© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells1
Department of Pharmacology and Toxicology, Karl-Franzens-University Graz, A-8010 Graz, Austria
Klaus.Groschner{at}kfunigraz.ac.at
* Corresponding author. Tel.: +43-316-380-5570; fax: +43-316-380-9890
Objective: Expression of homologues of the Drosophila transient receptor potential (Trp) protein has recently been demonstrated for vascular endothelium. Some Trp isoforms such as Trp3, are known to constitute cation conductances with biophysical properties similar to those of the endothelial oxidant-activated cation conductance. Therefore we tested whether Trp proteins provide the molecular basis of the oxidant-induced membrane currents in porcine aortic endothelial cells (ECAP). Methods: Expression of the Trp3 isoform in ECAP was tested by RT–PCR and subsequent southern blot analysis. In order to knock-out the function of endogenous Trp channels, ECAP were transiently transfected to express NTRP3, a dominant negative fragment of Trp3. Oxidative-stress was introduced by exposure of cells to tert-butylhydroperoxide (tBHP; 400 µM), and membrane currents as well as membrane potential were recorded using the conventional whole cell patch–clamp technique. Results: RT–PCR experiments demonstrated the expression of a Trp3 isoform in ECAP. The oxidant tert.-butylhydroperoxide (tBHP) completely depolarized endothelial cells by activation of a cation conductance which allowed significant Na+ inward currents at negative potentials (mean inward current 462 pA at –80 mV). The tBHP-induced currents resembled Trp-related currents in terms of cation selectivity, La3+ sensitivity and lack of voltage dependence. Expression of the N-terminal fragment of hTrp3 (NTRP3), but not of a C-terminal fragment of hTrp3 (CTRP3), abolished the oxidant-induced cation current and reduced membrane depolarization. Conclusion: Our results strongly suggest Trp proteins as the molecular basis of endothelial oxidant-activated cation channels. It is concluded that Trp proteins play an important role in the redox sensitivity of the vascular endothelium.
KEYWORDS Endothelial functions; Ion channels; Oxidative stress; Trp
1 M. Balzer and B. Lintschinger contributed equally to this study.
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