Activation of calcium entry in human carcinoma A431 cells by store depletion and phospholipase C- dependent mechanisms converge on I_CRAC-like calcium channels

Abstract

Activation of phospholipase C in nonexcitable cells causes the release of calcium (Ca 2؉ ) from intracellular stores and activation of Ca 2؉ influx by means of Ca 2؉ release-activated channels (ICRAC) in the plasma membrane. The molecular identity and the mechanism of I CRAC channel activation are poorly understood. Using the patch-clamp technique, here we describe the plasma membrane Ca 2؉ channels in human carcinoma A431 cells, which can be activated by extracellular UTP, by depletion of intracellular Ca 2؉ stores after exposure to the Ca 2؉ -pump inhibitor thapsigargin, or by loading the cells with Ca 2؉ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetate. The observed channels display the same conductance and gating properties as previously described I min channels, but have significantly lower conductance for monovalent cations than the I CRAC channels. Thus, we concluded that the depletion-activated Ca 2؉ entry processes (1-3). These processes are mediated by plasma membrane Ca 2ϩ channels termed ''Ca 2ϩ release activated channels'' (I CRAC ) (4-7). The molecular identity of I CRAC remains unclear, with mammalian trp channels (mTrp) usually considered the most likely candidate for the role of I CRAC (1-3, 8, 9). When compared with I CRAC , mTrp channels display relatively low selectivity for divalent cations, higher single channel conductance, and different kinetic and pharmacological properties. In experiments with a human carcinoma A431 cell line, we previously described plasma membrane Ca 2ϩ channels (I min ) that are activated by application of uridine triphosphate and bradykinin to cell-attached patches or by application of inositol (1,4,5)-trisphosphate (IP 3 ) to excised inside-out (i͞o) patches (10-12). IP 3 -gated channels that share some common properties with I min have been also observed in experiments with human T cells (13), rat macrophages (12), and endothelial cells (14,15). Major functional properties of I min channels, such as small conductance (1 pS for divalent cations), high selectivity for divalent cations (P Ca/K Ͼ 1,000), inward rectification, and sensitivity to block by SKF95365 are similar to I CRAC channels (12, 16). Thus, we previously suggested that I min and I CRAC may in fact be the same channels (17).The mechanism of I CRAC activation remains similarly controversial (1-3). When studied in a heterologous expression system, activation of mTrp channels by IP 3 appear to be mediated by direct conformational coupling between the cytosolic carboxylterminal tail of mTrp and the amino-terminal ligand-binding domain of intracellular IP 3 receptor (IP 3 R) (18-21). However, whether mTrp can serve as an appropriate model system for understanding I CRAC activation is unresolved (18,21,22). In previous studies, we demonstrated that activity of I min in i͞o patches is potentiated by addition of IP 3 R-enriched microsomes as predicted by an I min -IP 3 R conformational coupling model (16). More recently, we discovered that anti-PIP 2 antibody (PIP 2 Ab) sensitizes I min to IP 3 a...

Suppression of TRPC3 Leads to Disappearance of Store-operated Channels and Formation of a New Type of Store-independent Channels in A431 Cells

“…TRPC3 Does Not Regulate I min and I max Cation ChannelsWe have previously shown that A431 cells express highly selective I min calcium channels that could be activated by extracellular UTP, intracellular InsP 3 and depletion of intracellular Ca 2ϩ stores (35,36,39, see also Fig. 5F).…”

“…We found that I SOC currents are at least partly mediated by I min channels, whose activity is controlled by downstream products of PLC (33)(34)(35)(36)(37)(38) and could be triggered by passive depletion of intracellular Ca 2ϩ stores with Tg and/or BAPTA-AM (39). In the present study, we aimed to determine the role of TRPC3 in mediating the receptor-and store-operated calcium influx in A431 cells.…”

Human Trp3 forms both inositol trisphosphate receptor-dependent and receptor-independent store-operated cation channels in DT40 avian B lymphocytes

“…Several studies also have suggested that Trp3 activation involves interaction with underlying IP 3 Rs or, in some instances, ryanodine receptors (34)(35)(36). There is also evidence that CCE may involve such an interaction (37,38), in support of the proposed conformational coupling model for the regulation of SOCs (7,8). Thus, despite the fact that expressed Trp3 apparently is not regulated by store depletion, its interaction with IP 3 Rs has been investigated as a model for understanding the conformational coupling mechanism.…”

Functional Properties of Endogenous Receptor- and Store-operated Calcium Influx Channels in HEK293 Cells

“…I min Channels Act as ROC Channels in HEK293 Cells-In the previous studies we utilized single channel patch clamp recordings to characterize Ca 2ϩ influx channels in A431 cells (13)(14)(15)(16)(17)(18)(19). What are the channels supporting Ca 2ϩ influx in HEK293 cells?…”

“…We reasoned that single channel patch clamp recordings provide unique and precise information about functional properties of individual types of channels supporting Ca 2ϩ influx in non-excitable cells. In our previous experiments we used single channel patch clamp recordings to perform detailed characterization of channels supporting Ca 2ϩ influx in A431 cells (13)(14)(15)(16)(17)(18)(19)). Now we extended our studies to HEK239 cell line, which is used extensively as heterologous expression host in functional studies of TRPC family members, such as TRPC1 (20), TRPC3 (21-23), TRPC4 (24), TRPC5 (20,23,25), TRPC6 (26,27), and TRPC7 (27).…”