Functional significance of charged amino acids on the wall of the inner vestibule of Kir2.1

Abstract

It has been known that the rectification property of inward rectifier K⁺ channel (Kir) is mainly due to block of outward current by cytoplasmic Mg²⁺ and polyamines. Crystal structure analysis of the cytoplasmic region of Kir3.1 has revealed that positively (R228, R260) and negatively (E224, D259, E299) charged residues are on the wall of the inner vestibule in Kir2.1. In this study, we aimed to approach to the functional significance of these charged residues, and compared the electrophysiological properties of single/double mutants of them. We examined the inward rectification property of the mutants expressed in Xenopus oocytes under two-electrode voltage clamp. We observed that E224Q, D259N and E229Q single point mutations weakened rectification, and that R228Q and R260Q single point mutations on the background of E224Q made the rectification property recovered. We next analyzed susceptibilities of the mutants to blockade by intracellular blockers using the inside-out patch clamp technique. We observed that the decrease in the net negative charge in the cytoplasmic pore reduced the blocking sensitivity to Mg²⁺ and spermine, and the voltage dependency of Mg²⁺ blocking. It also influenced K⁺ permeation; i.e. unexpectedly weakened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these results suggest that the net negative charge in the cytoplasmic pore increases the local concentration of cations such as Mg²⁺, spermine as well as K⁺, and also extend the electrostatic field to the inner vestibule. [Jpn J Physiol 54 Suppl:S132 (2004)]