Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels.

The outward component of the strong inward rectifier K(+) current (I(Kir)) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward I(Kir) and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current-voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 microm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current-voltage relations showed one peak, like those of native I(Kir), outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K(+)] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward I(Kir). When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers.

Different intracellular polyamine concentrations underlie the difference in the inward rectifier K(+) currents in atria and ventricles of the guinea-pig heart.

The outward component of the strong inward rectifier potassium current, I(K1), is significantly larger in ventricles than in atria of the heart, resulting in faster repolarization at the final phase of the action potential in ventricles. However, the underlying mechanism of the difference in I(K1) remains poorly understood. I(K1) channels are composed of subunits from the Kir2 subfamily, and I(K1) amplitude is determined by the voltage-dependent blockade of the channel by the intracellular polyamines spermine and spermidine, and by Mg(2+). Using a perforated patch-clamp method, which minimizes changes in the intracellular polyamine and Mg(2+) concentrations, we detected repolarization-induced outward I(K1) transients, which are caused by competition between Mg(2+) and spermine to block the channel, in ventricular but not in atrial myocytes from guinea-pig heart. The contribution of the Kir2.3 subunit to the I(K1) channel was found to be minor in the guinea-pig heart, because the activation time course of the Kir2.3 currents was approximately 10-fold slower than those of I(K1), and the marked external pH sensitivity of the Kir2.3 currents was not found in I(K1). Both the Kir2.1 and Kir2.2 currents recorded from inside-out patches exhibited outward transients similar to those of ventricular I(K1) in the presence of 5-10 microM spermine and 0.6-1.1 mM Mg(2+), and their amplitudes were diminished by increasing the spermine or spermidine concentrations. The total and free polyamine concentrations in guinea-pig cardiac tissues were higher in atria than ventricles. These results strongly suggest that different intracellular polyamine concentrations are responsible for the difference in atrial and ventricular I(K1) of the guinea-pig heart.

Two Kir2.1 channel populations with different sensitivities to Mg(2+) and polyamine block: a model for the cardiac strong inward rectifier K(+) channel.

The strong inward rectification of the whole cell Kir2.1 current, which is very similar to the cardiac inward rectifier K(+) current (I(K1)), is caused by voltage-dependent blockade of outward currents by the intracellular polyamines spermine and spermidine. We recently showed that macroscopic Kir2.1 currents obtained from inside-out patches in the presence of various concentrations of cytoplasmic polyamines are well explained by the sum of the currents through two populations of channels that show differing susceptibilities to polyamine blockade. The outward currents obtained with 5-10 microM cytoplasmic spermine showed current-voltage relationships similar to those of I(K1) and were considered to flow mostly through a small population of channels exhibiting lower spermine sensitivity. Here we used inside-out patches to examine the blockade of macroscopic Kir2.1 currents by cytoplasmic Mg(2+) in the absence and presence of cytoplasmic spermine. Outward currents were blocked by 0.6 and 1.1 microM Mg(2+) in a concentration-dependent manner, but a small fraction ( approximately 0.1) of the macroscopic conductance was resistant to Mg(2+) at those concentrations, suggesting there are two populations of Kir2.1 channels with different sensitivities to Mg(2+). Furthermore, at those concentrations, Mg(2+) blocked inward currents by inducing a shallow blocked state that differed from the deeper state causing the inward rectification. In the presence of 1.1 microM Mg(2+) + 5 microM spermine, Mg(2+) blocked a substantial current component during depolarizing pulses and generated transient outward components, which is consistent with findings from earlier whole-cell experiments. In the steady state, Mg(2+) blocked the currents at voltages around and negative to the reversal potential and induced sustained outward components. The steady-state and time-dependent current amplitudes and the fractional blockades caused by spermine and Mg(2+) could be quantitatively explained by a model in which Mg(2+) competes with spermine to block the high-affinity channel and induces three conductance states. The present results suggest that the outward I(K1) flows through two populations of channels with different sensitivities to cytoplasmic blockers.

Inward rectifier K(+) current under physiological cytoplasmic conditions in guinea-pig cardiac ventricular cells.

The outward current that flows through the strong inward rectifier K(+) (K(IR)) channel generates I(K1), one of the major repolarizing currents of the cardiac action potential. The amplitude and the time dependence of the outward current that flows through K(IR) channels is determined by its blockage by cytoplasmic cations such as polyamines and Mg(2+). Using the conventional whole-cell recording technique, we recently showed that the outward I(K1) can show a time dependence during repolarization due to competition of cytoplasmic particles for blocking K(IR) channels. We used the amphotericin B perforated patch-clamp technique to measure the physiological amplitude and time dependence of I(K1) during the membrane repolarization of guinea-pig cardiac ventricular myocytes. In 5.4 mM K(+) Tyrode solution, the density of the current consisting mostly of the sustained component of the outward I(K1) was about 3.1 A F(-1) at around -60 mV. The outward I(K1) showed an instantaneous increase followed by a time-dependent decay (outward I(K1) transient) on repolarization to -60 to -20 mV subsequent to a 200 ms depolarizing pulse at +37 mV (a double-pulse protocol). The amplitudes of the transients were large when a hyperpolarizing pre-pulse was applied before the double-pulse protocol, whereas they were small when a depolarizing pre-pulse was applied. The peak amplitudes of the transients elicited using a hyperpolarizing pre-pulse were 0.36, 0.63 and 1.01 A F(-1), and the decay time constants were 44, 14 and 6 ms, at -24, -35 and -45 mV, respectively. In the current-clamp experiments, a phase-plane analysis revealed that application of pre-pulses changed the current density at the repolarization phase to the extents expected from the changes of the I(K1) transient. Our study provides the first evidence that an outward I(K1) transient flows during cardiac action potentials.