Bugaj Vladislav, Alexeenko Vadim, Zubov Alexander, Glushankova Lyuba, Nikolaev Anton, Wang Zhengnan, Kaznacheyeva Elena, Bezprozvanny Ilya, Mozhayeva Galina N
Institute of Cytology RAS, 4 Tikhoretsky Avenue, St. Petersburg 194064, Russia.
J Biol Chem. 2005 Apr 29;280(17):16790-7. doi: 10.1074/jbc.M500192200. Epub 2005 Mar 1.
Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.
在非兴奋性细胞中，磷脂酶C（PLC）介导的信号通路激活会导致钙离子（Ca2+）从对肌醇1,4,5-三磷酸（InsP3）敏感的细胞内钙库释放，并通过质膜钙通道激活钙内流。非兴奋性细胞质膜钙内流通道的特性和分子身份是深入研究的重点。在之前的研究中，我们使用膜片钳电生理学来描述人癌细胞A431细胞系中钙内流通道的特性。现在我们将研究扩展到人胚肾HEK293细胞。通过结合钙离子成像、全细胞和单通道膜片钳记录，我们发现：1）HEK293细胞含有四种质膜钙内流通道：I(CRAC)、Imin、Imax和I(NS)；2）I(CRAC)通道具有高度的钙选择性（P(Ca/Cs)>1000），且I(CRAC)单通道电导太小，无法进行单通道分析；3）HEK293细胞中的Imin通道显示出与A431细胞中Imin通道相同的功能特性，二价阳离子的单通道电导为1.2 pS，一价阳离子为10 pS，二价阳离子选择性P(Ba/K)=20；4）HEK293细胞中的Imin通道被InsP3激活，被磷脂酰肌醇4,5-二磷酸抑制，但与钙库无关；5）与Imin相比，Imax通道对二价（17 pS）和一价（33 pS）阳离子具有更高的电导，但对二价阳离子的选择性较低（P(Ba/K)=4）；6）HEK293细胞中的Imax通道可被InsP3或钙库耗竭激活；7）I(NS)通道是非选择性的（P(Ba/K)=0.4），单通道电导为5 pS；8）I(NS)通道不受InsP3门控，但被细胞内钙库耗竭激活。我们的发现为支持HEK293细胞中受体介导和钙库操纵的钙内流途径的内源性钙通道提供了新信息。
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