Characterization of the PCMBS-dependent modification of KCa3.1 channel gating.
Intermediate conductance, calcium-activated potassium channels are gated by the binding of intracellular Ca(2+) to calmodulin, a Ca(2+)-binding protein that is constitutively associated with the C terminus of the channel. Although previous studies indicated that the pore-lining residues along the C-terminal portion of S6 contribute to the activation mechanism, little is known about whether the nonluminal face of S6 contributes to this process. Here we demonstrate that the sulfhydral reagent, parachloromercuribenze sulfonate (PCMBS), modifies an endogenous cysteine residue predicted to have a nonluminal orientation (Cys(276)) along the sixth transmembrane segment (S6). Modification of Cys(276) manipulates the steady-state and kinetic behavior of the channel by shifting the gating equilibrium toward the open state, resulting in a left shift in apparent Ca(2+) affinity and a slowing in the deactivation process. Using a six-state gating scheme, our analysis shows that PCMBS slows the transition between the open state back to the third closed state. Interpreting this result in the context of the steady-state and kinetic data suggests that PCMBS functions to shift the gating equilibrium toward the open state by disrupting channel closing. In an attempt to understand whether the nonluminal face of S6 participates in the activation mechanism, we conducted a partial tryptophan scan of this region. Substituting a tryptophan for Leu(281) recapitulated the effect on the steady-state and kinetic behavior observed with PCMBS. Considering the predicted nonluminal orientation of Cys(276) and Leu(281), a simple physical interpretation of these results is that the nonluminal face of S6 forms a critical interaction surface mediating the transition into the closed conformation, suggesting the nonluminal C-terminal portion of S6 is allosterically coupled to the activation gate.
Bailey
Mark A
MA
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Grabe
Michael
M
Devor
Daniel C
DC
D002731:4-Chloromercuribenzenesulfonate / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; D002731:4-Chloromercuribenzenesulfonate; D007930:Leucine; D003545:Cysteine; D002118:Calcium