Flexibility Between the Linker of the CD and G-Loops Determines the Gating Dynamics of Hte Kir2.1 Channel

Abstract

Inwardly rectifying K+ (Kir) channels are important regulators of the resting membrane potential and cell excitability. The activity of Kir channels is critically dependent on the integrity of channel-PIP2 (phosphatidylinositol 4, 5-bisphosphate). Molecular Dynamics (MD) simulations predict interactions of specific residues with PIP2. Mutagenesis data are in good agreement with the theoretical predictions.Here we measure Kir channel gating kinetics regulated by endogenous and exogenous PIP2. Our data show that one Kir2.1 channel mutation, V223L, which is not predicted to interact with PIP2 directly altered the PIP2 apparent affinity by approximately 3-fold (the EC50 was 2.75±0.12 μM for WT channel and 8.57±0.76μM for the V223L mutation). V223 is localized in the CD-loop that has been implicated in the control of PIP2-dependent gating. V223 is predicted to interact with M301 in the G-loop, thus providing a coupling mechanism between the CD and G-loops. Unlike mutations of residues that are predicted to affect directly or indirectly channel-PIP2 interactions, V223L accelerated both the rates of inhibition (PIP2 depletion) and reactivation (PIP2 replenishment). Based on MD simulation results, we show that the V223L mutation enhances the flexibility of the amino acids from 270 to 290 which are located between the CD and G loops. Increasing the viscosity of the bathing solution reduces the dynamics of proteins, and consistent with the MD simulation results also in slowing down the kinetics of V223L inhibition and reactivation. Our results offer for the first time a link between the CD and G-loops in transducing the PIP2 effect on gating the Kir2.1 channel.