Stable Activation of Single Ca2+ Release-activated Ca2+ Channels in Divalent Cation-free Solutions

Abstract

The regulation of store-operated, calcium-selective channels in the plasma membrane of rat basophilic leukemia cells (RBL-2H3 m1), an immortalized mucosal mast cell line, was studied at the single-channel level with the patch clamp technique by removing divalent cations from both sides of the membrane. The activity of the single channels in excised patches could be modulated by Ca 2؉ , Mg 2؉ , and pH. The maximal activation of these channels by divalent cation-free conditions occurred independently of depletion of intracellular Ca 2؉ stores, whether in excised patches or in whole cell mode. Yet, a number of points of evidence establish these single-channel openings as amplified store-operated channel events. Specifically, (i) the single channels are exquisitely sensitive to inhibition by intracellular Ca In a variety of cells, binding of ligands such as neurotransmitters, hormones, or growth factors to receptors on the cell surface generates intracellular calcium signals. Receptor-mediated release of Ca 2ϩ from IP 3 1 -sensitive intracellular stores triggers an influx of Ca 2ϩ into the cell via store-operated channels (SOCs) in the plasma membrane, a process termed "capacitative calcium entry" (1, 2). The resulting increase in intracellular Ca 2ϩ concentration regulates important processes, ranging from cell growth and differentiation to apoptosis and cell death. Molecular candidates for SOCs are the mammalian homologues of the Drosophila TRP (transient receptor potential) protein (3). Expression of the related genes, however, has resulted in varying patterns of response, including constitutive activity, augmentation of capacitative calcium entry, apparent direct activation by IP 3 , and activation by diacylglycerol (4). Therefore, the mechanism by which emptying of intracellular Ca 2ϩ stores activates SOCs remains elusive. Recent findings have favored a "direct coupling" model, whereby IP 3 receptors in the store membranes sense the Ca 2ϩ -filling status of the stores and transfer this information via direct coupling to subunits of SOCs in the plasma membrane (5-8).For the present, the best knowledge of SOCs comes from electrophysiological studies of a specific current associated with SOCs, known as the Ca 2ϩ release-activated calcium current (I crac ) (9). Despite extensive characterization of the macroscopic CRAC currents, the single-channel signature is poorly understood. This is because the channels are presumed to have a minute single-channel conductance, too low to be resolved at the single-channel level (10). During whole cell measurements in Jurkat T-lymphocytes, recently, Kerschbaum and Cahalan (11) were able to detect for the first time single-channel events which they attributed to CRAC channels. Their approach was to measure currents in the complete absence of intra-and extracellular divalent cations. They reasoned that under these conditions CRAC channels, like voltage-gated Ca 2ϩ channels should pass monovalent cations indiscriminately and with enhanced single-channel conductance (12)(13)(14). Ind...

Activation of Store-operated Calcium Channels

“…These observations provide strong evidence against a requisite role for SNARE-mediated membrane fusion in the coupling of SOC activation to calcium store depletion. This conclusion is consistent with the notion that the SOC channels, and the machinery necessary to activate them, exist in the plasma membrane prior to activation, as predicted by the observation of single store-operated channel openings in excised membrane patches (35).…”

Complex Actions of 2-Aminoethyldiphenyl Borate on Store-operated Calcium Entry

“…The inhibitory actions of 2-APB differed markedly, both quantitatively and qualitatively, among the three Orai species, consistent with but not proving a direct effect on the channels themselves. Earlier findings from a number of laboratories suggest an extracellular site of action of 2-APB on I CRAC (24,26,27), which is further consistent with a direct action on the channel. Both Orai1 and -3 could also be activated by 2-APB, and Orai3 channels additionally showed altered ion selectivity when activated by 2-APB.…”

Regulation of Store-Operated Calcium Entry by Calcium-Independent Phospholipase A2in Rat Cerebellar Astrocytes

“…It has been shown in rat hippocampal astrocytes that Ca 2ϩ oscillations were attributable to phospholipase C-mediated Ca 2ϩ release from intracellular stores (Zur Nieden and Deitmer, 2006). 2-APB effectively inhibits SOCE channels in many cell types (Bakowski et al, 2001;Braun et al, 2001;Broad et al, 2001;Gregory et al, 2001;Kukkonen et al, 2001;Prakriya and Lewis, 2001) but can also be a membranepermeable inhibitor of IP 3 receptors (Wu et al, 2000). We observed that the number of spontaneously active astrocytes as well as the frequency of oscillations were reduced by blocking SOCE channels and by inhibiting iPLA 2 activity.…”