Abstract
Inwardly rectifying K(+) (Kir) channels set the resting membrane potential and regulate cellular excitability. The activity of Kir channels depends critically on the phospholipid PIP(2). The molecular mechanism by which PIP(2) regulates Kir channel gating is poorly understood. Here, we utilized a combination of computational and electrophysiological approaches to discern structural elements involved in regulating the PIP(2)-induced gating kinetics of Kir2 channels. We identify a novel role for the cytosolic GH loop. Mutations that directly or indirectly affect GH loop flexibility (e.g. V223L, E272G, D292G) increase both the on- and especially the off-gating kinetics. These effects are consistent with a model in which competing interactions between the CD and GH loops for the N terminus regulate the gating of the intracellular G loop gate.
Highlights
The detailed mechanism of PIP2-induced Kir channel gating remains elusive
The Val-223 and Glu-272 Show High Correlativity with A306 in the G Loop Gate—To explore the mechanism by which the G loop gate relates to the rest of the cytosolic domain during PIP2induced Kir channel gating, we performed 10-ns Molecular Dynamics (MD) simulations of the cytosolic domains of the WT Kir2.1 and analyzed the relative movements between C-terminal residues and the G-loop residue Ala-306
PIP2-induced channel gating is an intrinsically dynamic process that is difficult to understand by the few available static crystal structures that are missing key transition steps connecting them
Summary
Results: Specific mutations increase the flexibility of the cytosolic GH loop and accelerate the PIP2-induced gating kinetics of Kir channels. 42278 JOURNAL OF BIOLOGICAL CHEMISTRY maintenance of the resting membrane potential, hormonal secretion, extracellular Kϩ buffering in the brain, and Kϩ secretion in the kidney These physiological processes are regulated by specific stimuli that control the gating of Kir channels. Functional studies and mutagenesis data have identified several residues as potential PIP2-interacting sites in both the N- and C termini of the cytoplasmic domain of the Kir2.1 channel. We set out to study Kir channel gating kinetics, focusing on the cytosolic channel regions using a voltage-gated phosphatase to control PIP2 levels [22] and computational, mutagenesis, and electrophysiological approaches to probe the role of specific secondary structural elements and specific residues within them. Mutations that decrease the GH loop interactions with the N terminus (e.g. V223L, E272G, D292G) increase the flexibility of the GH loop and allow the N terminus to gate the G loop with faster kinetics
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