J Biol Chem, Vol. 274, Issue 37, 25983-25985, September 10, 1999

COMMUNICATION
Regulation of the Miniature Plasma Membrane Ca²⁺ Channel I_min by Inositol 1,4,5-Trisphosphate Receptors^*

Alexander I. Zubov, Elena V. Kaznacheeva, Anton V. Nikolaev, Vadim A. Alexeenko, Kirill Kiselyov^§, Shmuel Muallem^§, and Galina N. Mozhayeva^¶

From the  Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia 194064 and the ^§ Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

	ABSTRACT

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I_min is a plasma membrane-located, Ca²⁺-selective channel that is activated by store depletion and regulated by inositol 1,4,5-trisphosphate (IP₃). In the present work we examined the coupling between I_min and IP₃ 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 IP₃. 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 IP₃ was lost upon extensive washes of the patch surface. The ability of IP₃ to activate I_min was restored by treating the patches with rat cerebellar microsomes reach in IP₃ 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 Ca²⁺-mobilizing agonists in intact cells and by IP₃ 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 IP₃ receptors.

	INTRODUCTION

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The Ca²⁺ signal evoked by agonists that stimulate phospholipase C is generated by Ca²⁺ release from intracellular stores and Ca²⁺ influx across the plasma membrane (1, 2). The two Ca²⁺ transporting events are linked through the regulation of Ca²⁺ influx by Ca²⁺ content in the stores, a gating behavior termed capacitative Ca²⁺ entry (CCE).¹ In many cells CCE is manifested as Ca²⁺ release-activated Ca²⁺ current (I_crac). I_crac is distinguished by its low conductance and very high selectivity for Ca²⁺ (3-5). Store depletion by agonist or IP₃-mediated Ca²⁺ release (5), inhibition of SERCA pumps (6), and/or intracellular infusion of Ca²⁺-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 single-channel recording of I_crac in Jurkat T cells estimated an I_crac single channel conductance of about 1 pS when transporting Ca²⁺ (7). This is remarkably similar to the conductance of a miniature, Ca²⁺-selective channel (I_min) we described in several cell types (8, 9). Moreover, similar to I_crac, I_min is highly selective for Ca²⁺ over K⁺ (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 Ca²⁺-mobilizing agonists or thapsigargin in intact cells, and I_min is activated by IP₃ in the same excised plasma membrane patch. Therefore studying the gating of I_min by IP₃ and IP₃R 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 Ca²⁺ content is not understood. The two leading hypotheses to explain gating of I_crac by stored Ca²⁺ are the soluble messenger (10) and the conformational coupling hypothesis (2, 11). The former proposes generation of a soluble messenger, such as the Ca²⁺ 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 IP₃R. Ca²⁺ release from internal stores causes a conformational change in the IP₃R, 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 events (13). Recently we studied gating of the store-operated, human homologue of the Drosophila Trp channel, hTrp3 by IP₃R (14). We found that hTrp3 channels are gated by coupling to IP₃R (14). These findings were corroborated by studies reported as unpublished observations (15), which identified sequences in hTrp3 and IP₃R that interacts with each other to influence Ca²⁺ influx.

Building upon our studies of hTrp3 gating by IP₃R (14) and identification of I_min as an I_crac-like channel (8, 9), in the present work we studied regulation of I_min by IP₃R. We report that I_min in excised plasma membrane patches is indeed gated by IP₃R, further supporting the coupling hypothesis and the possible identity of I_min with I_crac.

	MATERIALS AND METHODS

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Cells-- 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 to 3 days before use.

Electrophysiology-- Single-channel currents were recorded using the inside-out and cell-attached modes of the patch clamp technique (16). Currents filtered at 500 Hz were recorded using a PC-501A patch clamp amplifier (Warner Instruments, Hamden, CT) with a conventional feedback resistance in the headstage (10 G). During recording the currents were digitized at 2.5 kHz. For data analysis and presentation, currents were additionally digitally filtered. 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_min activity. These include free Ca²⁺ buffered at pCa 7 and membrane potential of 70 mV (8, 9). Experiments were carried out at room temperature (22-24 °C). I_min was activated by 2.5 µM IP₃.

Solutions-- The pipette solution contained (in mM): 105 BaCl₂ and 10 Tris/HCl (pH 7.4). The standard intracellular solution contained (in mM): 140 potassium glutamate, 5 NaCl, 1 MgCl₂, 10 HEPES/KOH, 1.13 CaCl₂ and 2 EGTA/KOH (pCa 7, pH 7.4). In cell-attached experiments, the bath solution contained (in mM): 140 KCl, 5 NaCl, 10 HEPES/KOH, 1 MgCl₂, and 2 CaCl₂. Drugs were applied to the patches either by bath perfusion or by brief pressure ejection. In both cases, the time required for a complete change of solution around the patch was less than 1 s.

Microsomes-- Cerebellar and forebrain microsomes were isolated from rat brains (Wistar 4-5 weeks old) as described (17) and stored at 70 °C. Microsomes were suspended at a protein concentration of 5 µg/µl. IP₃R content was determined by Western blot analysis with the use of polyclonal antibodies against type 1 IP₃R. As reported before (17) cerebellar microsomes contained at least 20-fold more IP₃R than forebrain microsomes at comparable microsomal protein content.

Chemicals-- HEPES was from Sigma, EGTA was from Fluka Chemie AG (Buchs, Switzerland), and IP₃ and SKF96365 were from Calbiochem (Behring Diagnostics, La Jolla, CA).

Data are given as mean ± S.E. Error bars denoting S.E. are shown where they exceed the symbol size.

	RESULTS AND DISCUSSION

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In previous work we reported that IP₃ activates a Ca²⁺-selective channel in excised plasma membrane patches which we termed I_min (8, 9). However, activation of I_min by IP₃ was successful only in about 50% of excised patches. Furthermore, in these studies we noticed that the likelihood of I_min activation by IP₃ was decreased with increasing patch washes. This is reminiscent of a similar observation noted when activation of hTrp3 by IP₃ and IP₃R was studied (14). In the case of hTrp3, it was shown that membrane washes prevented hTrp3 activation by IP₃ due to removal of IP₃R associated with the patch. Activation of hTrp3 by IP₃ could be restored by addition of native or recombinant IP₃R1 to the patches (14).

Suspecting that the loss of I_min regulation by IP₃ was due to dissociation and loss of IP₃R attached to I_min in a manner similar to that reported for hTrp3, we tested whether native IP₃R can restore regulation of I_min by IP₃. For these experiments we used the following experimental protocol. The cell attached mode of the patch clamp technique was obtained in A431 cells. Only patches showing channel activity in cell-attached mode were used for further experiments. In most cases patch excision into IP₃-free bath solution abolished channel activity observed in the cell-attached mode (8, 9). When patches were excised into a bath solution containing IP₃, I_min was observed in about 50% of patches. If I_min activity in response to IP₃ application was observed, the patches were washed until activation by IP₃ was lost. If the patches showed no I_min activity upon excision, they were not further washed. Figs. 1, A and B, show an example of an excised patch that did not respond to bath application of IP₃. After the control period patches were incubated with IP₃ and microsomes prepared either from rat cerebellum (rich source of IP₃R1, see Ref. 18) or rat forebrains (poor source of IP₃R (18)). The middle portion of Fig. 1A and the traces in Fig. 1B show that cerebellar microsomes restored the ability of IP₃ to activate I_min. Removal of IP₃ abolished channel activity, despite the continuous presence of microsomes. Hence, activation of IP₃R by IP₃ was needed to activate I_min. Fig. 1D shows that cerebellar microsomes restored activation of I_min by IP₃ in 26/41 (63%) experiments. On the other hand, no activation was observed in all 27 experiments with forebrain microsomes or in 6 experiments in which cerebellar microsomes without IP₃ were used.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Reconstitution of functional Imin/IP3R complexes in excised plasma membrane patches. A, a typical reconstitution experiment. Before patch excision channel activity was monitored in cell-attached mode to ensure the existence of an Imin channel in the patch. After excision the patch was exposed to 2.5 µM IP3. When the patch showed no IP3-induced channel activity, it was treated with cerebellar microsomes (cer.m/s). Removal of IP3 abolished the activity of Imin in the presence of cerebellar microsomes. B, segments of current recording in A presented in expanded time scale. Filtering was at 100 Hz. C, a patch excised from intact cells showing Imin activity was exposed to forebrain microsomes and IP3. Forebrain microsomes were not able to re-activate Imin. D, frequency of Imin activation.

To further establish the specificity of the effect of cerebellar microsomes, their activity was compared with forebrain microsomes in the same patches and in patches showing weak activity of I_min. Patches from five different cells exposed to IP₃ and microsomes prepared from forebrain were inactive. Subsequent exposure of the same patches to IP₃ and cerebellar microsomes activated I_min. Application of microsomes together with IP₃ to patches that show weak response to IP₃ increased the activity of I_min by about 7-fold. Thus, NP_o increased from 0.22 ± 0.09 to 1.55 ± 0.38 (n = 7).

The I_min current restored by IP₃R had properties identical to those reported previously for I_min (8, 9). Several properties of the reconstituted I_min are illustrated in Fig. 2. Specifically, the single channel conductance measured in the presence of 105 mM Ba²⁺ in the pipette was 1 pS (Fig. 2B). In six experiments the extrapolated reversal potential of the restored channel was close to +60 mV (Fig. 2B), indicating high selectivity for Ca²⁺ (=Ba²⁺) over K⁺. Furthermore, I_min open probability was highly voltage-dependent with increased activity at potentials below 40 mV (Fig. 2C). The channel mean open time was about 7.7 ms (Fig. 2D).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Kinetic properties of the re-activated Imin. A, the current induced by IP3 and cerebellar microsomes was recorded at the indicated membrane potentials. B, the mean current-voltage relationship of reconstituted Imin channels. Each point represents the average of six experiments. Error bars (S.E.) are smaller than symbols size. C, the dependence of channels NPo on membrane potential (average of seven experiments). NPo values were normalized with respect to the activity measured at 70 mV in each experiment. When not shown, error bars are smaller than symbol size. D, an open-time histogram for Imin. One channel was active in a given patch. Events with a time duration of less than 3 ms were discarded to exclude artifactual openings from the analysis. Total number of events was 1750. The line represents a single exponential fit to the data with a time constant of 7.7 ms. Channel activity was recorded at membrane potential of 70 mV.

SKF 96365 (SKF) is probably the best characterized I_crac inhibitor (19). This compound also inhibits Ca²⁺ influx and current mediated by the store-operated Trp family channels (20). Therefore, it was of interest to test the effect of SKF on the I_min activated by IP₃R. Fig. 3 shows that SKF strongly inhibited I_min activity after its restoration by IP₃ and IP₃R. Similar results were obtained in eight experiments.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Inhibition of reconstituted Imin by SKF. After reconstitution of Imin activity by IP3 and cerebellar microsomes the patch was incubated with microsomes, IP3, and 20 µM SKF. Similar results were obtained in eight experiments. The top trace was filtered at 50 Hz. The bottom trace shows the indicated segment of the top trace at an expanded time scale and at a filtering of 100 Hz. Current was measured at a holding potential of 70 mV.

The mechanism of I_crac gating by store depletion remained a mystery for a long time due to a lack of adequate experimental systems to study this question. Two recent findings, however, allowed the reprobing of this question. The first is the demonstration of the regulation of the store-operated hTrp3 channel by IP₃R (14), and the second is the measurement of I_crac single channel conductance (7). Previous studies estimated single channel conductance of I_crac to be below the resolution of a standard excised patch technique (4, 5). However, isolation of I_crac single channel conductance in whole-cell recording revealed that I_crac conductance is about 1 pS when transporting divalent ions, a conductance that can be comfortably resolved in excised patches. In previous studies, we measured the conductance of I_min to be about 1 pS (8, 9). In this study, we determined the channel mean open time to be about 7.7 ms (Fig. 2D). Channel mean open time is the most characteristic of channel properties and is commonly used as channel fingerprints (21). In this respect, we note that I_crac mean open time was reported to be about 8 ms (7). The findings that I_min open probability is regulated by voltage in a manner similar to that of I_crac, (7), both channels are highly selective for divalent ions (4, 5), and the two channels have almost identical mean open time suggest to us that I_min is a form of I_crac.

The similarity (and possible identity) of I_min and I_crac and our ability to record I_min in excised plasma membrane patches provided us with the opportunity to study the gating of native I_crac-like channels by IP₃R. The present work shows that I_min is gated by interaction with IP₃R, that this protein-protein interaction is relatively lose, and that the gating required occupation of IP₃R by IP₃. All of these characteristics are identical to those we found for the regulation of the store-operated hTrp3 channel by IP₃R (14). Gating of store-operated channels by IP₃R may be mediated by direct interaction between the channels. Thus, recent findings reported as unpublished observations (15) indicated that IP₃R/hTrp complexes can be co-immunoprecipated, and peptide sequences in IP₃R can bind to peptide sequences in hTrp3 in vitro. Interaction between I_crac and IP₃R may occur in vivo in microdomains rich in IP₃R. Such microdomains were reported in many cells (22) and probably facilitated excision of I_min with IP₃R attached to them in our experiments.

	ACKNOWLEDGEMENTS

We thank Dr. A. Arnautov for helping with Western blot analysis.

	FOOTNOTES

^* This work was supported by the Russian Basic Research Foundation Grant 98-04-49512.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: Institute of Cytology RAS, Tikhoretsky Ave 4., 194064 St. Petersburg, Russia. Tel.: 7-812-2471497; Fax: 7-812-2470341; E-mail: gnmozh@link.cytsbp.rssi.ru.

	ABBREVIATIONS

The abbreviations used are: CCE, capacitative Ca²⁺ entry; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptors; I_crac, Ca²⁺ release-activated Ca²⁺ current.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES