Barrett P Q, Lu H K, Colbran R, Czernik A, Pancrazio J J
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22903, USA.
Am J Physiol Cell Physiol. 2000 Dec;279(6):C1694-703. doi: 10.1152/ajpcell.2000.279.6.C1694.
The effect of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca(2+) channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of approximately 9 pS in 110 mM BaCl(2) or CaCl(2). In the cell-attached patch, elevation of bath Ca(2+) from 150 nM to 1 microM raised intracellular Ca(2+) in K(+)-depolarized (140 mM) cells and evoked an increase in the LVA Ca(2+) channel probability of opening (NP(o)) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP(o) elicited by elevated Ca(2+) concentration. Furthermore, administration of a mutant recombinant CaMKIIalpha exhibiting cofactor-independent activity in the absence of elevated Ca(2+) produced a threefold elevation in LVA channel NP(o). These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca(2+).
采用膜片钳技术测量了Ca(2+)/钙调蛋白依赖性蛋白激酶II（CaMKII）刺激对分离的牛肾上腺球状带（AG）细胞中单位低电压激活（LVA）T型Ca(2+)通道电流的影响。在细胞贴附式和内面向外式膜片中，通过电压依赖性失活以及在110 mM BaCl(2)或CaCl(2)中约9 pS的单通道电导来识别LVA通道活性。在细胞贴附式膜片中，将浴液Ca(2+)从150 nM升高至1 μM可使K(+)去极化（140 mM）细胞内的Ca(2+)升高，并使LVA Ca(2+)通道开放概率（NP(o)）增加2至6倍。这种增强与非空白扫描次数的增加、非空白扫描中通道开放频率的升高以及首次潜伏期缩短30%相关。单通道开放时间分布、爆发长度或每次爆发的开放次数均无明显变化。在细胞贴附式或切除式（内面向外式）构型中，用CaMKII的亲脂性或肽类抑制剂对AG细胞进行预孵育可防止Ca(2+)浓度升高引起的NP(o)升高。此外，在未升高Ca(2+)的情况下给予具有辅因子非依赖性活性的突变重组CaMKIIα可使LVA通道NP(o)升高三倍。这些数据表明，CaMKII活性对于Ca(2+)激活LVA通道既是必需的也是充分的。
