A Functional Analysis of NaK at the Single Channel Level

Abstract

NaK is a non-selective monovalent cation channel from Bacillus cereus. Despite being unable to discriminate between Na+ and K+, NaK shows high sequence similarity to other K+ channels. Based on recently solved crystal structures in the closed and putatively open state, NaK exhibits an overall architecture similar to that found in the pore domain of tetrameric K+ channels. Rb+ influx studies suggest the channel conducts cations, however net flux is unusually low for a channel. The absence of electrophysiological data from NaK precludes significant understanding of it's functional behavior. Using a random mutagenesis approach together with a K+ transport based screen, we have identified gain-of-function mutants in an attempt to develop a system for electrophysiological studies. One of these purified and reconstituted mutants was further studied by liposome patch-clamp. The channel displays non-selective conductances at 25 and 91 pS and is characterized by a low probability spiking behavior. In addition, we show that NaK undergoes a voltage-dependent inactivation process, which is functionally similar to that seen in K+ channels. This inactivation may contribute to the low flux of Rb+ through NaK. Our functional characterization, along with the known crystal structures, now allows us to use NaK as a model system to further investigate structure-function correlations in non-selective channels and related selectivity filters.