Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells
Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells
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DOI：
10.1113/jphysiol.2004.075051
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发表时间：
2004-12-01
影响因子：
5.5
通讯作者：
Inoue, R
中科院分区：
文献类型：
--
作者：
Shi, J;Mori, E;Inoue, R
我们调查，通过使用膜片钳技术，钙离子介导的调节异源表达TRPC 6和TRPC 7蛋白在HEK 293细胞，两个密切相关的同源物的瞬时受体电位（TRP）家庭和分子候选人的天然受体操作的钙离子进入通道。与制霉菌素穿孔记录，卡巴胆碱（CCh; 100 μ M）激活的TRPC 6电流（I-TRPC 6）的激活和失活的幅度和时间过程分别增强和加速，由细胞外Ca 2+（Ca-O（2+）），无论是连续存在或应用后受体刺激。相反，Ca-o（2+）仅抑制TRPC 7电流（I-TRPC 7）。在常规的全细胞钳夹下，细胞内Ca ~（2+）（Ca ~（2+））的剧烈缓冲消除了Ca ~（2+）的缓慢增强（即加速激活）和失活作用，揭示了I-TRPC 6的快速增强（EC_（50）：类似于0.4 mm）和抑制（IC_（50）：4 mm）以及I-TRPC 7的快速抑制（IC_（50）：类似于0.4 mm）。I-TRPC 6和I-TRPC 7的这种抑制似乎与单通道水平的单位电导和开放概率的电压依赖性降低有关，而I-TRPC 6的增强几乎没有电压依赖性，并且被Sr 2+而不是Ba 2+模仿。I-TRPC 6的激活过程或其由Ca 2+的加速可能涉及钙调蛋白（CaM）依赖性激酶II（CaMK II）的磷酸化，作为预处理与calmidazolium（3 μ m），Ca 2 +-insesentive突变体CaM的共表达，和细胞内灌注的不可水解的ATP类似物AMP-PNP和CaMK II特异性抑制肽都有效地防止通道激活。然而，对于TRPC 7没有观察到这一点。相反，单个CCh激活的TRPC 7通道活性被浓度依赖性抑制纳摩尔Ca 2+通过钙调素，并反过来增强IP 3。此外，I-TRPC 6的失活时间过程被蛋白激酶C（PKC）的药理学抑制显著延迟。这些结果共同表明，TRPC 6和7通道是由Ca 2+从膜的两侧通过不同的Ca 2 +-CaM依赖性和非依赖性机制的多重调节。
We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 muM)-activated TRPC6 currents (I-TRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca-o(2+)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca-o(2+) solely inhibited TRPC7 currents (I-TRPC7). Vigorous buffering of intracellular Ca2+ (Ca-i(2+)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca-o(2+), disclosing fast potentiation (EC50: similar to0.4 mm) and inhibition (IC50: 4 mm) of I-TRPC6 and fast inhibition (IC50: similar to0.4 mm) of I-TRPC7. This inhibition of I-TRPC6 and I-TRPC7 seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I-TRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I-TRPC6 or its acceleration by Ca2+ probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 mum), coexpression of Ca2+-insesentive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I-TRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.