Role of conserved peptide in taurine transporter inactivation modulated by protein kinase C

Xiaobin Han, Andrea M. Budreau, Russell W. Chesney

Research output: Contribution to journalArticle

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Abstract

Activation of protein kinase C (PKC) by the active phorbol ester 12- myristate 13-acetate (PMA, 100 nM) or phorbol-12,13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine up-take by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity (K(m)) (from 5.6 μM to 18.1 μM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (Ab(S4)) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of Ab(S4) was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of Ab(S4) (12 ng/oocyte). In contrast, downregulation of the transporter by PMA could not be restored by Ab(S4) when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.

Original languageEnglish (US)
Pages (from-to)2088-2096
Number of pages9
JournalJournal of the American Society of Nephrology : JASN
Volume7
Issue number10
StatePublished - Oct 1996

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Protein Kinase C
Taurine
Oocytes
Peptides
Complementary RNA
Madin Darby Canine Kidney Cells
Down-Regulation
Phosphorylation
Phorbol 12,13-Dibutyrate
Antigens
Staurosporine
Antibodies
Phorbol Esters
Acetates
Immunoglobulin G
taurine transporter
Injections
calphostin C

All Science Journal Classification (ASJC) codes

  • Nephrology

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Role of conserved peptide in taurine transporter inactivation modulated by protein kinase C. / Han, Xiaobin; Budreau, Andrea M.; Chesney, Russell W.

In: Journal of the American Society of Nephrology : JASN, Vol. 7, No. 10, 10.1996, p. 2088-2096.

Research output: Contribution to journalArticle

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abstract = "Activation of protein kinase C (PKC) by the active phorbol ester 12- myristate 13-acetate (PMA, 100 nM) or phorbol-12,13-dibutyrate (100 nM) reduced taurine uptake by 80{\%} in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine up-take by 20{\%} and 400{\%}, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity (K(m)) (from 5.6 μM to 18.1 μM) with minimal effect on the maximal velocity (25{\%} decrease) of the transporter. A polyclonal antibody (Ab(S4)) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of Ab(S4) was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of Ab(S4) (12 ng/oocyte). In contrast, downregulation of the transporter by PMA could not be restored by Ab(S4) when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.",
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