13 Universitetsparken

Copenhagen, Denmark

13 Universitetsparken

Copenhagen, Denmark
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Jorgensen S.T.,Copenhagen University | Eriksen H.N.,Copenhagen University | Dausell J.,Copenhagen University | Jespersen T.,Copenhagen University | And 3 more authors.
Cellular Physiology and Biochemistry | Year: 2013

Background/Aims: Signalling via CysLT1 is involved in activation of volume sensitive K+ channels and homologous desensitization of the LTD 4 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 LTD4-evoked, Ca2+-activated Cl- currents recorded by two-electrode voltage clamp. Results: Repetitive LTD4 administration (100 nM) desensitized the LTD4-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 LTD4 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 LTD4- pretreatment on RVD in Ehrlich ascites tumour cells. Conclusion: These data indicate that simultaneous PKC-mediated phosphorylation at the 2nd inner loop (Thr151) and at the C-terminal domain (Thr323) leads to mCysLT1 receptor desensitization and abrogates the RVD response following osmotic cell swelling. © 2013 S. Karger AG, Basel.


Juul C.A.,13 Universitetsparken | Grubb S.,13 Universitetsparken | Poulsen K.A.,13 Universitetsparken | Kyed T.,13 Universitetsparken | And 4 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2014

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6's physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich-Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca2+ is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium. © 2014 The Author(s).

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