Interference In Coiled-coil Mediated Coupling Between Stim1 And Orai Channels

Abstract

STIM1 and ORAI1, the two limiting components in the CRAC signalling cascade, have been reported to couple tightly upon store-depletion culminating in CRAC current activation. Based on the homology within the ORAI protein family, an analogous scenario might be assumed for ORAI2 as well as ORAI3 channels as both are activated in a similar store- and STIM1-dependent manner. A combined approach of electrophysiology and confocal Förster Resonance Energy Transfer (FRET) microscopy revealed a general mechanism in the communication of STIM1 with ORAI proteins that involved the predicted second coiled-coil motif in STIM1 C-terminus and the conserved putative coiled-coil domain in the respective ORAI C-terminus. Of the latter, a much higher coiled-coil probability is predicted for ORAI2 as well as ORAI3 than for ORAI1, compatible with our observation that a single point coiled-coil mutation in ORAI1 C-terminus abrogated communication with STIM1 C-terminus, while an analogous mutation in ORAI2 and ORAI3 still allowed for their moderate activation. Conversely, destabilizing the second coiled-domain of STIM1 C-terminus by a single point mutation still enabled partial stimulation of ORAI2 and ORAI3 channels but not of ORAI1. A double mutation within the second coiled-coil motif of STIM1 C-terminus fully disrupted communication with all three ORAI channels. In aggregate, the impairment in the overall communication between STIM1 and ORAI channels upon mutual destabilization of putative coiled-coil domains in either C-terminus would be compatible with their heteromeric interaction.Supported by FWF P18169.