State-dependent Cysteine Modification during pH and PIP2 Gating in Kir Channels

Abstract

Inhibition by intracellular H+ (pH-gating) and activation by phosphoinositides such as PIP2 (PIP2-gating) are key regulatory mechanisms in the physiology of inwardly-rectifying potassium (Kir) channels. Our recent findings suggest that PIP2 gating and pH gating underlie similar conformational change at the helix bundle crossing, however, little is known about the structural changes in the cytoplasmic domains. Here we explore the state-dependent changes in accessibility of three endogenous cysteines (C175, C49, C308) in Kir1.1 channels occurring during PIP2 and pH gating. C175 in the inner pore cavity is modified by MTSET in the open state, but protected from modification in the closed state induced by either low intracellular pH or PIP2 depletion. This confirms the concept that the helix bundle crossing represents the gate controlled by pH and PIP2. C49 in the N-terminus is protected from modification in the open state but can be modified in the closed state induced by either low pH or PIP2 depletion indicating a similar conformational change in this region. C308 in the C-terminus can only be modified in the closed state induced by PIP2 depletion but is protected in the open state and as well in the pH-inhibited closed state. A homology model of Kir1.1 shows that C308 is located in close proximity to the PIP2 binding site indicating that PIP2 either directly, or by a conformational change at C308 protects this residue from modification. The lack of C308 modification in the pH inhibited state suggests that pH induced channel closure occurs with PIP2 tightly bound (thereby protecting C308), which is also consistent with our measurements on the kinetics of pH and PIP2 induced channel activation.