Dual effect of calmodulin on store-operated Ca2+ -permeable cation channels in rabbit portal vein myocytes.
We have previously described a Ca(2+)-permeable non-selective cation channel in freshly dispersed rabbit ear artery myocytes, which is activated by agents that deplete internal Ca(2+) stores and also by protein kinase C (PKC). In the present study, we investigated the effect of calmodulin (CaM) on store-operated channels (SOCs) with electrophysiological techniques. Bath application of the CaM inhibitor calmidazolium (CMZ) to quiescent cells produced transient activation of SOC activity in cell-attached patches. CMZ produced a dual effect on cyclopiazonic acid (CPA)-evoked SOCs by initially inducing an increase in mean open probability (NP(o)) and subsequently producing a marked inhibition of SOC activity. In contrast, SOCs activated by the cell-permeable Ca(2+) chelator 1,2-bis (2-aminophenoxy)ethane-N-N,N',N'-tetraacetic acid (BAPTA-AM) were inhibited by CMZ. In inside-out patches where SOCs were activated by CPA or the PKC activator phorbol-12,13-dibutyrate (PDBu), bath application of CaM induced an initial inhibition followed by an increase in SOC activity. In contrast, CaM only enhanced BAPTA-AM-evoked SOC activity in inside-out patches. Bath application of CaM to the cytoplasmic surface of quiescent inside-out patches evoked single channel currents with biophysical properties similar to SOCs. The inhibitory action of CaM on PDBu-induced SOC activity was inhibited by the calmodulin-dependent kinase II (CaM kinase II) inhibitor autocamtide-related inhibitory peptide (AIP) but not by inhibitors of calcineurin or myosin light chain kinase (MLCK). In addition, CaM-evoked channel currents were inhibited by coapplication of purified CaM kinase II but not by inhibitors of CaM kinase II, calcineurin or MLCK. With whole-cell and cell-attached recording, bath application of the CaM kinase II inhibitors KN93 and AIP evoked SOCs in unstimulated myocytes. These results indicate that CaM has pronounced dual inhibitory and excitatory actions on SOCs with the inhibitory effect of CaM mediated by CaM kinase II. Moreover, the present work provides strong evidence that CaM has an important role in activating SOCs, possibly through a direct action on channel/associated proteins.
Albert
Anthony P
AP
Ion Channel and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 0RE. aalbert@sgul.ac.uk
Liu
Min
M
Large
William A
WA
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D002412:Cations / Q000378:metabolism; D002469:Cell Separation; D004594:Electrophysiology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism; D011169:Portal Vein / Q000166:cytology / Q000187:drug effects*; D011817:Rabbits
D002147:Calmodulin; D002412:Cations; D007473:Ion Channels; D015240:Phorbol 12,13-Dibutyrate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D002118:Calcium