Molecular identity and calmodulin-mediated regulation of the taurine transporter in a human retinal pigment epithelial cell line.
The molecular identity and calmodulin-mediated regulation of the taurine transporter were investigated in a human retinal pigment epithelial cell line (HRPE). Reverse transcription-polymerase chain reaction amplification of HRPE cell mRNA using primer specific for a taurine transporter cloned from human placenta yielded a product of expected size (approximately 0.9 kb) which hybridized to the placental cDNA probe under high stringency conditions. The nucleotide sequence of the product was identical to the sequence of the portion of the placental taurine transporter cDNA flanked by the specific primers. The taurine transporter expressed in the HRPE cell line thus appears to be identical to the transporter cloned from the placenta. Treatment of the HRPE cells with a selective calmodulin antagonist CGS 9343 B (CGS) led to a marked decrease in taurine transport activity. This effect could be reproduced with W-7, another calmodulin antagonist. The inhibition caused by CGS occurred rapidly (t1/2 approximately 10 min). Treatment of the cells with CGS did not affect the transport of leucine, and amino acid not recognized by the taurine transporter as a substrate. The CGS-induced inhibition of taurine transport was accompanied by a decrease in the maximal velocity of the transporter with no detectable change in the substrate affinity. The steady state levels of the transporter mRNA however remained unaffected by CGS treatment. It is concluded that the HRPE cell line expressed a taurine transporter identical to the transporter describe in the human placenta and that the function of this transporter is regulated by calmodulin-dependent processes.
Ramamoorthy
S
S
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Del Monte
M A
MA
Leibach
F H
FH
Ganapathy
V
V
D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology; D015152:Blotting, Northern; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008967:Molecular Biology; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism*; D000099247:Taurine Transporters
D001562:Benzimidazoles; D002147:Calmodulin; D002352:Carrier Proteins; D017931:DNA Primers; D007930:Leucine; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D013449:Sulfonamides; D013654:Taurine; D000099247:Taurine Transporters; C052087:CGS 9343B; C017967:W 7