Regulation of the Miniature Plasma Membrane Ca2+ Channel Imin by Inositol 1,4,5-Trisphosphate Receptors

Abstract

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...

Plasma Membrane Calcium Channels in Human Carcinoma A431 Cells Are Functionally Coupled to Inositol 1,4,5-Trisphosphate Receptor-Phosphatidylinositol 4,5-Bisphosphate Complexes

“…3) are all highly reminiscent of recently described effects of PIP 2 Ab and PIP 2 on intracellular InsP 3 R (23). We recently demonstrated direct I min -InsP 3 R functional coupling (12). The data described in the present report can be explained under the assumption that the I min in A431 cells are directly coupled to InsP 3 R-PIP 2 complexes proposed to exist underneath the plasma membrane (Fig.…”

“…Evidence for a "conformational coupling" model comes from the highly compartmentalized connection between InsP 3 -induced Ca 2ϩ release and Ca 2ϩ influx processes (15)(16)(17)(18)(19). Direct InsP 3 R-I CRAC conformational coupling has been experimentally supported by recent data obtained with recombinantly expressed mammalian trp channels (20 -22) and with I min in human carcinoma A431 cells (12).…”

“…Channels that share some common properties with I min have also been observed in experiments with human T-cells (9) and rat macrophages (8) and in endothelial cells (10,11). Major functional properties of I min channels, such as small conductance (1 picosiemens for divalent cations), high selectivity for divalent cations (P Ca/K Ͼ 1,000), inward rectification, and sensitivity to block by SKF95365 are very similar to properties of I CRAC channels (8,12). I min activity is induced in cell-attached patches by the addition of thapsigargin to the pipette solution, 2 further supporting the notion that I min and I CRAC are in fact the same channel.…”

“…It is generally believed that inside-out patch is not a bare bilayer but rather a membrane-covered bleb of cytoplasm (26) that may include InsP 3 R-containing organelles and cytoskeleton. We reason that PIP 2 Ab and PIP 2 exert an influence on InsP 3 R as described previously (23); change in InsP 3 R conformation is in turn transmitted to changes in I min behavior via the conformation coupling mechanism (1,12,14,20,21). PIP 2 binds to the amino-terminal ligand binding domain of the InsP 3 R (InsP 3 R-N) (23), 3 the same region that has been implicated in regulation of recombinant human trp3 activity (21).…”

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.…”