Molecular basis of the effect of potassium on heterologously expressed pacemaker (HCN) channels

Abstract

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels modulate the firing rates of neuronal and cardiac pacemaker cells. HCN channels resemble voltage-gated K+ channels structurally, but much less is known about their structure-function correlation. Although modulation of K+ channel gating by external K+ is a well-known phenomenon, such a link has not been established for HCN channels. Here we examined the effects of external permeant (K+, Na+ and Li+) and non-permeant (NMG+) ions on HCN1 and HCN2 gating. Substituting 64 of 96 mM external K+ with Na+, Li+ or NMG+ positively shifted steady-state activation (approximately 13 mV), and preferentially slowed activation of HCN1. Mutating the pore variant C-terminal to the GYG motif in HCN1, A352, to the analogous conserved Asp in K+ channels or Arg in HCN2 produced a significant hyperpolarizing activation shift (by 5-15 mV), slowed gating kinetics (up to 6-fold), and abolished or attenuated gating responses to external K+. Whereas Na+, Li+ and NMG+ substitutions produced depolarizing activation shifts of HCN2 similar to those of HCN1, deactivation but not activation of HCN2 was exclusively decelerated. We conclude that gating and permeation of HCN channels are coupled, and that modulation of this 'pore-to-gate' coupling by external K+ is isoform-specific.

Figure 1. Effect of K⁺, Na⁺, Li⁺ and NMG⁺ on HCN1 gating

Representative currents (A) through HCN1 channels in 96 mm K⁺ (n = 9), 32 mm K⁺-64 mm Na⁺ (n = 10), 32 mm K⁺-64 mm Li⁺ (n = 6) and 32 mm K⁺-64 mm NMG⁺ (n = 14) (a, −120 mV; b, −100 mV; c, −80 mV; d, −70 mV) normalized to the maximum currents recorded, and the corresponding steady-state activation curves (B). Summary of τ_act (open) and τ_deact (filled) (n= 10 (K⁺), 8 (Na⁺), 10 (NMG⁺), 6 (Li⁺)) (C), and normalized activation and deactivation tracings (D) recorded under different ionic conditions as noted. Thickened x-axes indicate voltage ranges over which all 3 ionic conditions differ significantly from 96 mm K⁺.

Figure 4. Summary of the effects on V_1/2 (A), and open and close rates (B) of HCN1, HCN1-A352D, HCN1-A352R and HCN2 channels by ion substitutions

Asterisks in A indicate significant V_1/2 shifts from 96 mm K⁺ (P < 0.05). Filled and open symbols in B represent α₀ and β₀, respectively.

Figure 2. HCN1-A352D abolished the effects of permeant ions on gating

Typical current tracings from the same protocol as shown in Fig. 1 (A), steady-state activation curves (n= 14 (K⁺), 7 (Na⁺), 11 (NMG⁺), 4 (Li⁺)) (B), and gating kinetics (n= 7 (K⁺), 8 (Na⁺), 7 (NMG⁺), 6 (Li⁺)) (C) of HCN1 A352D channels recorded under the same ionic conditions described in Fig. 1. D, E and F, same descriptions as A, B and C, respectively, but for A352R channels (n= 4 (K⁺), 4 (Na⁺)). Thickened x-axes indicate voltage ranges over which all 3 ionic conditions differ significantly from 96 mm K⁺.

Figure 3. Effect of K⁺, Na⁺, Li⁺ and NMG⁺ on HCN2

Same descriptions as Fig. 1 but for HCN2 channels. Unlike HCN1, Na⁺, Li⁺ and NMG⁺ substitutions decelerated deactivation without affecting activation. For activation curves, n = 14 (K⁺), 7 (Na⁺), 11 (NMG⁺), 4 (Li⁺). For kinetics, n = 7 (K⁺), 9 (Na⁺), 6 (NMG⁺), 7 (Li⁺).