Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709

Edited by Lutz Birnbaumer, University of California, Los Angeles, CA, and approved July 17, 2001 (received for review May 14, 2001)

Abstract

Mammalian Trp proteins are candidates for plasma membrane calcium channels regulated by receptor activation or by intracellular calcium store depletion [capacitative calcium entry (CCE)]. One extensively investigated member of the Trp family, the human Trp3 (hTrp3), behaves as a receptor-activated, calcium-permeable, nonselective cation channel when expressed in cell lines and does not appear to be activated by store depletion. Nonetheless, there is good evidence that Trp3 can be regulated by interacting with inositol trisphosphate receptors (IP₃Rs), reminiscent of the conformational coupling mode of CCE. To investigate the role of Trp3 in CCE, and its regulation by IP₃R, we transiently expressed hTrp3 in the wild-type DT40 chicken B lymphocyte cell line and its variant lacking IP₃R. Expression of hTrp3 in either wild-type or IP₃R-knockout cells did not increase basal membrane permeability, but resulted in a substantially greater divalent cation entry after thapsigargin-induced store depletion. This hTrp3-dependent divalent cation entry was significantly greater in the wild type than in IP₃R-knockout cells. Thus, it appears that in this cell line, hTrp3 forms channels that are store-operated by both IP₃R-dependent and IP₃R-independent mechanisms. Trp3, or one of its structural relatives, is a candidate for the store-operated, nonselective cation channels observed in smooth muscle cells and other cell types.

Footnotes

↵ * To whom reprint requests should be addressed at: National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. E-mail: vasquez{at}niehs.nih.gov.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

CCE,

capacitative calcium entry;

hTrp3,

human transient receptor potential 3;

SOC,

store-operated Ca2+ channel(s);

IP3,

inositol trisphosphate;

IP3R,

IP3 receptor;

Icrac,

calcium release-activated calcium current;

OAG,

1-oleoyl-2-acetyl-sn-glycerol;

IP3R-KO,

DT40 B lymphocytes with all three IP3Rs knocked out;

T3-WT,

wild-type DT40 transiently transfected with hTrp3;

T3-KO,

IP3R-KO transiently transfected with hTrp3;

PLC,

phospholipase C;

eGFP,

enhanced green fluorescent protein