Identification of a proton sensor that regulates conductance and open time of single hERG channels

Figures

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  Figure 1. hERG1a- and 1b-mediated current is inhibited by extracellular acidosis. (ai) HERG1a-mediated current evoked by the protocol in inset is reduced in amplitude and current deactivation is accelerated by addition of extracellular solution with a pH of 6.3. (aii and iii) Extracellular acidification causes a shift in voltage-dependence of activation of hERG1a-mediated current, with V0.5 of −10.0 ± 1.2 mV (k = 6.8 ± 0.2 mV) at pH 7.4 (n = 15 cells) shifted to a V0.5 of −7.0 ± 2.8 mV (k = 8.3 ± 1.1 mV) at pH 6.3 (n = 8 cells). (aii) Shows mean current-voltage (I–V) relations for IhERG tails at pH 7.4 and 6.3, expressed in pA/pF, whilst in (aiii) currents following each test potential were normalized to the maximum current (Imax) elicited by the protocol. (aiv) Graphs showing acceleration by acidic pHe of the two time -course components of hERG1a-mediated current with hyperpolarization. Data are shown as mean ± S.E.M for the fast time constant (τFast) (left) at pH 7.4 (•)(n = 15 cells) and 6.3 (○)(n = 8 cells) and slow time constant (τSlow) (right) of deactivation at pH 7.4 (•)(n = 15 cells) and 6.3 (○)(n = 8 cells). The fast component of current deactivation changed e-fold in 26 mV.…

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  Figure 2. Extracellular acidosis reduces single hERG1a and 1b channel conductance. (ai and bi). Representative traces of single hERG1a (bi) and hERG1b (bi) channels at -80 mV, originating from recording with pipette solution pH of 7.4 or pH 6.3. Channel activity was observed following a voltage step to +20 mV, imposed as indicated by the arrows. Openings are shown as downward deflections, with dashed lines to represent the closed (C) and open (O) state. (ai) Single channel openings from two patches containing hERG1a channels, one with an electrode solution of pH 7.4 (upper 2 traces, from a single recording) and another with an electrode solution of pH 6.3 (lower 2 traces, from a single recording). (bi) hERG1b channel openings from two patches, one with an electrode solution of pH 7.4 (upper 2 traces from a single recording) and another with an electrode solution of pH 6.3 (lower 2 traces from a single recording). Horizontal scale bar represents 500 ms and vertical scale bar represents 1 pA. Single-channel current-voltage (I-V) relationships for hERG1a (aii) and hERG1b (bii) channels at the two pH values. Channel amplitudes were derived from amplitude histograms fitted with a single…

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  Figure 3. Extracellular acidosis shortens hERG1 channel burst duration. (a,b) Closed-time distributions for hERG1a (a) and hERG1b (b) in pH 7.4 at -80 mV. Distributions for both channel isoforms were best described by four exponentials with time constants (% contribution) of 0.48 ms (89.2%), 0.98 ms (5.7%), 44.2 ms (2.5%) and 129 ms (2.6%) for hERG1a. The closed time distribution for hERG1b was 0.51 ms (86.3%), 0.86 ms (8.1%), 20.5 ms (3.8%) and 384 ms (1.8%). τcrit values were calculated between the third and fourth component of closed-time distributions as indicated by an arrow. (c) Burst durations of hERG1a channels were obtained from histograms comprising of 10 patches for pH 7.4 and 9 patches for pH 6.3 and plotted against the respective membrane potential. The plot was fit with an exponential function, showing the hERG1a channel burst duration accelerates e-fold in 40 mV. This voltage-dependence is largely lost in pH 6.3. (d) hERG1b burst durations obtained from histograms comprising of 7 patches for pH 7.4 and 6 patches for pH 6.3. Data were fitted with an exponential function, showing the hERG1b channel burst duration accelerates e-fold in 30 mV, a voltage dependence retained at pH…

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  Figure 4. A possible role of histidine residues in the effect of extracellular acidosis. (a) Representative hERG1a currents in control (black trace) and after 10 minutes of 2 mM DEPC application (grey trace), showing a significant reduction in the amplitude and acceleration of current deactivation by the histidine residue modifier. (b) Steady-state activation plots from hERG1a ITail upon repolarisation to −40 mV. For control and 2 mM DEPC, values were normalized to maximal ITail in each condition and plotted against membrane potential. Relations were fit with a Boltzmann function to give V0.5 values of −9.1 ± 3.2 mV (k = 7.1 ± 0.4 mV) in control (n = 7 cells) and a V0.5 of −4.2 ± 4.2 mV (k = 8.0 ± 1.9 mV) with 2 mM DEPC treatment (n = 7 cells). (c) Representative current traces of hERG1a current when subjected to extracellular solutions with pH values decreasing from 8.0 to 5.0. (d) Concentration-inhibition curve for reduction of hERG1a current evoked by a step from −80 mV to +20 mV by decreasing extracellular pH. Data are shown as mean ± SEM and fit with the Hill equation to derive a pKa value of 5.5 ± 0.2 with a slope (k) of −0.6 ± 0.1 (n = 7). (e,f) hERG1a tail current deactivation was…

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  Figure 5. Structural context of target titratable residues. (a) Single subunit of the cryo-EM structure of the hERG1a channel24 with transmembrane domains labelled. Regions of extracellular loops colored white are absent in the cryoEM structure (PDB:5VA1)24 and were modelled into the structure using Modeller v9.1734 (https://salilab.org/modeller/). The region enclosed in the dashed box comprises most of the extracellular turret between helices S5 and S6, and is expanded in (b). (c) Location of the E575/H578 pair mapped onto the electrostatic surface (at pH 7.4; 0.15 mM NaCl) on the extracellular side of the hERG1a channel. (d) The E575/H578 motif is linked via an E575 (on S5) –T634 (S6) charge/hydrogen bond interaction to a hydrogen-bonded network involving N633, S631 with N629 on adjacent subunits of the hERG1a channel that forms a ring around the top of the selectivity filter (SF).

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  Figure 6. Extracellular acidosis affects hERG1a single point mutants. The effects of extracellular acidosis on the wildtype (WT) hERG1a and channels derived with single point mutations. The vertical dashed line represents the effect seen on WT hERG1a channels. (a–d) The effects of extracellular acidosis on WT and single point mutations. % decrease of hERG channel characteristics were observed: IEnd Pulse (a), ITail (b), and the fast (τFast, c) and slow (τSlow, d) components of deactivation. The data derive from IhERG measured using depolarizing voltage commands to +20 mV, followed by repolarization to −40 mV. Example current traces are included in Supplementary Fig. 2. Numbers in brackets indicate cell numbers. * and ** denote statistical significance of P < 0.01 and P < 0.001 respectively, when compared with values obtained from WT channel current (one-way ANOVA with Bonferroni post-test).

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  Figure 7. Effects of extracellular acidosis on current amplitude abolished by double mutation of E575 and H578. (ai-ci) Membrane current traces of single point mutant (ai and bi) hERG1a channel currents and the double mutant E575Q/H578N, evoked by step depolarization to +20 mV and repolarization to −40 mV (holding potential −80 mV). Application of pH 6.3 to hERG1a-E575Q had little effect on macroscopic current amplitude positive to +20 mV, but significantly accelerated current deactivation. Greater inhibition of macroscopic current by pH 6.3 of current mediated by hERG1a-H578N was observed, with less effect on the time-course of current deactivation. In contrast, hERG1a-E575Q/H578N-mediated current was augmented by extracellular acidosis at +20 mV, with a clear acceleration of current deactivation. (aii-bii) Current-voltage relationships for the two single point mutants and the double mutant of hERG1a channels. Extracellular acidosis reduced the amplitude of evoked current at potentials negative to +20 mV, with little effect at more depolarized levels. In contrast, current amplitude mediated by hERG1a-E575Q/H578N was unaffected at potentials negative to +20 mV, but was augmented at more…

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  Figure 8. Single channel conductance of hERG1a E575Q/H578N is resistant to extracellular acidosis. (a) Representative traces of single hERG1a E575Q/H578N channels at -80 mV, recorded with a pipette solution pH of 7.4 (upper panel, 2 traces from a single recording) and pH 6.3 (lower panel, 2 traces from a single recording). Channel openings were evoked by a prepulse to +20 mV (VH −80 mV) and observed following repolarization to -80 mV. Horizontal scale bar represents 500 ms and vertical scale bar represents 1 pA. (b) Single channel current-voltage relationship of hERG1a E575Q/H578N channels in pH 7.4 (•) and 6.3 (○). Channel conductance was not affected by extracellular acidosis, with a slope conductance of 11.7 ± 0.4 pS for pH 7.4 (n = 5 cells) and 12.0 ± 0.5 pS for pH 6.3 (n = 6 cells; P = 0.7032, unpaired student’s t-test). (ci) Open-time duration histogram derived from 5 patches with at pH 7.4. Channel activity at -80 mV was fit with a single exponential function with time constant 5.9 ms. (cii). Open duration histogram from 6 patches with at pH 6.3. Channel activity at −80 mV was not affected by acidic pH, being fit with a single exponential component with time constant 5.7 ms. (d) The…