Identification of Sodium Sensitive Site and Chloride Sensitive Site on the C-Terminus of Rat KCNT1 Channel

Abstract

KCNT1 channel is a sodium and chloride activated potassium channel that is widely expressed in nervous system. Our previous study had identified D818 mutants can dramatically decrease the sodium sensitivity of KCNT1 channel. But the full co-ordinate sodium sensitive site and chloride site of this channel remain unknown. In this abstract, we reported that identification of a sodium binding site of this channel by fully screening 109 negatively charged amino acids mutants on the C-terminus of Slack channel. Results show several mutants of these negative charged channel increased Kd of sodium sensitivity to more than 1 M while several mutants did not show currents. Subsequently, we screened mutants that did not show currents on the background of mutant M335A because the mutant M335A of KCNT1 channel can be activated without intracellular sodium. Thus, we got either macroscopic current or single channel current from all mutants. Combined with electrophysiology experimental data and molecular dynamic simulation, we found that one subunit of Slack channel could bind two sodium ions. One dominant sodium sensitive site is located on RCK1 domain of the Slack channel. Another weak sodium binding pocket that is composed of 6 spatially closed negative charged residues. In the meantime, by molecular dynamic prediction and alignment with C.elegan Slo2 channel, we screened mutants of some positive charged residues on the C-terminus of the rat KCNT1 channel. One chloride binding site was identified from RCK1 domain of KCNT1 channel. In conclusion, our results dissected sodium and chloride sensitive sites of KCNT1 channel.