title: Identification of an N-terminal TRPC2 splice variant which inhibits calcium influx.
abstract: TRPC2 is a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable channels expressed in nonexcitable cells. TRPC2 is involved in a number of physiological processes including sensory activation of the vomeronasal organ, sustained Ca2+ entry in sperm, and regulation of calcium influx by erythropoietin. Here, a new splice variant of TRPC2, called "Similar to mouse TRPC2" (smTRPC2), was identified consisting of 213 amino acids, largely coincident with the N-terminus of TRPC2 clone 17. This splice variant lacks all six TRPC2 transmembrane domains and the calcium pore. Expression of smTRPC2 was found in all tissues examined by RT-PCR and in primary erythroid cells by RT-PCR and Western blotting. Confocal microscopy of CHO-S cells transfected with TRPC2 clone 14 and smTRPC2 demonstrated that TRPC2 clone 14 and smTRPC2 both localize at or near the plasma membrane and in the perinuclear region. Cell surface localization of TRPC2 was confirmed with biotinylation, and was not substantially affected by smTRPC2 expression. Coassociation of TRPC2 c14 and alpha with smTRPC2 was confirmed by immunoprecipitation. To examine the functional significance of smTRPC2 expression, a CHO-S model was used to study its effect on calcium influx stimulated by Epo through TRPC2. Single CHO-S cells which express transfected Epo-R were identified by detection of green fluorescent protein (GFP). Cells that express transfected TRPC2 c14 or alpha were identified by detection of blue fluorescent protein (BFP). [Ca]i was quantitiated with Fura Red fluorescence using digital video imaging. Epo stimulated calcium influx through TRPC2 isoforms c14 and alpha, which was inhibited by coexpression of smTRPC2. These data demonstrate that a short splice variant of TRPC2 exists in many cell types, which associates with and modifies the activity of functional TRPC2 splice variants.
authors.affiliation: The Henry Hood Research Program, The Sigfried and Janet Weis Center for Research, The Geisinger Clinic, Danville, PA 17822, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Chu
authors.forename: Qin
authors.initials: Q
authors.lastname: Tong
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Wozney
authors.forename: Wenyi
authors.initials: W
authors.lastname: Zhang
authors.forename: Joseph Y
authors.initials: JY
authors.lastname: Cheung
authors.forename: Kathleen
authors.initials: K
authors.lastname: Conrad
authors.forename: Virginia
authors.initials: V
authors.lastname: Mazack
authors.forename: Richard
authors.initials: R
authors.lastname: Stahl
authors.forename: Dwayne L
authors.initials: DL
authors.lastname: Barber
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Miller
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D050052:TRPC Cation Channels
chemical_list: D008565:Membrane Proteins; D012333:RNA, Messenger; D050052:TRPC Cation Channels; C491858:Trpc2 protein, mouse; D002118:Calcium