The Store-operated Calcium Entry Pathways in Human Carcinoma A431 Cells: Functional Properties and Activation Mechanisms

Activation of phospholipase C (PLC)-mediated signaling pathways in nonexcitable cells causes the release of Ca 2 from intracellular Ca 2 stores and activation of Ca 2 influx across the plasma membrane. Two types of Ca 2 channels, highly Ca 2 –selective I CRAC and moderately Ca 2 –selective I SOC , support store-operated Ca 2 entry process. In previous patch-clamp experiments with a human carcinoma A431 cell line we described store-operated I min /I CRACL plasma membrane Ca 2 influx channels. In the present paper we use whole-cell and single-channel recordings to further characterize store-operated Ca 2 influx pathways in A431 cells. We discovered that (a) I CRAC and I SOC are present in A431 cells; (b) I CRAC currents are highly selective for divalent cations and fully activate within 150 s after initiation of Ca 2 store depletion; (c) I SOC currents are moderately selective for divalent cations (P Ba/ P Cs 14.5) and require at least 300 s for full activation; (d) I CRAC and I SOC currents are activated by PLC-coupled receptor agonists; (e) I SOC currents are supported by I min /I CRACL channels that display 8.5–10 pS conductance for sodium; (f) I CRAC single channel conductance for sodium is estimated at 0.9 pS by the noise analysis; (g) I min /I CRACL channels are activated in excised patches by an amino-terminal fragment of InsP 3 R1 (InsP 3 R1N); and (h) InsP 3 binding to InsP 3 R1N is necessary for activation of I min /I CRACL channels. Our findings provide novel information about store-operated Ca 2 influx pathways in A431 cells. key words: calcium signaling • patch-clamp • inositol 1,4,5-trisphosphate • calcium channels • whole-cell I N T R O D U C T I O N Activation of PLC-mediated signaling pathways in nonexcitable cells causes the release of Ca 2 from intracellular Ca 2 stores and promotes Ca 2 influx across the plasma membrane via capacitative Ca 2 entry (CCE)* or store-operated Ca 2 entry (SOC) processes (Berridge, 1995; Parekh and Penner, 1997; Putney et al., 2001; Venkatachalam et al., 2002). Two types of Ca 2 channels have been implicated in store-operated Ca 2 entry in nonexcitable cells. Highly Ca 2 -selective channels (P D/M 1,000) named “Ca 2 release activated channels” (I CRAC ) have been initially discovered in studies of Jurkat and RBL cells (Hoth and Penner, 1992; Zweifach and Lewis, 1993; Premack et al., 1994). Moderately Ca 2 selective channels have been later identified in a number of cells and grouped under the name I SOC (Berridge, 1995; Parekh and Penner, 1997; Nilius and Droogmans, 2001; Putney et al., 2001; Venkatachalam et al., 2002). When compared with I CRAC , I SOC channels display lower selectivity for divalent cations (P D/M 10), at least 10-fold higher single channel conductance for divalent and monovalent cations, and different kinetic and pharmacological properties (Berridge, 1995; Parekh and Penner, 1997; Nilius and Droogmans, 2001; Putney et al., 2001; Venkatachalam et al., 2002). The molecular identity of I CRAC remains unclear (Clapham, 1996; Parekh and Penner, 1997; Putney et al., 2001; Venkatachalam et al., 2002). Mammalian trp channels of TRPC family are the most likely candidates for the role of I SOC channels (Birnbaumer et al., 1996; Clapham et al., 2001; Nilius and Droogmans, 2001; Montell et al., 2002; Venkatachalam et al., 2002; Zitt et al., 2002). The mechanisms of I SOC and I CRAC channel activation have been under intense investigation (Birnbaumer et al., 1996; Clapham et al., 2001; Nilius and Droogmans, 2001; Montell et al., 2002; Venkatachalam et al., 2002; Zitt et al., 2002). Activation of I SOC /I CRAC channels by a diffusible messenger CIF (Ca 2 -influx factor) released by depleted Ca 2 stores (Putney and Bird, 1993; Randriamampita and Tsien, 1993; Kim et al., 1995; Csutora et al., 1999; Trepakova et al., 2000), via “conformational coupling” with the intracellular InsP 3 R (Irvine, 1990; Kiselyov et al., 1998; Zubov et al., 1999), by cleavage of PIP 2 (Kaznacheyeva et al., 2000; Estacion et al., 2001), and by regulated insertion of channels into plasma membrane (Patterson et al., 1999; Yao et al., 1999) have been considered. Direct association of Address correspondence to Dr. Ilya Bezprozvanny, Department of Physiology, K4.112, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Fax: (214) 6482974; E-mail: Ilya.Bezprozvanny@UTSouthwestern.edu * Abbreviations used in this paper: CCE, Ca 2 entry; DVF, divalentfree; SOC, store-operated Ca 2 entry. on April 27, 2019 jgp.rupress. org Downloaded from http://doi.org/10.1085/jgp.200308815 Published Online: 30 June, 2003 | Supp Info: