□) or +60 mV (□) was measured using the current amplitude at 300 ms after the start of the +60 mV voltage step (red dashed line in e and f). The current amplitude was plotted versus the cumulative MTSET exposure and fitted with an exponential. The fitted second-order rate constant is shown. k_open = 5620 ± 1843 M⁻¹s⁻¹ (n = 4).

Figure 2

State-dependent modification of S4 residues by internal MTSET. (a-b) Currents from an excised patch containing N264C channels in response to voltage steps from −60 mV to +60 mV (in 20 mV increments), (a) before and (b) after 1mM MTSET was applied. pH_i = 5.5 and pH_o =7. (b) Currents in response to a +100-mV voltage step every 10 seconds during MTSET application on open N264C channels. Open-state modification was tested by applying 1 mM MTSET at +100 mV for 1 sec, followed by wash-out at +100 mV. Wash-in and wash-out was monitored by the fast changes in current amplitude induced by changes in internal pH: pH_i = 5.5 in rinse and pH_i = 7.0 in MTSET solution. pH_o = 7.0. (c) Currents in response to a +100-mV voltage step every 10 seconds during MTSET application on closed N264C channels. Closed-state modification was tested by applying 1 mM MTSET at −60 mV for 1 sec, followed by wash-out. (e) The rate of MTSET modification was measured using the current amplitude at the end of the +100 mV voltage step (red dashed line in c and d). The current amplitude was plotted versus the cumulative MTSET exposure at +100 mV and at −60 mV), and fitted with an exponential. The fitted second-order rate constant is shown. k_open = 290 ± 32 M⁻¹s⁻¹ (n = 4). (f) Cysteine accessibility data (Supplementary Figs. 1-5) mapped onto the voltage-sensing domains from the crystal structure of the Kv1.2-2.1 chimera channel and the closed-state model of Shaker K channels. Charged S4 residues (ball and stick), S4 residues more accessible to internal MTSET at negative potentials (yellow) and to external MTSET at positive potentials (purple), S4 residues not modified by either internal or external MTSET (green). Solid lines indicate proposed lipid bilayer boundaries and dashed line indicate proposed MTSET accessibility due to water-filled crevices.

Figure 3

Kinetics of S4 movement. (a) Currents from an oocyte containing S242C H_V channels in response to a voltage protocol (top) with symmetrical 100 mM HEPES in both intra- and extracellular solutions (see Methods). Inset: currents during voltage ramps to measure reversal potentials. pH_i = 7 and pH_o =7.4. (b) I/V curves during voltage ramps from a. Currents reversed at −21.8 mV, close to the expected proton equilibrium potential E_H = −23 mV. Leak and capacitive currents were subtracted off-line using the currents from the ramp following the −60 mV episode. (c) Current (black) and fluorescence (red) from Alexa488-labeled Ci-H_V S242C channels for voltage steps between (−80) mV to +100 mV, V_h= −60 mV. (d) Normalized current (black) and fluorescence (red) from c for steps to +40 mV, +60 mV, and +80 mV. Current and fluorescence traces were normalized to their steady-state amplitudes, or fitted steady-state amplitudes (see Methods). Shown in green are the fluorescence signals raised to the power of 2.

Figure 4

Kinetics in monomeric ΔNΔC Ci-H_V channels. (a-b) Fluorescence spectra from (a) S242C Ci-H_V channels and (b) ΔNΔC S242C Ci-H_V channels labelled with Alexa488- maleimide (black) or Alexa488-maleimide and tetramethylrhodamine-carboxylamino-ethyl-methanethiosulfonate (TMR-MTS; red). Note that the donor (Alexa488) fluorescence at 510 nm does not change in ΔNΔC S242C with the addition of the acceptor (TMR), in contrast to the donor fluorescence from S242C. The absence of FRET in b shows that ΔNΔC S242C Ci-H_V channels are monomeric channels. (c) Normalized current (black) and fluorescence (red) from Alexa488-labeled ΔNΔC S242C Ci-H_V channels for voltage steps as indicated, V_h= −60 mV. 100 mM HEPES in both intra- and extracellular solutions. pH_i = 7 and pH_o =7.4. Fluorescence traces raised to a power of 2 (green). Inset: Initial part of the traces shown at extended time scale. Scale bar = 50 ms. The small difference between the fluorescence and current traces might be due to a second fast conformational step in the activation of the proton permeation pathway (See Supplementary Fig. 7b & d).

Figure 5

Estimates of the effective gating charge in wt H_V channels. (a) Currents from an excised patch containing wt H_V channels in response to a triple pulse protocol (top). Only a subset of voltage steps is shown for clarity. Inset: currents during voltage ramps to measure reversal potentials. pH_i = 6 and pH_o =7. (b) I/V curves during voltage ramps from a. Currents reversed at −54 mV, close to the expected proton equilibrium potential E_H = −57 mV. Leak and capacitive currents were subtracted off-line using the currents from the ramp following the −80 mV step. (c) Corrected G(V), measured at V_tail = 0 mV (arrow 2 in a). Fit with G(V)/G_max = 1/(1+exp(−zδ(V−V_1/2)/kT); V_1/2 = 2 mV and zδ = 4.55 e₀. (d) Normalized conductance measured from slow ramps between −60 and 0 mV for wt H_V channels in pH_i = 5.5 and pH_o = 7. The lower pH_i during the ramps relative to the G(V) measurements increases the currents to better resolve the conductance at negative potentials, but also shifts the V_1/2 by approximately −20 mV. Fit with G(V)/G_max= exp(zδ(V−V_1/2)/kT), where zδ = 6.1 e₀. (e) Fit of the slope factor zδ for different voltages (from d). (f) Normalized conductance measured from slow ramps between −20 and +80 mV for ΔNΔC H_V channels. zδ = 2.2 e₀. (g) Fit of the slope factor zδ for different voltages (from f).

Figure 6

Channel opening in monomeric and dimeric H_V channels. (a) Dimeric H_V channel with dimer interactions between transmembrane domains and in the cytosolic domains as shown earlier-. Each subunit has its own proton permeation pathway as shown previously-. The activation movement of the two S4s are assumed to be independent in the two subunits. In this simple model, one S4 in the resting position inhibits the proton currents through both subunits. Not until both S4s have activated can the proton current flow through both subunits. (b) In monomeric H_V channel (ΔNΔC construct), the activation of one S4 is enough to allow the proton flow, because there is no inhibition from a second subunit.