Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native I_f at physiological calcium concentrations

The hyperpolarization-activated cyclic nucleotide-gated (HCN) current I_f/I_HCN is generally thought to be carried by Na⁺ and K⁺ under physiological conditions. Recently, Ca²⁺ influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca²⁺ permeation through I_f/I_HCN is still lacking.

To possibly provide direct evidence of Ca²⁺ influx through I_HCN/I_f, we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human I_f, since Ca²⁺-mediated I_f/I_HCN currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca²⁺ inward currents upon hyperpolarization with a single-channel amplitude of −0.87 ± 0.06 pA, a low open probability of 3.02 ± 0.48% (at −110 mV, n = 6, Ca²⁺ 2 mmol/L), and a Ca²⁺ conductance of 8.9 ± 1.2 pS. I_HCN2-Ca2+ was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific I_f inhibitor ivabradine, clearly confirming that Ca²⁺ influx indeed was conducted by HCN2 channels. Changing [Na⁺] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca²⁺ caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na⁺/Ca²⁺ conductance, whereas the K⁺ conductance of HCN2 increased significantly in the absence of external Ca²⁺ with increasing K⁺ concentrations. The mixed K⁺–Ca²⁺ conductance, however, was unaffected by the external K⁺ concentration. Notably, we could also record hyperpolarization-activated Ca²⁺ permeation of single native I_f channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca²⁺ conductance of human I_f, 9.19 ± 0.34 pS; amplitude, −0.81 ± 0.01 pA; open probability, 1.05 ± 0.61% at −90 mV).

We directly show Ca²⁺ permeability of native rat and, more importantly, human I_f at physiological extracellular Ca²⁺ concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased I_f density and HCN expression.