A synergistic blocking effect of Mg²⁺ and spermine on the inward rectifier K⁺ (Kir2.1) channel pore.

Chiung-Wei Huang,Chung-Chin Kuo

doi:10.1038/srep21493

Chiung-Wei Huang, Chung-Chin Kuo

Open Access

https://doi.org/10.1038/srep21493

Copy DOI

Abstract

Inward rectifier K+ channels (Kir2.1) exhibit an extraordinary rectifying feature in the current–voltage relationship. We have previously showed that the bundle–crossing region of the transmembrane domain constitutes the crucial segment responsible for the polyamine block. In this study, we demonstrated that the major blocking effect of intracellular Mg2+ on Kir2.1 channels is also closely correlated with K+ current flow, and the coupled movements of Mg2+ and K+ seem to happen in the same flux–coupling segment of the pore as polyamines. With a preponderant outward K+ flow, intracellular Mg2+ would also be pushed to and thus stay at the outermost site of a flux–coupling segment in the bundle–crossing region of Kir2.1 channels to block the pore, although with a much lower apparent affinity than spermine (SPM). However, in contrast to the evident possibilities of outward exit of SPM through the channel pore especially during strong membrane depolarization, intracellular Mg2+ does not seem to traverse the Kir2.1 channel pore in any case. Intracellular Mg2+ and SPM therefore may have a synergistic action on the pore–blocking effect, presumably via prohibition of the outward exit of the higher–affinity blocking SPM by the lower–affinity Mg2+.

Highlights

SPM, a blocker with an apparently much higher affinity than Mg2+ upon moderate depolarization, but an evident tendency of outward exit of the pore with large driving forces
We have seen that intracellular Mg2+ blocks the Kir2.1 channel pore in both voltage– and flow– dependent manners (Figs 1 and 2)
The blocking effect and especially the flow–dependence is markedly abolished by specific mutations involving E224, E299, and the bundle–crossing region (I176–A184), with double mutations either offsetting the effect of each single mutation or showing a strong negative cooperative effect (Figs 5–7)

Summary

Results

Inhibition of WT Kir2.1 currents by intracellular Mg2+ in symmetrical 100 mM K+ solution. Despite quite evident variations in the absolute values of the off rates, the voltage dependence remained similar among the WT and different mutant channels, suggesting a significantly altered height but not location and shape of the major barrier to the blocking Mg2+ exiting the pore These data are very similar to SPM12, and are consistent with the view that mutation of E224 and at least residues M183 change the height of the same barrier for Mg2+ permeation between the blocking site and intracellular melieu. The decay of outward currents at +100 mV again is very similar to the development of slow tails in these double–mutant channels (Fig. 6b) These findings collectively indicate that the E224Q mutation changes the pore size at the bundle–crossing region of transmembrane domain (TM2) (e.g., M183 and/or A178), and the blocking/unblocking kinetics of intracellular Mg2+. In the later phase of depolarization, on the other hand, Mg2+ could further decrease the steady–state K+ effluxes by prohibition of the outward exit of SPM ( see DISCUSSION and Fig. 10)

Discussion

Data analysis

Additional Information