ER localized bestrophin1 activates Ca2+ dependent ion channels TMEM16A and SK4

René Barro-Soria,Ralph Witzgall,Jiraporn Ousingsawat,Karl Kunzelmann,Joana Almaça,Rainer Schreiber,Fadi Aldehni

doi:10.1038/npre.2009.2881.1

Abstract

AbstractBestrophins form Ca2+ activated Cl- channels and regulate intercellular Ca2+ signaling1. We demonstrate that bestrophin 1 is localized in the endoplasmic reticulum (ER), where it physically interacts with stromal interacting molecule 1 (Stim1), the ER-Ca2+ sensor2,3. Intracellular Ca2+ transients in HEK293 cells elicited by stimulation of purinergic P2Y2-receptors were augmented but more transient after expression of hBest1, in contrast to dominant negative hBest1-R218C, which attenuated Ca2+ increase. The p21-activated protein kinase Pak2 was found to phosphorylate hBest1, thereby enhancing Ca2+ signaling and activation of Ca2+ dependent Cl- (TMEM16A)4 and K+ (SK4)5 channels. Lack of bestrophin 1 expression in respiratory epithelial cells of mBest1 knockout mice caused expansion of ER cisterns and induced Ca2+ deposits. We propose that hBest1 is important for Ca2+ handling of the ER store, probably by controlling the function of Stim1 and by acting as a counter-ion channel to balance transient membrane potentials occurring through inositol trisphosphate (IP3) induced Ca2+ release and refill of the ER-Ca2+ store. Thus bestrophin 1 controls activation of Ca2+ dependent ion channels by regulation of compartmentalized Ca2+ signaling.

Highlights

Compelling evidence has been provided that bestrophins are Ca2+ activated and DIDSsensitive Cl- channels (CaCC)[6,7], but much controversy exists about the question whether these proteins form apical Cl- channels in epithelial cells[8]
We found that hBest[1] expressed in HEK293 cells co-localizes with endoplasmic reticulum (ER) markers such as calnexin, Stim[1] (Fig. 1a) or calreticulin. hBest[1] was coimmunoprecipitated with Stim[1] (Fig. 1b), but not with other proteins involved in ER-Ca2+ signaling such as IP3-receptors, the Ca2+-ATPase sarcoendoplasmic reticulum calcium ATPase (SERCA) or components of the store operated Ca2+ influx (SOCE) pathway such as TRPC1
We measured the intracellular Ca2+ concentration [Ca2+]i in hBest[1] transfected HEK293 cells, using Fura[2] as Ca2+-senstive dye and we found that hBest[1] largely augments Ca2+ signals induced by stimulation of purinergic receptors with ATP (100 μM) (Fig. 1c)

Summary

Introduction

Compelling evidence has been provided that bestrophins are Ca2+ activated and DIDSsensitive Cl- channels (CaCC)[6,7], but much controversy exists about the question whether these proteins form apical Cl- channels in epithelial cells[8]. We measured the intracellular Ca2+ concentration [Ca2+]i in hBest[1] transfected HEK293 cells, using Fura[2] as Ca2+-senstive dye and we found that hBest[1] largely augments Ca2+ signals induced by stimulation of purinergic receptors with ATP (100 μM) (Fig. 1c). We examined the functional impact of Pak[2] on endogenous xBest[119] and activation of Ca2+ dependent Clchannels, by expression of P2Y2 receptors in Xenopus oocytes and stimulation with ATP.

Results

Conclusion