Calcium Inhibition and Calcium Potentiation of Orai1, Orai2, and Orai3 Calcium Release-activated Calcium Channels

Rebecca R Boyles,Jeremy T Smyth,James W Putney,Wayne I Dehaven

doi:10.1074/jbc.m611374200

Abstract

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.

Highlights

Store-operated or capacitative calcium entry channels subtend or support important Ca2ϩ signaling pathways in a wide variety of cell types [1]
In the course of this work, we developed a technique that permits reproducible assessment of very small in CRAC current (Icrac), such as encountered in wild type HEK293 cells or in cells transfected with Stim1 and Orai3
With the divalent free solution (DVF) switching technique, a clear increase in store-operated Naϩ current was seen in Orai3transfected cells

Summary

Introduction

Store-operated or capacitative calcium entry channels subtend or support important Ca2ϩ signaling pathways in a wide variety of cell types [1]. Extracellular Ca2ϩ has two effects on CRAC channels It reduces conductance, or blocks the channels, by binding to a selectivity filter in the channel pore [10], but it augments channel function by binding to an undefined extracellular site, a process known as potentiation [11]. Blocks the channels, by binding to a selectivity filter in the channel pore [10], but it augments channel function by binding to an undefined extracellular site, a process known as potentiation [11] The latter role of extracellular Ca2ϩ can be revealed by protocols in which extracellular divalent cations are removed. We find that Orai differs strikingly from Orai and -2 in being relatively resistant to the process of depotentiation

Methods

Results

Conclusion