Albert Anthony P, Liu Min, Large William A
Ion Channel and Cell Signalling Research Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London SW17 0RE.
Br J Pharmacol. 2006 Aug;148(7):1001-11. doi: 10.1038/sj.bjp.0706797. Epub 2006 Jun 12.
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.
我们之前曾描述过，在新鲜分离的兔耳动脉肌细胞中存在一种Ca(2+)通透性非选择性阳离子通道，该通道可被耗尽细胞内Ca(2+)储存的试剂以及蛋白激酶C（PKC）激活。在本研究中，我们运用电生理技术研究了钙调蛋白（CaM）对储存操纵性通道（SOCs）的影响。将CaM抑制剂氯氮平（CMZ）浴用处理静息细胞，可使细胞贴附膜片上的SOC活性产生短暂激活。CMZ对环匹阿尼酸（CPA）诱发的SOCs产生双重作用，最初诱导平均开放概率（NP(o)）增加，随后显著抑制SOC活性。相反，由细胞可渗透的Ca(2+)螯合剂1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA-AM）激活的SOCs则被CMZ抑制。在细胞内面向外膜片中，当SOCs由CPA或PKC激活剂佛波醇-12,13-二丁酸酯（PDBu）激活时，浴用CaM会先诱导抑制，随后使SOC活性增加。相反，CaM仅增强细胞内面向外膜片中BAPTA-AM诱发的SOC活性。将CaM浴用处理静息细胞内面向外膜片的细胞质表面，可诱发具有与SOCs相似生物物理特性的单通道电流。CaM对PDBu诱导的SOC活性的抑制作用可被钙调蛋白依赖性激酶II（CaM激酶II）抑制剂自钙调蛋白相关抑制肽（AIP）抑制，但不能被钙调神经磷酸酶或肌球蛋白轻链激酶（MLCK）抑制剂抑制。此外，CaM诱发的通道电流可被共同应用纯化的CaM激酶II抑制，但不能被CaM激酶II、钙调神经磷酸酶或MLCK抑制剂抑制。采用全细胞和细胞贴附记录法时，浴用CaM激酶II抑制剂KN93和AIP可在未受刺激的肌细胞中诱发SOCs。这些结果表明，CaM对SOCs具有明显的双重抑制和兴奋作用，其中CaM的抑制作用由CaM激酶II介导。此外，本研究提供了有力证据，表明CaM在激活SOCs中具有重要作用，可能是通过对通道/相关蛋白的直接作用来实现的。
