J Biol Chem, Vol. 275, Issue 7, 4561-4564, February 18, 2000

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

Elena Kaznacheyeva, Alexander Zubov, Anton Nikolaev, Vadim Alexeenko, Ilya Bezprozvanny^§, and Galina N. Mozhayeva^¶

From the  Institute of Cytology RAS, 4 Tikhoretsky Ave., St. Petersburg 194064, Russia and the ^§ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In most nonexcitable cells, calcium (Ca²⁺) release from inositol 1,4,5-trisphosphate (InsP₃)-sensitive intracellular Ca²⁺ stores is coupled to Ca²⁺ 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. InsP₃-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 (PIP₂Ab) and diminished in the presence of PIP₂. Anti-PIP₂ antibody induced a greater than 6-fold increase in I_min sensitivity for InsP₃ activation and an almost 4-fold change in I_min maximal open probability. The addition of exogenous PIP₂ vesicles to the cytosolic surface of inside-out patches inhibited I_min activity. These results lead us to propose an existence of a Ca²⁺ influx pathway in nonexcitable cells activated via direct conformational coupling with a selected population of InsP₃ receptors, located just underneath the plasma membrane and coupled to PIP₂. The described pathway provides for a highly compartmentalized Ca²⁺ influx and intracellular Ca²⁺ store refilling mechanism.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Stimulation of nonexcitable cells by agonists acting via the phospholipase C-mediated signaling pathway causes the depletion of InsP₃¹-sensitive intracellular Ca²⁺ stores and activation of Ca²⁺ influx across the plasma membrane, the process known as capacitative Ca²⁺ entry (1-3). Capacitative Ca²⁺ entry is mediated by plasma membrane Ca²⁺ channels termed "Ca²⁺ release activated channels" (I_CRAC) (4, 5). Despite intense investigation, the molecular identity of I_CRAC remain unclear (1-3). In experiments with the human carcinoma A431 cell line, we have previously described miniature Ca²⁺ channels (I_min), which are activated by application of uridine trisphosphate and bradykinin to cell-attached patches or by application of InsP₃ to excised inside-out patches (6-8). 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 > 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.

The mechanism of I_CRAC activation remains similarly controversial (1-3), with attention in the field primarily focused on two major hypotheses. In the first scenario, I_CRAC is activated by a "diffusible messenger" released by depleted Ca²⁺ stores (13). In the second scenario, I_CRAC is activated via a direct mechanical connection with the intracellular InsP₃R via a "conformational coupling" mechanism (1, 14). Evidence for a "conformational coupling" model comes from the highly compartmentalized connection between InsP₃-induced Ca²⁺ release and Ca²⁺ influx processes (15-19). Direct InsP₃R-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).

InsP₃R located just underneath the plasma membrane must exist in cells to support formation of InsP₃R-I_min complexes. It has been recently proposed that a subpopulation of intracellular InsP₃R forms tight complexes with phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasma membrane (23). Can the same InsP₃R interact with both I_min and PIP₂? Can the I_min-InsP₃R-PIP₂ complex mediate signal transduction? To answer these questions, here we tested the effects of monoclonal anti-PIP₂ antibody (PIP₂Ab) (24) and exogenous PIP₂ on I_min activity in patches excised from A431 cells. We found that PIP₂Ab activated and exogenous PIP₂ inhibited I_min activity. Based on these results, we propose a novel I_min-InsP₃R-PIP₂ functional coupling model.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Electrophysiological Recordings-- Human carcinoma A431 cells (Cell Culture Collection, Institute of Cytology, St. Petersburg, Russia) were kept in culture as described elsewhere (8). For patch clamp experiments, cells were seeded onto coverslips and maintained in culture for 1-3 days before use. Single-channel currents were recorded using the inside-out configuration of the patch clamp technique (25). Currents filtered at 500 Hz were recorded using a PC-501A patch clamp amplifier (Warner Instruments, USA) with a conventional feedback resistance in the head stage (10 gigaohms). During recording, the currents were digitized at 2.5 kHz. For data analysis and presentation, currents were additionally digitally filtered as indicated. I_min open probability (NP_o) was determined using the following equation: NP_o = <I>/i where <I> and i are the mean channel current and unitary current amplitude, respectively. <I> was estimated from the time integral of the current above the base line, and i was determined from current records and all-point amplitude histograms. Data were collected from 20-s current records after channel activity reached steady state. Unless otherwise specified, all experiments were performed at standard conditions optimal for I_Ca activity at 70 mV holding potential. The pipette solution contained 105 mM BaCl₂ and 10 mM Tris/HCl (pH 7.4). The standard intracellular solution contained 140 mM potassium glutamate, 5 mM NaCl, 1 mM MgCl₂, 10 mM HEPES/KOH, 1.13 mM CaCl₂, and 2 mM EGTA (pCa 7, pH 7.4). Experiments were carried out at room temperature (22-24 °C).

Materials-- PIP₂Ab (24) was from PerSeptive Biosystems. PIP₂Ab was reconstituted in phosphate-buffered saline (titer 1:1500), diluted 1:100 by intracellular solution, and used for chamber perfusion. Stearoyl-arachidonyl 4,5-PIP₂ was from Roche Molecular Biochemicals. PIP₂ vesicles were prepared as described previously (23) and applied to the patch at the 10 µM final concentration by rapid bath perfusion. 4-4-bromophenacylbromide and HEPES were from Sigma; EGTA was from Fluka Chemie AG (Buchs, Switzerland). InsP₃ and U73122 were from Calbiochem.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Anti-PIP₂ Antibody Activates I_min-- In inside-out patches excised from the plasma membrane of human carcinoma A431 cells, the application of 5 µM InsP₃ to the cytoplasmic side of the patch in approximately 50% of experiments induced activation of I_min (Fig. 1A). At 5 µM InsP₃, I_min activity in excised patches was transient due to InsP₃-dependent inactivation (8), and channel activity rapidly subsided (Fig. 1, A and B). We found that I_min activity can be restored and further enchanced by the application of PIP₂Ab (24) to the cytosolic side of the patch membrane (Fig. 1, A and B). PIP₂Ab activated I_min in all experiments (11 of 11), and, on average, increased channel NP_o in the presence of 2.5 µM InsP₃ from 0.8 ± 0.2 (n = 13) to 3.0 ± 0.2 (n = 7). The addition of boiled PIP₂Ab had no effect on I_min (n = 5). By reversing the order of InsP₃ and PIP₂Ab additions to the patch, we established that InsP₃ was absolutely required to register channel activity (Fig. 1C). Moreover, current-voltage relationship and voltage-dependence of channels observed in the presence of PIP₂Ab and InsP₃ were indistinguishable from the corresponding properties of I_min channels recorded in the presence of InsP₃ alone (Fig. 1, D and E).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   PIP2Ab enchances Imin activity. A, current traces in inside-out patches recorded in control, after application of InsP3 and PIP2Ab at 70 mV holding potential. The fragments of current records are shown at the bottom on an expanded time scale. The unitary current amplitude of Imin in utilized recording conditions is 0.18 pA. Filtering is 100 Hz. B, Imin NPo plot for the experiment shown in A. NPo was averaged over 0.5-s intervals and plotted versus time in the experiment. Mean NPo was 0.71 after InsP3 application and 3.31 after PIP2Ab addition. Data are representative for 11 experiments. C, activation of Imin by InsP3 in the presence of PIP2Ab. Current traces were recorded at a compressed time scale (top) and an expanded time scale (bottom). D and E, current-voltage relationship and NPo voltage dependence of Imin channels are unaffected by PIP2Ab antibodies. Open circles, InsP3 alone; filled triangles, InsP3 plus PIP2Ab. Average data are shown as mean ± S.E. (n  6). NPo values were normalized to the value obtained at 70 mV in the same experiment.

Anti-PIP₂ Antibody Increases Sensitivity of I_min to InsP₃ Activation-- To gain insight into the mechanism of PIP₂Ab action, we characterized the effect of PIP₂Ab on I_min sensitivity to activation by InsP₃. In this series of experiments, we determined the mean NP_o of I_min in inside-out patches as a function of InsP₃ concentration. Fit to the data obtained in control conditions (Fig. 2, filled circles) yielded apparent affinity (K_app) of 0.51 µM InsP₃, maximal NP_o (NP_o^max) of 0.87 and a Hill coefficient (n_H) of 1.05 (curve). In contrast, fit to the data obtained in the presence of PIP₂Ab (Fig. 2, open circles) resulted in K_app of 0.08 µM InsP₃, NP_o^max of 3.21, and n_H of 0.8 (curve). Thus, on average, PIP₂Ab induced a greater than 6-fold change in I_min apparent affinity for InsP₃ and a 3.7-fold change in NP_o^max. PIP₂Ab did not activate I_min in the absence of InsP₃ (Fig. 1C) but instead dramatically increased the potency of InsP₃ to activate I_min (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   PIP2Ab increases the apparent affinity of Imin for InsP3. Filled circles, InsP3 alone; open circles, InsP3 plus PIP2Ab. Average data at each InsP3 concentration are shown as mean ± S.E., with the number of experiments used to generate each data point as indicated. Both sets of data were fit with the equation NPo = NPomax [InsP3]nH/([InsP3]nH + KappnH). The values of parameters resulting in the best fit (smooth curves) are given under "Results." The estimate of the curve position in the presence of InsP3 plus PIP2 (filled triangles) is shown by a dotted line.

PIP₂ Inhibits I_min-- PIP₂Ab acts by specifically chelating PIP₂ (24). The addition of exogenous PIP₂ is expected to have an effect opposite to that of PIP₂Ab. Indeed, the addition of PIP₂ vesicles to the cytosolic surface of inside-out patches inhibited InsP₃-activated I_min (Fig. 3A) (15 of 18) and shifted InsP₃ dose-dependence curve toward lower apparent affinity for InsP₃ (Fig. 2, triangles). The inhibitory influence of PIP₂ could be partially overcome by raising InsP₃ concentration to 20 µM (Figs. 2 and 3A). The addition of high InsP₃ concentrations prior to PIP₂ was not possible due to InsP₃-induced desensitization of I_min (8). An ability of PIP₂ to inhibit I_min was not significantly affected by phospholipase A₂ inhibitor 4-bromophenacylbromide (Fig. 3B) (9 of 13) or phospholipase C inhibitor U73122 (Fig. 3C) (4 of 4). Thus, it is unlikely that exogenous PIP₂ needs to be converted to arachidonic acid (8) or diacylglycerol to inhibit I_min. Moreover, diacylglycerol had no inhibitory effect on I_min activity (6 of 6) (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Exogenous PIP2 inhibits the activity of Imin. A, 10 µM PIP2 inhibits activity of Imin channels activated by 2.5 µM InsP3. The data are shown as an NPo versus time plot. A similar effect was observed in 15 of 18 experiments. The addition of 20 µM InsP3 partially restored channel activity following PIP2 inhibition. B and C, pretreatment of cells with 1 µM 4-bromophenacylbromide (4-BPB) (9 of 13) or 10 µM U73122 (4 of 4) and their presence in the internal solution does not prevent PIP2 inhibition of Imin.

I_min-InsP₃R-PIP₂ Coupling Model-- Activation of I_min by PIP₂Ab in inside-out patches (Fig. 1), a dramatic shift in I_min InsP₃ dose dependence induced by PIP₂Ab (Fig. 2), and the ability of PIP₂ to exert the opposite effects (Fig. 3) are all highly reminiscent of recently described effects of PIP₂Ab and PIP₂ on intracellular InsP₃R (23). We recently demonstrated direct I_min-InsP₃R 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 InsP₃R-PIP₂ complexes proposed to exist underneath the plasma membrane (Fig. 4A). 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 InsP₃R-containing organelles and cytoskeleton. We reason that PIP₂Ab and PIP₂ exert an influence on InsP₃R as described previously (23); change in InsP₃R conformation is in turn transmitted to changes in I_min behavior via the conformation coupling mechanism (1, 12, 14, 20, 21). PIP₂ binds to the amino-terminal ligand binding domain of the InsP₃R (InsP₃R-N) (23),³ the same region that has been implicated in regulation of recombinant human trp3 activity (21).

View larger version (56K): [in this window] [in a new window]   Fig. 4.   Model of Imin functional coupling to InsP3R-PIP2 complex. A, in the resting state, a population of InsP3R localized underneath the plasma membrane forms a tight inhibitory complex with PIP2 in the juxtaposed membrane (23). We propose that the same InsP3Rs are directly or indirectly coupled to Imin Ca2+ influx channels in the plasma membrane. B, hormonal stimulation leads to activation of PLC, cleavage of InsP3R-tethered PIP2, generation of InsP3 "on site," and the conformational change of the InsP3R (23). We propose that the induced conformational change of the InsP3R is coupled to the opening of Imin channels, activation of localized Ca2+ influx across the plasma membrane, and compartmentalized Ca2+ store refilling process.

Cleavage of InsP₃R-tethered PIP₂ has been previously proposed to lead to localized Ca²⁺ release from the "trigger" stores and Ca²⁺ wave initiation (23). "On-site" conversion of PIP₂ into InsP₃ should induce large scale conformational changes of the InsP₃R-N domain (27). Conformational change of the InsP₃R-N domain, which plays an organizing role in I_min-InsP₃R-PIP₂ complex formation, leads to opening of the InsP₃R channel gate and to activation of I_min via direct protein-protein interaction (Fig. 4B). Indeed, only InsP₃-ligated InsP₃R-N is able to evoke human trp3 channel activity (21), in general agreement with our model. The proposed model also agrees with much of experimental evidence supporting a highly compartmentalized connection between InsP₃-induced Ca²⁺ release and Ca²⁺ influx (15-18).

The proposed coupling model (Fig. 4) implies that InsP₃R is essential for capacitative Ca²⁺ entry channel activation. The removal of all three InsP₃R isoforms by genetic means in chicken B lymphocytes had no effect on thapsigargin-induced Ca²⁺ influx (28), apparently in direct conflict with the conformational coupling model. The existence of at least one more Ca²⁺ influx pathway in nonexcitable cells can potentially account for these results. This additional pathway may be stimulated by the global intracellular Ca²⁺ store depletion and does not require InsP₃R for activation. Additional studies are needed to clarify which channel type is responsible for an InsP₃R-independent Ca²⁺ influx in nonexcitable cells.

	FOOTNOTES

^* This work was supported by a grant from the Russian Basic Research Foundation (to G. N. M) and by National Institutes of Health Grant NS38082 (to I. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 812-247-1497; Fax: 812-247-0341; E-mail: gnmozh@link.cytspb.rssi.ru.

² E. Kaznacheyeva, A. Zubov, A. Nikolaev, V. Alexeenko, I. Bezprozvanny, and G. N. Mozhayeva, unpublished results.

³ L. Glouchankova and I. Bezprozvanny, unpublished data.

	ABBREVIATIONS

The abbreviations used are: InsP₃, inositol 1,4,5-trisphosphate; I_CRAC, calcium release-activated channel(s); I_min, InsP₃-dependent miniature calcium channel(s); InsP₃R, InsP₃ receptor; PIP₂, phosphatidylinositol 4,5-bisphosphate; PIP₂Ab, anti-PIP₂ monoclonal antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES