Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway.
Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.
Brough
G H
GH
Department of Pharmacology, University of South Alabama, College of Medicine, Mobile, Alabama 36688, USA.
Wu
S
S
Cioffi
D
D
Moore
T M
TM
Li
M
M
Dean
N
N
Stevens
T
T
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004553:Electric Conductivity; D004730:Endothelium, Vascular; D015870:Gene Expression; D006801:Humans; D008175:Lung Neoplasms; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011651:Pulmonary Artery; D012333:RNA, Messenger / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D002118:Calcium