High affinity associations with α-SNAP enable calcium entry via Orai1 channels.

Ramanagouda Ramanagoudr-Bhojappa,Monika Vig,Yong Miao,Eugene A Permyakov

doi:10.1371/journal.pone.0258670

Abstract

Molecular steps that activate store-operated calcium entry (SOCE) via Orai channel supramolecular complex remain incompletely defined. We have earlier shown that α-SNAP regulates the on-site functional assembly and calcium selectivity of Orai1 channels. Here we investigate the molecular basis of its association with Orai, Stim and find that the affinity of α-SNAP for Orai and Stim is substantially higher than previously reported affinities between Stim and Orai sub-domains. α-SNAP binds the coiled-coil 3 (CC3) sub-domain of Stim1. Mutations of Tryptophan 430 in Stim1-CC3 disrupted α-SNAP association and SOCE, demonstrating a novel α-SNAP dependent function for this crucial subdomain. Further, α-SNAP binds the hinge region near the C-terminus of Orai1 and an additional broad region near the N-terminus and Valine 262 and Leucine 74 were necessary for these respective interactions, but not Orai, Stim co-clustering. Thus, high affinity interactions with α-SNAP are necessary for imparting functionality to Stim, Orai clusters and induction of SOCE.

Highlights

The genome-wide RNAi screens initially identified Stim and Orai as two crucial components of the store-operated calcium release activated calcium (CRAC) channel complex [1,2,3,4,5,6] and the past decade has seen rapid growth in the structure-function analysis of these two proteins
We have previously shown that α-SNAP binds the Stim Orai activation region (SOAR)/CRAC activation domain (CAD) domain of Stim1 [20]
We have identified the minimal sub-domains of Orai1 and Stim1 involved in associating with α-SNAP, a calcium release activated calcium (CRAC) channel component necessary for the functional assembly and selectivity of Orai1 multimers

Summary

Introduction

The genome-wide RNAi screens initially identified Stim and Orai as two crucial components of the store-operated CRAC channel complex [1,2,3,4,5,6] and the past decade has seen rapid growth in the structure-function analysis of these two proteins. The depletion of calcium stores induces oligomerization and translocation of Stim to the peripheral/ junctional endoplasmic reticulum (ER) tubules localized in close proximity with plasma membrane (PM) [7]. Stim oligomerization remains equated with the functional exposure and binding of the Stim Orai activation region (SOAR)/ CRAC activation domain (CAD) of Stim with Orai1 [9, 10]. SOAR/ CAD is a ~100 amino-acid long minimal domain of Stim necessary for activating SOCE [11, 12] and previous reports have assigned multiple different roles to it. It is thought to be responsible for Stim oligomerization, entrapment of freely diffusing Orai in PM [13, 14], via direct interactions with the C-terminus [15], and induce allosteric shifts in Orai for activating SOCE. How Stim single-handedly performs all these roles remains

Methods

Results

Conclusion