A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2
A functional link between store-operated and TRPC channels revealed by the 3,5-bis(trifluoromethyl)pyrazole derivative, BTP2
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DOI：
10.1074/jbc.m411797200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Gill, DL
中科院分区：
文献类型：
--
作者：
He, LP;Hewavitharana, T;Gill, DL
受体介导的Ca 2+库释放和“库操作”Ca 2+进入通道的激活之间的耦合是一个重要的但迄今为止知之甚少的机制。瞬时受体电位（TRP）通道超家族包含几个成员，可能服务于存储操作通道（SOC）的功能。3，5-双（三氟甲基）吡唑衍生物，BTP 2，是最近描述的T淋巴细胞中SOC活性的抑制剂。我们比较了它在许多细胞类型中对SOC激活的作用，并评估了它对三种特定TRP通道（典型瞬时受体电位3（TRPC 3），TRPC 5和TRPV 6）的修饰，以揭示SOC和TRP通道功能之间的任何联系。使用HEK 293细胞、DT 40 B细胞和A7 r5平滑肌细胞，BTP 2在10分钟内阻断钙池操纵的Ca 2+内流，IC 50为0.1-0.3 μ M。钙泵阻断或响应毒蕈碱或B细胞受体激活诱导的钙池操作性钙内流对BTP 2同样敏感。使用稳定表达TRPC 3通道的T3-65克隆HEK 293细胞系，由毒蕈碱受体激活的TRPC 3介导的Sr 2+进入也被BTP 2阻断，IC 50 < 0.3 μ M。重要的是，二酰基甘油对TRPC 3通道的直接激活也被BTP 2阻断（IC 50类似于0.3 μ M）。在用N-甲基-D-葡糖胺-氯化物替代Na+的培养基中，BTP 2仍然阻断TRPC 3，表明BTP 2不阻断通过激活单价阳离子进入通道诱导的去极化的二价阳离子进入。而全细胞卡巴胆碱诱导的TRPC 3电流被3 μ M BTP 2阻断，单个TRPC 3通道记录显示持续的短开口，表明BTP 2降低了通道的开放概率，而不是其孔特性。在HEK 293细胞中瞬时表达的TRPC 5通道被BTP 2阻断，阻断范围与TRPC 3相同。然而，具有许多类似于SOC的通道特性的高度Ca 2+选择性TRPV 6通道的功能完全不受BTP 2的影响。结果表明，表达的TRPC通道和内源性SOC活性的操作之间的强大的功能联系。
The coupling between receptor-mediated Ca2+ store release and the activation of "store-operated" Ca2+ entry channels is an important but so far poorly understood mechanism. The transient receptor potential (TRP) superfamily of channels contains several members that may serve the function of store-operated channels (SOCs). The 3,5-bis(trifluoromethyl) pyrazole derivative, BTP2, is a recently described inhibitor of SOC activity in T-lymphocytes. We compared its action on SOC activation in a number of cell types and evaluated its modification of three specific TRP channels, canonical transient receptor potential 3 (TRPC3), TRPC5, and TRPV6, to throw light on any link between SOC and TRP channel function. Using HEK293 cells, DT40 B cells, and A7r5 smooth muscle cells, BTP2 blocked store-operated Ca2+ entry within 10 min with an IC50 of 0.1-0.3 mu M. Store-operated Ca2+ entry induced by Ca2+ pump blockade or in response to muscarinic or B cell receptor activation was similarly sensitive to BTP2. Using the T3-65 clonal HEK293 cell line stably expressing TRPC3 channels, TRPC3-mediated Sr2+ entry activated by muscarinic receptors was also blocked by BTP2 with an IC50 of < 0.3 mu M. Importantly, direct activation of TRPC3 channels by diacylglycerol was also blocked by BTP2 ( IC50 similar to 0.3 mu M). BTP2 still blocked TRPC3 in medium with N-methyl-D- glucamine-chloride replacing Na+, indicating BTP2 did not block divalent cation entry by depolarization induced by activating monovalent cation entry channels. Whereas whole-cell carbachol-induced TRPC3 current was blocked by 3 mu M BTP2, single TRPC3 channel recordings revealed persistent short openings suggesting BTP2 reduces the open probability of the channel rather than its pore properties. TRPC5 channels transiently expressed in HEK293 cells were blocked by BTP2 in the same range as TRPC3. However, function of the highly Ca2+-selective TRPV6 channel, with many channel properties akin to SOCs, was entirely unaffected by BTP2. The results indicate a strong functional link between the operation of expressed TRPC channels and endogenous SOC activity.