Time-dependent Outward Currents through the Inward Rectifier Potassium Channel IRK1 : The Role of Weak Blocking Molecules

Outward currents through the inward rectifier K⁺ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg²⁺ (0.44–1.1 mM) or putrescine (∼0.4mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K⁺. Without internal Mg²⁺, small outward currents flowed only at potentials between −80 (the reversal potential) and ∼−40 mV during voltage steps appliedfrom −110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating,which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg²⁺, small outwardcurrents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between −40 and 0 mV were concurrent with the contribution of Mg²⁺ to blocking channels at these potentials,judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to −50 mV after short depolarizing steps (>0 mV), a transient increase appeared in outward currents at −50 mV. Since the peak amplitude depended on the fraction of Mg²⁺-blocked channels in thepreceding depolarization, the transient increase was attributed to the relief of Mg²⁺ block, followed by a re-blockof channels by spermine. Shift in the holding potential (−110 to −80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg²⁺-blocked channels in depolarization,which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects asMg²⁺. When both spermine (1 μM, an estimated free spermine level during whole-cell recordings) and putrescine(300 μM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Ourstudy indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg²⁺ (putrescine), elicits atransient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.