Ion Conductance of Orai1 Channel Selectivity-Filter Heterodimers

Abstract

Store Operated Ca2+ Entry (SOCE) controls a wide spectrum of physiological functions. Recent studies have identified the ER Ca2+ sensor STIM1 and channel forming unit Orai1 as two main players in this process. However, the mechanism by which the Orai1 channel is gated has remained elusive. In this study, we focused on the selectivity filter of the Orai1 channel, the Glu 106 residue. Previous studies reported that the single E106D point mutation diminishes Ca2+ selectivity, and E106Q or E106A mutations result in non-conductive channels. Here, using a concatemer strategy, we utilized heterodimers of Orai1 consisting of a wildtype Orai1 (O) and a mutated Orai1E106Q (Q) moiety. We observed that such dimers form nonselective channels with increased Cs+ permeability when transiently expressed in HEK Orai1 knockout cells stably expressing STIM1-YFP. These results were also repeated using the concatenated hexamers of Orai1, OQOQOQ and QOQOQO. Similar to the Orai1 E106D monomer, the aspartate-substituted OD or DO heterodimers still function as non-selective channels. Based on the side chain size of different amino acids, we tested the Orai1 E106N monomer, which results in a non-conductive channel. However, the ON and NO heterodimers of Orai1 can form non-selective channels. Other substitutions of the E106 residue, such as glycine, valine, and leucine failed to restore significant Ca2+ influx after store depletion either as monomers or heterodimers with wildtype Orai1.Using different ammonium ion derivatives of increasing size, we found all the nonselective heterodimers showed enlarged pore size compared with the wildtype dimer. Therefore, we conclude that the E106Q, E106N and E106D mutations in the heterodimer alter the geometry of the channel pore. Both the charge and size of the Glu106 residue are critical to function of the tightly regulated and highly Ca2+ selective Orai1 channel.