Abstract

Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K<sup>+</sup> channels and homologous desensitization of the LTD<sub>4</sub> receptor impairs regulatory volume decrease (RVD). The aim is to illustrate the effect of mutation of putative PKC consensus phosphorylation sites in the CysLT1R on desensitization and RVD. Methods: mCysLT1 contains 4 putative PKC consensus phosphorylation sites, and four mutants were created: Thr151Gly, Thr323Gly, Thr151Gly plus Thr323Gly, and Thr236Gly plus Ser243Gly. Functional mCysLT1 receptor activity after injection of in vitro transcribed cRNA into Xenopus laevis oocytes was visualized as a LTD<sub>4</sub>-evoked, Ca<sup>2+</sup>-activated Cl<sup>-</sup> currents recorded by two-electrode voltage clamp. Results: Repetitive LTD<sub>4</sub> administration (100 nM) desensitized the LTD<sub>4</sub>-evoked currents in oocytes expressing wild type CysLT1. Single mutations as well as the double mutation Thr236Gly plus Ser243Gly had no or a slight effect on the LTD<sub>4</sub> induced desensitization. However, double mutation Thr323Gly plus Thr151Gly prevented the desensitization. As a functional consequence we find that inhibition of PKC accelerates RVD and prevents the inhibitory effect of LTD<sub>4</sub>-pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2<sup>nd</sup> inner loop (Thr<sup>151</sup>) and at the C-terminal domain (Thr<sup>323</sup>) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling.

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