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

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 betweenI min and IP3 receptors in excised plasma membrane patches from A431 cells. I minwas 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 minby IP3. In the remaining patches activation ofI min by IP3 was lost upon extensive washes of the patch surface. The ability of IP3 to activateI min was restored by treating the patches with rat cerebellar microsomes reach in IP3 receptors but not by control forebrain microsomes. The re-activatedI min had the same kinetic properties asI 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 ofI crac and is gated by IP3receptors.

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). The two Ca 2ϩ transporting events are linked through the regulation of Ca 2ϩ influx by Ca 2ϩ content in the stores, a gating behavior termed capacitative Ca 2ϩ entry (CCE). 1 In many cells CCE is manifested as Ca 2ϩ release-activated Ca 2ϩ current (I crac ). I crac is distinguished by its low conductance and very high selectivity for Ca 2ϩ (3)(4)(5). Store depletion by agonist or IP 3 -mediated Ca 2ϩ release (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)(8)(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 events (13). Recently we studied gating of the store-operated, human homologue of the Drosophila Trp channel, hTrp3 by IP 3 R (14). We found that hTrp3 channels are gated by coupling to IP 3 R (14). These findings were corroborated by studies reported as unpublished observations (15), which identified sequences in hTrp3 and IP 3 R that interacts with each other to influence Ca 2ϩ influx.
Building upon our studies of hTrp3 gating by IP 3 R (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 IP 3 R. We report that I min in excised plasma membrane patches is indeed gated by IP 3 R, further supporting the coupling hypothesis and the possible identity of I min with I crac .

MATERIALS AND METHODS
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 ϭ ϽIϾ/i, where ϽIϾ and i are the mean channel current and unitary current amplitude, respectively. ϽIϾ 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 Ca 2ϩ buffered at pCa 7 and membrane potential of Ϫ70 mV (8,9). Experiments were carried out at room temperature (22-24°C). Microsomes-Cerebellar and forebrain microsomes were isolated from rat brains (Wistar 4 -5 weeks old) as described (17) and stored at Ϫ70°C. Microsomes were suspended at a protein concentration of 5 g/l. IP 3 R content was determined by Western blot analysis with the use of polyclonal antibodies against type 1 IP 3 R. As reported before (17) cerebellar microsomes contained at least 20-fold more IP 3 R than forebrain microsomes at comparable microsomal protein content.
Data are given as mean Ϯ S.E. Error bars denoting S.E. are shown where they exceed the symbol size.

RESULTS AND DISCUSSION
In previous work we reported that IP 3 activates a Ca 2ϩselective channel in excised plasma membrane patches which we termed I min (8,9). However, activation of I min by IP 3 was successful only in about 50% of excised patches. Furthermore, in these studies we noticed that the likelihood of I min activation by IP 3 was decreased with increasing patch washes. This is reminiscent of a similar observation noted when activation of hTrp3 by IP 3 and IP 3 R was studied (14). In the case of hTrp3, it was shown that membrane washes prevented hTrp3 activation by IP 3 due to removal of IP 3 R associated with the patch. Activation of hTrp3 by IP 3 could be restored by addition of native or recombinant IP 3 R1 to the patches (14).
Suspecting that the loss of I min regulation by IP 3 was due to dissociation and loss of IP 3 R attached to I min in a manner similar to that reported for hTrp3, we tested whether native IP 3 R can restore regulation of I min by IP 3 . 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 IP 3 -free bath solution abolished channel activity observed in the cell-attached mode (8,9). When patches were excised into a bath solution containing IP 3 , I min was observed in about 50% of patches. If I min activity in response to IP 3 application was observed, the patches were washed until activation by IP 3 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 IP 3 . After the control period patches were incubated with IP 3 and microsomes prepared either from rat cerebellum (rich source of IP 3 R1, see Ref. 18) or rat forebrains (poor source of IP 3 R (18)). The middle portion of Fig. 1A and the traces in Fig. 1B show that cerebellar microsomes restored the ability of IP 3 to activate I min . Removal of IP 3 abolished channel activity, despite the continuous presence of microsomes. Hence, activation of IP 3 R by IP 3 was needed to activate I min . Fig. 1D shows that cerebellar microsomes restored activation of I min by IP 3 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 IP 3 were used.
To further establish the specificity of the effect of cerebellar After excision the patch was exposed to 2.5 M IP 3 . When the patch showed no IP 3 -induced channel activity, it was treated with cerebellar microsomes (cer.m/s). Removal of IP 3 abolished the activity of I min 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 I min activity was exposed to forebrain microsomes and IP 3 . Forebrain microsomes were not able to re-activate I min . D, frequency of I min activation. 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 Ϫ70 mV. 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 IP 3 and microsomes prepared from forebrain were inactive. Subsequent exposure of the same patches to IP 3 and cerebellar microsomes activated I min . Application of microsomes together with IP 3 to patches that show weak response to IP 3 increased the activity of I min by about 7-fold. Thus, NP o increased from 0.22 Ϯ 0.09 to 1.55 Ϯ 0.38 (n ϭ 7).
The I min current restored by IP 3 R 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 Ba 2ϩ in the pipette was 1 pS (Fig. 2B). In six experiments the extrapolated reversal potential of the restored channel was close to ϩ60 mV (Fig. 2B), indicating high selectivity for Ca 2ϩ (ϭBa 2ϩ ) over K ϩ . Furthermore, I min open probability was highly voltage-dependent with increased activity at potentials below Ϫ40 mV (Fig. 2C). The channel mean open time was about 7.7 ms (Fig. 2D).
SKF 96365 (SKF) is probably the best characterized I crac inhibitor (19). This compound also inhibits Ca 2ϩ 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 IP 3 R. Fig. 3 shows that SKF strongly inhibited I min activity after its restoration by IP 3 and IP 3 R. Similar results were obtained in eight experiments.
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 IP 3 R (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 IP 3 R. The present work shows that I min is gated by interaction with IP 3 R, that this protein-protein interaction is relatively lose, and that the gating required occupation of IP 3 R by IP 3 . All of these characteristics are identical to those we found for the regulation of the store-operated hTrp3 channel by IP 3 R (14). Gating of store-operated channels by IP 3 R may be mediated by direct interaction between the channels. Thus, recent findings reported as unpublished observations (15) indicated that IP 3 R/hTrp complexes can be co-immunoprecipated, and peptide sequences in IP 3 R can bind to peptide sequences in hTrp3 in vitro. Interaction between I crac and IP 3 R may occur in vivo in microdomains rich in IP 3 R. Such microdomains were reported in many cells (22) and probably facilitated excision of I min with IP 3 R attached to them in our experiments.