Calcium inhibits the funny current (If)in sinoatrial myocytes.

Abstract

Hyperpolarization-activated cyclic nucleotide sensitive (HCN4) channels are the molecular correlate of the funny current (If), which is required for spontaneous action potentials in cardiac pacemaker cells. Activation of β-adrenergic receptors on pacemaker cells increases cAMP, which shifts HCN4 activation to more depolarized potentials. The resulting increase in current, along with activation of Ca2+ cycling proteins, increases action potential firing rate and, thus, heart rate. Here we show that Mg2+ and Ca2+ regulate the effects of cAMP on If and HCN4. We found that physiological concentrations of intracellular Ca2+ inhibited cAMP-dependent potentiation of native If channels in excised membrane patches from sinoatrial node myocytes (IC50 ≈ 9 μM). This inhibition was recapitulated in heterologously expressed HCN4 channels, where intracellular Ca2+ inhibited the channels with a similar IC50 (≈ 5 μM) and limited the cAMP-dependent shift in channel activation (4 mV in 100 µM Ca2+ vs. 12 mV in zero Ca2+). Interestingly, Mg2+ had opposing effects to Ca2+. Mg2+ has been shown to interact with cAMP-bound HCN4 channels at a metal coordination site composed of two residues in the C-linker and two residues in the S4-S5 linker. We found that Mg2+ is also required to functionally couple the inhibitory, unbound CNBD to the pore in the absence of cAMP — removal of Mg2+ mimicked the effects of cAMP-dependent disinhibition. Although the metal coordination site is conserved among mammalian HCN channel isoforms, Mg2+ and Ca2+ regulation appear to be specific for HCN4 — cAMP regulation of HCN2 was insensitive to divalents. These results suggest that divalent cations directly regulate HCN4, raising the possibility that If in pacemaker cells is dynamically regulated in response to beat-to-beat changes in intracellular Ca2+.