Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene include s two pore-loop sequences that are thought to form the hydrophilic lining o f the pore. Gating of the channel is promoted by membrane depolarisation an d is regulated by the extracellular K+ concentration ([K+](0)) both in the yeast and when expressed in Xenopus oocytes, Our previous work showed that substitutions of equivalent residues L293 and A428,within the pore-loops ha d qualitatively similar effects on both the [K+](0)-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equiva lent residues N275 and N410, N-terminal from the K+ channel signature seque nces of the two pores, have very different actions on channel gating and, i n this case, are without effect on its voltage-sensitivity. The mutation N4 10D slowed current activation in a [K+](0)-dependent manner and it accelera ted deactivation, but without significant effect on the apparent affinity f or K+. The N275D mutant, by contrast, had little effect on the [K+](0) sens itivity for activation and it greatly altered the [K+](0) dependence of cur rent deactivation. Neither mutant affected the voltage-dependence of the st eady-state current nor the ability for other alkali cations to substitute f or K+ in regulating gating, The double mutant N410D-N275D showed characteri stics of N410D in the [K+](0)-sensitivity of current activation and of N275 D in the [K+](0)-sensitivity of deactivation, suggesting that little intera ction occurs between pore domains with mutations at these sites. The result s indicate that the two pore domains are not functionally equivalent and th ey suggest that the regulation of gating by external K+ is mediated by K+ b inding at two physically distinct sites with different actions, (C) 1999 Fe deration of European Biochemical Societies.