Activation Mechanism of the Calcium Release-Activated Calcium Channel Revealed by the Gating Competence of Constitutively Open Orai Mutants

Abstract

Calcium release-activated calcium (CRAC) channels in the plasma membrane are integral membrane proteins that is critical in cellular signaling by generating the sustained influx of calcium. The crystal structure of Orai [1], the pore unit of a CRAC channel, provides the first insight into the architecture of this transmembrane protein at atomic level. However, the gating mechanism of CRAC channel remains elusive. Previously, we used the crystal structure as the starting point to compare the wild type protein and its V174A mutant in fully hydrated lipid bilayers. We identified that both pore-waters and counterions are actively involved in regulating the channel conductance [2-3]. Nevertheless, the intrinsic flexibility of the channel, especially the pore-forming helices, is more relevant to its gating triggered by the endoplasmic reticulum calcium sensor, the stromal interaction molecule (STIM). In this work, we probe the channel's conduction properties by exploring the wild type Orai with a more hydrated pore, as well as the STIM1-independent constitutively open Orai mutants, G170P and P288A, with combined molecular dynamics and free-energy calculations. The insertion of water molecules is to study the inherent rigidity of the pore, and mutations target at conserved residues on and off the pore-forming helices; all of the three systems were found to have significant pore expansion. In accordance with the motions of the pore-lining residues, ion permeation through the pore of the mutant has a much lower free energy barrier than that in the wild type protein. Therefore the open state structures of Orai obtained from computer simulations provide models at atomic level to study the STIM-activated CRAC gating.[1] Science, 2012, 338, 1308-1313.[2] PNAS, 2013, 110, 17332−17337.[3] J. Phys. Chem. B, 2014, 118, 9668−9676.