Regulation of the Kir4 subfamily of inwardly rectifying potassium channels by extracellular cations.

摘要

Potassium inwardly rectifying (Kir) channels are a family of K +-selective channels. They are expressed in a wide variety of tissues and are involved in control of cell excitability and vectoral K+-transport. The work presented here studies the characteristics and mechanisms involved in regulation of Kir4.1 and Kir4.2 by extracellular cations. Using two-electrode voltage clamp on Xenopus leavis oocytes exogenously expressing the channels, I found that Kir4.1 channels are sensitive to changes in the [K+]o. Increasing the [K+]o leads to a slow increase in the whole-cell currents. This effect is reversible when the [K+]o is decreased. The regulation is not coupled to the pHi-sensitivity of the channels as evident from the apparent pKa:s being independent of the [K +]o and from introduction of a point mutation that shifts the pHi-sensitivity of the channels, does not effect the K +o-dependence. Studying the selectivity of this process in Kir4.1, I demonstrate that other ions that interact with the pore, either permeant (Rb+) or blocking (Cs+, Ba2+ ), are able to mimic the effect of W. However, NH4 +, also permeant through Kir4.1, is not able to substitute for K +. The dose dependence of the voltage-dependent block by Cs+ and regulation by Cs+ are found to be similar. This is consistent with the idea that block and the regulatory effect of CC occurring at the same binding site, i.e. the selectivity filter. Using a kinetic model of permeation I show the plausibility that the channel senses changes in the ionic environment through changes in the pore occupancy. Measurement of the surface expression of Kir4.2, using biotinylation, shows that this form of regulation does not involve a change in surface expression. Nor does it involve a change in the measurable open probability PO, as determined by patch clamp. Examining the dose dependency of the rate of activation I show that the data is consistent with the idea that Kir4.2 exists at the plasma membrane in inactive, intermediate unstable, and active states and that K + affects the rate of transition between the intermediate and active states.