Zhou Y et al. (2021), External Cd2+ and protons activate the hyperpol...

XB-ART-57589

J Gen Physiol 2021 Jan 04;1531:. doi: 10.1085/jgp.202012647.

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External Cd2+ and protons activate the hyperpolarization-gated K+ channel KAT1 at the voltage sensor.

Zhou Y , Assmann SM , Jegla T .

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The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.

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Species referenced: Xenopus laevis
Genes referenced: dtl elk1 hcn1 kcnh1 kcnh2 kyat1 tstd1

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	Figure 1. KAT1 and EAG channels have opposite voltage-gating modes despite sharing a highly conserved VSD. (A and B) Schematic diagrams show the relationship between VSD conformation and pore status for depolarization-gated metazoan EAG family K+ channels (left, Eag/Elk/Erg) and the hyperpolarization-gated plant K+ channel KAT1 (right) at hyperpolarized and depolarized voltages. VSD transmembrane domains (S1–S4) are shown in gray, and pore domain transmembrane domains (S5 and S6) are shown in cyan. The light gray box shows the approximate position of the membrane, with the extracellular side at the top. S4 basic residues are depicted as blue circles, and internal and external clusters of acidic residues in S1–S3 are depicted with magenta circles and labeled 1–6 by position according to previous convention (Zhang et al., 2009). Residue identities are given for the external cluster in KAT1, and black circles are used for residues that contain neutral rather than acidic amino acids in KAT1. Green circles represent a divalent cation occupying a divalent cation binding site that is available in the hyperpolarized S4-down conformation of EAG channels and hypothesized here for KAT1. Only two of four subunits of these tetrameric channels are depicted in the schematic diagrams, and the cytoplasmic C-linker and CNBD domains have been removed for simplicity. (C) Amino acid sequence alignment of VSDs for Arabidopsis thaliana KAT1 (NM_123993), human HCN1 (NM_021072), and the human EAG family channels Eag1 (NM_002238), Elk1 (NM_144633) and Erg1 (NM_000238). Residue numbers are shown at the right margin. Transmembrane domains S1–S4 are underlined, and residues identically or conservatively substituted are shaded. Magenta shading is used for conserved acidic residue positions 1–6 in S1–S3, and blue shading is used for conserved basic residues in S4. Amino acid positions identically conserved in all sequences are noted with asterisks. An extended string of basic residues (blue) at the extracellular end of S4 in HCN1 is marked with a dotted underline; these residues are conserved in the HCN family, and we speculate that they may block divalent ion binding in the S4-down state.
	Figure 2. External Cd2+ activates KAT1. (A) Example KAT1 currents recorded in 20 mM external K+ in response to a 2-s voltage ramp from +30 mV to −150 mV from a holding potential of −20 mV, in extracellular bath solutions with 200 µM (red) and 1 mM (black) Ca2+. KAT1 activation was minimally sensitive to Ca2+; 1 mM Ca2+ was used in the recording solutions for all subsequent experiments. (B) Example KAT1 currents recorded in control solution (black solid), in the presence of 300 µM Cd2+ (red solid) and after wash-off with control solution (black dotted). (C–F) Example KAT1 currents recorded in control solution (black solid) and other indicated divalent cations. Cd2+ (B) uniquely increased KAT1 currents both during the ramp and at the −20-mV holding voltage (arrows). Each panel (A–F) was recorded from a different oocyte. Gray dashed lines indicate zero-current level, the inset at the left middle indicates the voltage protocol, and the scale bar (right middle) applies to all panels.
	Figure S1. Effect of external Ca2+ on KAT1 gating and Cd2+ activation. Changing [Ca2+] of bath solution from 200 µM (open circles) to 1 mM (solid circles) had no significant effect on KAT1 V50 measured with (red) and without (gray) 300 µM Cd2+. V50 values of KAT1 under indicated conditions are plotted as mean ± SEM, and circles show individual measurements (n = 5).
	Figure 3. Extracellular Cd2+ depolarizes the voltage activation curve of KAT1. (A and B) Example traces of KAT1 currents recorded from a single oocyte at pH 7.0 with 20 mM external K+ in the absence (A) and presence (B) of 300 µM Cd2+. Currents were elicited by 2-s voltage steps in a family from −160 mV to either 40 mV (control, black dots in voltage protocol) or 120 mV (Cd2+, red dots in voltage protocol) from a holding potential of 0 mV. Tail currents were recorded at −100 mV. We applied voltage steps in 10-mV increments for G-V analysis, but for clarity only every fourth sweep (40-mV increments) is shown in these examples. Gray dashed lines show the zero-current level, and scale bars indicate current amplitude and time for both A and B. Note outward current at the holding potential and a large instantaneous component to the tail in Cd2+ (arrows). (C) Normalized voltage activation curves recorded from isochronal tail currents at −100 mV following 2-s steps to the indicated voltages in 10-mV increments for KAT1 at various Cd2+ concentrations. Data were recorded at pH 7. Control data are shown in black, and 300 µM Cd2+ data are shown in red. Normalized data are plotted as mean ± SEM (n = 10–16). The smooth lines show single Boltzmann distribution fits of the points, and Boltzmann fit parameters are given in Table 1. (D) ΔV50 (V50[Cd2+] − V50[control]) plotted versus external Cd2+ concentration for KAT1 at pH 7. Data were recorded as described in C; gray open circles represent individual measurements (n = 10–16), and whiskers show mean ± SEM. The smooth red curve shows the Hill fit with a Cd2+ Kd of 114.7 µM, a ΔV50max (end − start) of 170.4 mV, and a slope of 2.2.
	Figure S2. Native outward oocyte currents contribute to total current at extreme depolarizations. Peak magnitude of currents recorded during 2-s voltage steps to the indicated voltages from a holding potential of 0 mV in the presence of 300 µM Cd2+ for uninjected oocytes (black) and oocytes injected with KAT1 WT RNA (red). Data points show mean ± SEM (n = 10); some error bars are not visible because they are smaller than the symbols.
	Figure 4. State dependence of Cd2+ modification. (A) Example traces of KAT1 currents recorded at holding potentials of 0 mV (black) or −80 mV (brown) during application of 300 µM Cd2+ (arrows). The dotted line indicates the zero-current level for both traces, and the inset shows an overlay of the absolute value of the traces after normalization to highlight the rate difference for Cd2+ modification. (B) Comparison of the time at which currents induced by 300 µM Cd2+ reached 50% of the maximal change in amplitude (T0.5) for holding potentials of 0 mV (black) and −80 mV (brown). Gray open circles show individual data points, and whiskers show mean ± SEM. The rate of modification at −80 mV was significantly increased by approximately threefold compared with the rate at 0 mV (n = 12, t test, P = 3.4 × 10−15).
	Figure 5. Cd2+ reduces the voltage dependence of the activation rate in KAT1. (A) Normalized example traces for KAT1 activated by a voltage step to −180 mV following a 2-s depolarizing pulse at 0 or 80 mV in the absence (black) or presence (red) of 300 µM Cd2+. Cyan line overlays on the normalized traces show the dual exponential fits of the activation time course. The inset shows the raw currents for the two normalized traces. Cyan boxes denote the normalized region used for exponential fitting, and the gray dashed lines indicate the zero-current level. Inset scale bars indicate 10 μΑ and 100 ms. Capacitive transients have been clipped, and the two traces were recorded from different oocytes. (B and C) Time constants for the fast (τF; B) and slow (τS; C) components of activation with (red) and without (black) 300 µM Cd2+ are plotted versus voltage and fitted with a single exponential functions (lines) to estimate apparent gating charge (z). (D) Plot of the fractional amplitude of τF as a function of voltage for control (black) and 300 µM Cd2+ (red) experiments. Data in B–D show means ± SEM (n = 8).
	Figure 6. Cd2+ slows KAT1 deactivation. (A) KAT1 deactivation time constant (τDeact) at pH 7.0 calculated from a single exponential fit of the deactivation time course at the indicated voltages. A 1.35-s voltage step to −140 mV was used to activate channels before the voltage step used to measure deactivation. The smooth curve shows a single exponential fit of τDeact versus voltage, used to calculate equivalent gating charge (z). The inset shows an example trace (black) for KAT1 deactivating at +60 mV following a −140 mV step. The capacitive transients have been clipped. The cyan line shows an overlay of the exponential fit and highlights the region used for fitting. (B) Plot as shown in A for KAT1 deactivation in the presence of 300 µM Cd2+. Two exponentials (τDeact-F and τDeact-S) were needed to describe the deactivation time course in the presence of Cd2+. The inset shows example KAT1 current (red) deactivating at 100 mV after a 1.35-s step to −140 mV pulse overlaid with a dual exponential fit (cyan line). (C) 50% deactivation time (T0.5 deactivation) compared for control (black) and 300 µM Cd2+ (red). The Cd2+ data are shifted by −150 mV (see top voltage scale for actual voltages) to account for the G-V shift observed in 300 µM Cd2+. Data in A–C are shown as mean ± SEM (n = 8).
	Figure 7. KAT1 D95N is not activated by Cd2+. (A and B) Example traces for KAT1 D95N currents recorded from a single oocyte at pH 7.0 in the absence (A) and presence (B) of 300 µM Cd2+. The inset below B shows the voltage protocol for both panels, a universal scale bar is provided for current and time, and the gray dashed lines mark the zero-current level. (C) Voltage activation curves for KAT1 D95N in control solution (black) and 300 µM external Cd2+ (red) at pH 7. Conductance was measured using isochronal tail currents recorded at −100 mV after 2-s steps to the indicated voltages. Data are plotted as normalized mean ± SEM (n = 8), and curves show single Boltzmann fits (parameters reported in Table 1).
	Figure 8. Extracellular protons potentiate voltage activation of KAT1. (A and B) Example traces of KAT1 currents recorded from a single oocyte at pH 7.0 (A) and pH 4.0 (B) using the voltage step protocol from the inset. A universal scale bar is provided, and the gray dashed lines mark the zero-current level. (C) KAT1 G-V curves fitted with single Boltzmann functions for pH 8.0 to pH 4. Conductance was measured using isochronal tail currents (recorded at −100 mV) following 2-s steps to the indicated voltages by 10-mV increments. Normalized data are plotted as mean ± SEM (n = 8), and curves show single Boltzmann distribution fits (parameters reported in Table 2). (D) Plot of ΔV50 (V50[pH X] − V50[pH 8]) for pH 7.0 to pH 4.0 relative to pH 8. Asterisks indicate significant difference from pH 8.0 (n = 8, t test; , P = 0.03; **, P = 1.5 × 10−13). Box plots show mean and middle quartiles, and whiskers show SD.
	Figure 9. Effect of extracellular protons on KAT1 activation and deactivation kinetics. (A) Normalized example traces of KAT1 activation during a voltage step to −180 mV from a holding potential of 0 mV at pH 7.0 (black) and pH 4.0 (blue). Cyan line overlays show the dual exponential fits of the activation time course. The inset shows the raw currents, with the normalized region used for exponential fitting marked with cyan boxes. The gray dashed lines indicate zero-current, and the inset scale bars indicate 10 μΑ and 100 ms. Capacitive transients have been clipped. (B and C) Comparison of the fast (τF) and slow (τs) time constants versus voltage for activation at pH 4.0 and pH 7. Data were measured from 2-s steps to the indicated voltages from a holding potential of 0 mV and show mean ± SEM (n = 8). Curves show single exponential fits of the data used to estimate apparent gating charge (z). (D) Fractional amplitude of τF plotted versus voltage for pH 7.0 (black) and pH 4.0 (blue; mean ± SEM; n = 8). (E) Normalized example traces for KAT1 at pH 7.0 (black) and pH 4.0 (blue) for deactivation at 60 mV following a 1.35-s test step to −140 mV overlaid with single exponential fits (cyan lines). Insets show raw current traces, with the normalized regions used for exponential fitting marked with cyan boxes. Dashed lines indicate zero-current level, and inset scale bars show 5 μΑ and 100 ms. The two traces were recorded from different oocytes. (F) Plot of τDeact versus voltage for pH 7.0 and pH 4.0 fitted with single exponential functions to estimate apparent gating charge (z). Data shown are mean ± SEM (n = 8).
	Figure 10. The overlap of Cd2+ and proton binding sites. (A and B) Example traces of KAT1 D95N currents recorded from a single oocyte at pH 7.0 (A) and pH 4.0 (B) using the voltage step protocol from the inset. The scale bars for time and current apply to both panels, and the gray dashed line marks the zero-current level. (C) KAT1 D95N voltage activation curves fitted with single Boltzmann functions for pH 8–4. Data (mean ± SEM; n = 8) are normalized for comparison and were measured from isochronal tail currents recorded at −100 mV following 2-s steps from a holding potential of 0 mV to the indicated voltages. Curves show single Boltzmann distribution fits; parameters of the fits are reported in Table 2. (D) Plots of ΔV50 at pH 7.0 to pH 4.0 relative to pH 8.0 (V50[pH X] − V50[pH 8]) for KAT1 WT and D95N. Box plots show mean and middle quartiles, and whiskers show SD. Significant difference from pH 8, t test: , P < 0.05; , P < 0.01; **, P < 0.001. Significant difference between WT and D95N (t test; ###, P < 0.001). (E) Comparison of KAT1 voltage activation curves for control (black) and 300 µM Cd2+ (red) at pH 7.0 (dotted) and pH 5.0 (solid). Data were collected and presented as for C (n = 8); Boltzmann fit parameters are provided in Table 1. (F) Comparison of Cd2+ affinity across pH 9.0 to pH 5. The ΔV50 values for different Cd2+ concentrations at indicated pHs were collected as described in Fig. 3 D, and open circles represent individual measurements (n = 6–16). The smooth curves show the Hill plot fits; we did not attempt to fit data for pH 5. The Hill fit parameters are reported in Table 3.
	Figure 11. Characterization of pH and Cd2+ sensitivity in KAT N99D and Q149E. (A and B) Example traces of KAT1 mutants N99D and Q149E recorded at pH 7.0 using the voltage step protocol shown in the inset. The scale bars apply to both panels. (C) G-V curves for KAT1 WT (black), N99D (green), and Q149E (orange). Data (mean ± SEM; n = 8) were recorded at pH 7.0 from −100-mV isochronal tail currents following 2-s voltage steps to the indicated potentials. Curves show single Boltzmann fits of the data with parameters reported in Table 1. (D) Plots of ΔV50 at pH 7.0 to pH 4.0 relative to pH 8.0 (V50[pH X] − V50[pH 8]) for KAT1 mutants N99D and Q149E compared with WT. Box plots show mean and middle quartiles, and whiskers show SD (n = 8). Significant difference from pH 8, t test, , P < 0.05; **, P < 0.001. Significant difference from WT, t test, ##, P < 0.01; ###, P < 0.001. (E and F) G-V comparisons for N99D and Q149E at pH 7.0 in the presence (red) or absence (black) of 300 µM Cd2+. Data show means ± SEM (n = 8), and curves show single Boltzmann fits.

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