Intracellular Mg is a voltage-dependent pore blocker of HCN channels

Vemana S, Pandey S, Larsson HP. Intracellular Mg is a voltage-dependent pore blocker of HCN channels. Am J Physiol Cell Physiol 295: C557–C565, 2008. First published June 25, 2008; doi:10.1152/ajpcell.00154.2008.—Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg functions as a voltagedependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg is in the physiological range, with Mg binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at 50 mV. The effective electrical distance for the Mg binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg , suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.