Invited Symposium: Na-H Exchangers and Intracellular pH Regulation



Materials & Methods


Discussion & Conclusion



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Regulation of Sodium-Hydrogen Exchange Activity by Protein Kinase D

Haworth, RS (Cardiovascular Research, St. Thomas' Hospital, UK)
Rozengurt, E (Department of Medicine, UCLA, USA)
Avkiran, M (Cardiovascular Research, St. Thomas' Hospital, UK)

Contact Person: Robert Haworth (r.haworth@umds.ac.uk)


The regulation of Na+/H+ exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. A fusion protein containing the regulatory domain of NHE1 was phosphorylated in vitro by wild-type PKD (PKD-WT), but not by the dominant-negative kinase-inactive mutant PKD-K618M. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells transfected with empty vector (control), PKD-WT or PKD-K618M together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux at pHi 7.0 (JH), was determined in single GFP-positive cells following intracellular acidification. Overexpression of PKD-WT had no effect on JH (3.78±0.24 mM/min v 3.48±0.25 mM/min in control). In contrast, overexpression of PKD-K618M increased JH (5.31±0.57 mM/min; p < 0.05 v control). Thus, native PKD appears to have a tonic inhibitory effect on NHE, which is relieved by the dominant-negative action of PKD-K618M. Exposure to phorbol ester significantly increased JH in control cells but failed to do so in cells overexpressing either PKD-WT (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal JH was already near-maximal). Overall, these results suggest that PKD inhibits NHE activity, and represents a novel pathway in the regulation of the exchanger.

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Presentation Number SAhaworth0246
Keywords: PKD, PKCµ, NHE1, COS-7, GFP

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Haworth, RS; Rozengurt, E; Avkiran, M; (1998). Regulation of Sodium-Hydrogen Exchange Activity by Protein Kinase D. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/fliegel/haworth0246/index.html
© 1998 Author(s) Hold Copyright