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

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Abstract
The hyperpolarization-activated cyclic nucleotide-gated (HCN) current If/IHCN 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 If/IHCN is still lacking. To possibly provide direct evidence of Ca2+ influx through IHCN/If, we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human If, since Ca2+-mediated If/IHCN 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. IHCN2-Ca2+ was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific If 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 If 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 If, 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 If at physiological extracellular Ca2+ concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased If density and HCN expression.

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