The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I-f)/neuronal (I-h) time-and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and Ih channels in neurons. This raises the possibility of Ca2+ permeation in If, the Ih counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca2+ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I-f in rat ventricular myocytes. We observed Ca2+ influx when HCN/I-f channels were activated. Ca2+ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an If channel blocker, inhibited If and Ca2+ influx at the same time. Quantitative analysis revealed that Ca2+ flux contributed to similar to 0.5% of current produced by the HCN2 channel or If. The associated increase in Ca2+ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which If current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca2+ chelator), preactivation of If channels significantly reduced the action potential duration, and the effect was blocked by another selective I-f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I-f channels had no effects on action potential duration. Our data extend our previous discovery of Ca2+ influx in Ih channels in neurons to If channels in cardiac myocytes.
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