Activation of phospholipase C (PLC)-mediated signaling pathways in non-

I min is a plasma membrane-located, Ca 2؉ -selective channel that is activated by store depletion and regulated by inositol 1,4,5-trisphosphate (IP 3 ). In the present work we examined the coupling between I min and IP 3 receptors in excised plasma membrane patches from A431 cells. I min was recorded in cell-attached mode and the patches were excised into medium containing IP 3 . In about 50% of experiments excision caused the loss of activation of I min by IP 3. In the remaining patches activation of I min by IP 3 was lost upon extensive washes of the patch surface. The ability of IP 3 to activate I min was restored by treating the patches with rat cerebellar microsomes reach in IP 3 receptors but not by control forebrain microsomes. The re-activated I min had the same kinetic properties as I min when it is activated by Ca 2؉ -mobilizing agonists in intact cells and by IP 3 in excised plasma membrane patches and it was inhibited by the I crac inhibitor SKF95365. We propose that I min is a form of I crac and is gated by IP 3 receptors.The Ca 2ϩ signal evoked by agonists that stimulate phospholipase C is generated by Ca 2ϩ release from intracellular stores and Ca 2ϩ influx across the plasma membrane (1, 2 (5), inhibition of SERCA pumps (6), and/or intracellular infusion of Ca 2ϩ -chelating agents (4) can activate I crac . The molecular identity of I crac is not known. However, its functional characteristics began to emerge. An elegant singlechannel recording of I crac in Jurkat T cells estimated an I crac single channel conductance of about 1 pS when transporting Ca 2ϩ (7). This is remarkably similar to the conductance of a miniature, Ca 2ϩ -selective channel (I min ) we described in several cell types (8, 9). Moreover, similar to I crac , I min is highly selective for Ca 2ϩ over K ϩ (3,8,9). The open probability, but not the conductance of both channels, is increased by membrane hyperpolarization (7-9). I crac and I min are activated by store depletion with Ca 2ϩ -mobilizing agonists or thapsigargin in intact cells, and I min is activated by IP 3 in the same excised plasma membrane patch. Therefore studying the gating of I min by IP 3 and IP 3 R may be relevant to understanding the gating of I crac .Although widely documented (for review, see Refs. 1-3), the way I crac is gated by store Ca 2ϩ content is not understood. The two leading hypotheses to explain gating of I crac by stored Ca 2ϩ are the soluble messenger (10) and the conformational coupling hypothesis (2, 11). The former proposes generation of a soluble messenger, such as the Ca 2ϩ influx factor (10), in response to store depletion that diffuses to the plasma membrane to activate I crac . The conformational coupling model proposes gating of I crac by direct interaction with IP 3 R. Ca 2ϩ release from internal stores causes a conformational change in the IP 3 R, which is transduced to and is sensed by the I crac to regulate its activity (2). Additional suggestions include gating of I crac by agonist-generated lipid mediators (12) or by vesicle fusion e...

In most nonexcitable cells, calcium (Ca 2؉ ) release from inositol 1,4,5-trisphosphate (InsP 3 )-sensitive intracellular Ca 2؉ stores is coupled to Ca 2؉ influx (calcium release-activated channels (I CRAC )) pathway. Despite intense investigation, the molecular identity of I CRAC and the mechanism of its activation remain poorly understood. InsP 3 -dependent miniature calcium channels (I min ) display functional properties characteristic for I CRAC . Here we used patch clamp recordings of I min channels in human carcinoma A431 cells to demonstrate that I min activity was greatly enchanced in the presence of anti-phosphatidylinositol 4,5-bisphosphate antibody (PIP 2 Ab) and diminished in the presence of PIP 2 . Anti-PIP 2 antibody induced a greater than 6-fold increase in I min sensitivity for InsP 3 activation and an almost 4-fold change in I min maximal open probability. The addition of exogenous PIP 2 vesicles to the cytosolic surface of inside-out patches inhibited I min activity. These results lead us to propose an existence of a Ca 2؉ influx pathway in nonexcitable cells activated via direct conformational coupling with a selected population of InsP 3 receptors, located just underneath the plasma membrane and coupled to PIP 2 . The described pathway provides for a highly compartmentalized Ca 2؉ influx and intracellular Ca 2؉ store refilling mechanism.

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...