TRP4 (CCE1) Protein Is Part of Native Calcium Release-activated Ca2+-like Channels in Adrenal Cells

Stephan Philipp,Claudia Trost,Jan Warnat,Julia Rautmann,Nina Himmerkus,Gregor Schroth,Oliver Kretz,Wolfgang Nastainczyk,Adolfo Cavalié,Markus Hoth,Veit Flockerzi

doi:10.1074/jbc.m003408200

Abstract

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca(2+) entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry that TRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2), and TRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca(2+) (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Highlights

Receptor-operated Ca2ϩ signaling has been described in a variety of non-excitable cells and has been attributed to basic and specific cell functions like cell growth, differentiation, and hormone release
The channels formed by heterologously expressed TRP1, TRP4, and TRP5 appear to be activated by maneuvers known to deplete Ca2ϩ stores and represent store-operated Ca2ϩ channels (19 –23) but store independent activation of cation channels has been reported after heterologous expression of TRP4 and TRP5 [24]
The first evidence connecting the TRP4 protein to store-operated Ca2ϩ entry channels came from heterologous expression of the TRP4 cDNAs in mammalian cells lines like HEK 293

Summary

Introduction

Receptor-operated Ca2ϩ signaling has been described in a variety of non-excitable cells and has been attributed to basic and specific cell functions like cell growth, differentiation, and hormone release (reviewed by Refs. 1–3). The best studied storeoperated Ca2ϩ channels in terms of function and biophysical properties are calcium release-activated Ca2ϩ (CRAC) channels which have been originally described in mast cells and T-lymphocytes [16, 17]. This prototype of a store-operated Ca2ϩ channel is characterized by its inward rectifying currents and by its high selectivity for Ca2ϩ. Some recombinant mammalian TRP channels are sensitive to store depletion and form ion channels mainly permeable to Ca2ϩ whereas others appear to be non-selective cation channels insensitive to store depletion. The activity of recombinant TRP channels expressed in a foreign cellular environment does not necessarily correspond to the activity of native store-operated Ca2ϩ channels

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Conclusion