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 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) 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 had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.
Vergani
P
P
Laboratory of Plant Physiology and Biophysics, University of London, Wye College, Kent, UK.
Blatt
M R
MR
D000818:Animals; D005656:Fungal Proteins / Q000235:genetics*; D015640:Ion Channel Gating / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014982:Xenopus laevis
D005656:Fungal Proteins; D015221:Potassium Channels; D029701:Saccharomyces cerevisiae Proteins; C095220:TOK1 protein, S cerevisiae; D011188:Potassium