Phospholipase cgamma1 is required for activation of store-operated channels in human keratinocytes.
Store-operated calcium entry depicts the movement of extracellular Ca2+ into cells through plasma membrane Ca2+ channels activated by depletion of intracellular Ca2+ stores. The members of the canonical subfamily of transient receptor potential channels (TRPC) have been implicated as the molecular bases for store-operated channels (SOC). Here we investigate the role of phospholipase C (PLC) in regulation of native SOC and the expression of endogenous TRPC in human epidermal keratinocytes. Calcium entry in response to store depletion with thapsigargin was reversibly blocked by 2-aminoethoxydiphenyl borane, an effective SOC inhibitor, and suppressed by the diacylglycerol analoge, 1-oleoyl-2-acetyl-sn-glycerol. Inhibition of PLC with U73122 or transfection of a PLCgamma1 antisense cDNA construct completely blocked SOC activity, indicating a requirement for PLC, especially PLCgamma1, in the activation of SOC. RT-PCR and immunoblotting analyses showed that TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 are expressed in keratinocytes. Knockdown of the level of endogenous TRPC1 or TRPC4 inhibited store-operated calcium entry, indicating they are part of the native SOC. Co-immunoprecipitation studies demonstrated that TRPC1, but not TRPC4, interacts with PLCgamma1 and the inositol 1,4,5-trisphosphate receptor (IP3R). The association of TRPC1 with PLCgamma1 and IP3R decreased in keratinocytes with higher intracellular Ca2+, coinciding with a downregulation in SOC activity. Our results indicate that the activation of SOC in keratinocytes depends, at least partly, on the interaction of TRPC with PLCgamma1 and IP3R.
Tu
Chia-Ling
CL
Endocrine Unit, Veteran Affairs Medical Center and Department of Medicine, University of California, San Francisco, California 94121, USA. ctu@itsa.ucsf.edu
Chang
Wenhan
W
Bikle
Daniel D
DD
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D016373:DNA, Antisense; D018076:DNA, Complementary; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D051966:Phospholipase C gamma; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D050052:TRPC Cation Channels; D014162:Transfection; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D016373:DNA, Antisense; D018076:DNA, Complementary; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007473:Ion Channels; D018160:Receptors, Cytoplasmic and Nuclear; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; C096351:transient receptor potential cation channel, subfamily C, member 1; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium