Novel Trans-Membrane Mutation Switches Orai1 to a Constitutively Active and Ca2+ Selective Channel

Abstract

The endoplasmic reticulum Ca2+ sensor STIM1 forms together with the Ca2+ channel Orai1 the molecular basis for Ca2+ release activated Ca2+ (CRAC) channels. The recent crystal structure of Orai from Drosophila melanogaster shows a unique Ca2+ channel composed of a hexameric subunit complex. The pore structure is formed by transmembrane (TM) 1 helices, surrounded by two ring-like structures, formed by TM2 and TM3 as well as TM4. Employing a combined approach of patch-clamp, molecular biology, biochemical techniques, molecular modeling and structure guided mutagenesis; we discovered a novel key mutation in the second trans-membrane helix of Orai1 that results in a Ca2+ selective, STIM1 independent, constitutively active current. Substitution of this essential residue to a hydrophobic amino-acid retained store-operated activation, yet with largely reduced Orai1 currents. In addition, we took advantage of the constitutively active Orai1 mutant, to evaluate reorientation of the gate located within the cytosolic region of TM1 helices. Cysteine scanning mutagenesis within the TM1 helix enabled identification of gating residues, the dimerization of which was altered in the constitutively opened and closed Orai1 channel conformation. In molecular dynamic simulations of an all-atom model of human Orai1 we will show the interaction network of these identified residues and predict implication of mutations on conformational changes. Our experiments will be summarized in a unique gating model, and we will moreover discuss how STIM1 binding might trigger the open channel conformation. This work was supported by the Austrian Science Foundation (FWF): P26067 to R.S. and P25172 to C.R. Irene Frischauf is an Elise Richter Scholarship holder: V286.