Pacemaker activity of the human sinoatrial node: an update on the effects of mutations in HCN4 on the hyperpolarization-activated current

Abstract

Since 2003, several loss-of-function mutations in the HCN4 gene, which encodes the HCN4 protein, have been associated with sinus node dysfunction. In human sinoatrial node (SAN), HCN4 is the most abundant of the four isoforms of the HCN family. Tetramers of HCN subunits constitute the ion channels that conduct the hyperpolarization-activated "funny" current (If), which plays an important modulating role in SAN pacemaker activity. Voltage-clamp experiments on HCN4 channels expressed in COS-7, CHO and HEK-293 cells, as well as in Xenopus oocytes have revealed changes in the expression and kinetics of mutant channels, but the extent to which especially the kinetic changes would affect If flowing during a human SAN action potential often remains unresolved. In our contribution to the Topical Collection on Human Single Nucleotide Polymorphisms and Disease Diagnostics, we provide an updated review of the mutation-induced changes in the expression and kinetics of HCN4 channels and provide an overview of their effects on If during the time course of a human SAN action potential, as assessed in simulated action potential clamp experiments. Future research may solve apparent inconsistencies between data from clinical studies and data from in vitro and in silico experiments.

Figure 1

Schematic topology of the HCN4 and MiRP1 proteins. The HCN4 α-subunit has six transmembrane segments (S1–S6), a pore-forming loop (P) and intracellular N- and C-termini. The voltage sensor of the channel is formed by the positively-charged S4 helix. The C-terminus comprises the C-linker (dotted line) and the cyclic nucleotide-binding domain (cNBD), which is known to mediate cyclic AMP (cAMP)-dependent changes in HCN channel gating. The MiRP1 β-subunit has a single transmembrane segment with an extracellular N-terminus and intracellular C-terminus. Red dots indicate the location of the 23 known HCN4 and MiRP1 mutation sites associated with clinically established or potential sinus node dysfunction. The split dots indicate the truncations resulting from the 573X and 695X non-sense (truncating) mutations.

Figure 2

Membrane currents of a human sinoatrial node (SAN) pacemaker cell assessed by a simulated action potential clamp. (A) Action potentials recorded from a human SAN pacemaker cell used for the action potential clamp; (B) associated rate of change of the membrane potential (dV_m/dt); (C) global intracellular calcium concentration ([Ca²⁺]_i) in a different cell with a highly similar cycle length; and (D) numerically reconstructed membrane current (I_m): L-type calcium current (I_Ca,L), I_f, delayed rectifier potassium current (I_Kr) and net membrane current (I_net). See the text for details.

Figure 3

Effect of mutations in HCN4 on I_f in a human SAN pacemaker cell assessed by simulated action potential clamp. (A) Action potentials recorded from a human SAN pacemaker cell used for action potential clamp; (B) computed wild-type (WT) I_f of a human SAN pacemaker cell during the action potentials of (A) under control conditions (“control”, blue line) and upon adrenergic stimulation (“cAMP”, red line); (C–H) computed I_f of a human SAN pacemaker cell carrying heterozygous mutation in HCN4, as indicated, during the action potentials of (A) under control conditions (solid blue line) and upon adrenergic stimulation (solid red line). Wild-type I_f of (B) under control conditions (dashed blue line) and upon adrenergic stimulation (dashed red line) are shown for reference. G482R traces, labelled “a” and “b”, are based on data from Milano et al. [27] and Schweizer et al. [28], respectively.

Figure 4

Contribution of I_f to diastolic depolarization for each of the heterozygous mutations in HCN4 or KCNE2. The charge carried by I_f (Q_f) during the 25-mV, 550-ms spontaneous depolarization from the maximum diastolic potential of −63 mV of the human SAN action potential is as indicated in Figure 3A. The blue bars are computed from the I_f traces under control conditions (“control”). The red bars are computed from the I_f traces upon adrenergic stimulation (“cAMP”). The dashed grey line indicates the charge of 0.025 pC/pF carried by the net membrane current (Q_net) during the 25-mV depolarization. (A) Mutations of Figure 3; (B) mutations assessed in previous publication [30].