Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1

Min Seuk Kim,Paul F. Worley,Weizhong Zeng,Dong Min Shin,Shmuel Muallem,Joseph P. Yuan

doi:10.1074/jbc.m808097200

Abstract

With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca(2+) influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.

Highlights

STIM1 is the endoplasmic reticulum (ER) Ca2ϩ content sensor
The present work shows that TRPC1 can function as a bona fide store-operated Ca2ϩ channels (SOCs) and that the channel function of TRPC1 and Orai1 is required for native SOCs
Several studies showed that disruption of TRPC1 expression reduces SOC activity, and we reported that TRPC1 is regulated by the ER Ca2ϩ sensor STIM1 [12, 13]

Summary

Introduction

STIM1 is the ER Ca2ϩ content sensor. It has an N-terminal EF hand, which resides in the ER lumen, as well as cytoplasmic C-terminal Ezrin-Radixin-Moesin (ERM), serine/proline, and lysine-rich domains [8, 9]. Co-expression of Orai, TRPC1, and STIM1 in HEK cells resulted in a larger SOC-mediated Ca2ϩ influx than expression of the individual channels with STIM1 [30]. Further studies reported that transfection of low levels of Orai in HEK cells stably transfected with TRPC3 or TRPC6 converted the TRPC channels from store-independent to storeoperated channels [15] and increased the CRAC current [14]. These findings suggest that ectopically expressed Orai and TRPC channels interact, their contribution to the native SOCs and whether the channel function of Orai and of TRPC channels is required for the native SOC are not known. These findings have implications for how receptor stimulation regulates Ca2ϩ signaling and the role of TRPC channels in Ca2ϩ signaling

Methods

Results

Conclusion