The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus

Abstract

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

“…We also observed that after a few seconds MthK channel activity declined ( Supplementary Fig. 2 ), similar to what was previously reported using electrophysiological recordings with MthK from Escherichia coli spheroplasts 43 , but not observed in steady-state single-channel recordings in lipid bilayers 6 7 8 . This process did not interfere with our blocker studies, which were performed within a few hundred millisecond of channel activation (see below).…”

Molecular mechanism of a potassium channel gating through activation gate-selectivity filter coupling

“…Inspired by these observations, we wondered whether the same conformational change could occur in MthK. In fact, a similar inactivation process was identified in MthK 36,37,48 , with the SF being postulated as the inactivation gate; 35 intriguingly, however, the constricted conformation of the SF was never captured in structural studies of MthK 38 . Although inactivation in MthK is enhanced by the presence of certain divalent cations, it has also been shown that their presence is not mandatory for the channel to undergo inactivation 36 , which occurs at positive (depolarizing) voltages and in a voltage-dependent manner 35 .…”

Structure of the MthK RCK in complex with cadmium

“…This technique has been used extensively to study the function of bacterial mechanosensitive channels (Blount et al, 1999; Martinac et al, 1987) and has recently been applied very successfully in studying gating of potassium channels including MthK (Kuo et al, 2007a; Kuo et al, 2007b; Kuo et al, 2008). MthK undergoes desensitization via its N-terminus portion corresponding to positions 2-17 of its sequence, deletion of which completely removes desensitization (Kuo et al, 2008). This sequence does not influence the channel conductance and in fact complicates the analysis of the activation by Ca 2+ or Cd 2+ .…”

“…Other biochemical data is inconsistent with the gating-ring model of activation, showing that isolated RCK exists as hexamer at pH ∼8.0 and dissociates to monomers at pH ∼9.0, at which MthK can still be effectively activated by Ca 2+ (Kuo et al, 2007a; Kuo et al, 2008). This inconsistency may stem from the oligomerization data limited to isolated (“free”) RCK in solution, rather than the full-length channel in the membrane.…”