INTRODUCTION

The consensus model of glucose-stimulated insulin secretion is that closure of ATP-sensitive K⁺ channels (K⁺_ATP)¹ permits membrane depolarization, activation of voltage-dependent Ca²⁺ channels (VDCCs) and the influx of Ca²⁺ (1). Individual -cells, however, are often unresponsive to glucose alone, but can become responsive by combined stimulation with glucose and hormones, such as insulinotropic hormone glucagon-like peptide-1 (GLP-1; Ref. 2), that elevate intracellular cAMP levels (3-5). One mechanism underlying this increased responsiveness is the enhanced closure of K⁺_ATP channels (2). A second mechanism by which GLP-1, pituitary adenylate cyclase-activating polypeptide (PACAP), and cAMP can induce -cell depolarization is through the activation of voltage-independent, non-selective cation currents (6, 7). Similar cation currents are also activated by MTX, a polyether toxin isolated from dinoflagellates that in -cells has been shown to stimulate insulin secretion and inositol trisphosphate (Ins(1,4,5)P₃) production (8) and to enhance the influx of monovalent cations (9).

MTX-sensitive currents are activated by depletion of intracellular Ca²⁺ stores (10), and GLP-1 enhances intracellular Ca²⁺ mobilization through the potentiation of ryanodine-sensitive Ca²⁺-induced Ca²⁺ release in TC3 cells (11, 12). Increased cAMP levels stimulate Ca²⁺ release from secretory granules and reduce mitochondrial Ca²⁺ uptake in -cells (13, 14). These observations raise the possibility that the activation of non-selective cation currents by PACAP and GLP-1 may be a secondary consequence of glucose- and cAMP-dependent intracellular Ca²⁺ release. The physiological role of Ca²⁺ release-activated currents in -cells remains controversial, but such currents have been suggested to play a role in the cholinergic modulation of electrical bursting activity (15), and may control the membrane potential and intracellular Ca²⁺ ([Ca²⁺]_i) oscillations in response to nutrient stimulation (10).

Both PACAP (16) and GLP-1 (17) are potent insulin secretagogues in the presence of slightly elevated glucose levels. The activation of a voltage-independent, non-selective cation current by these hormones under conditions that stimulate insulin secretion suggests that this current may play an important role in depolarizing -cells to initiate insulin secretion. The aim of this study is to examine the mechanism of activation of the MTX-sensitive current and to compare the properties of I_Ca-NS with the current activated by GLP-1, PACAP, and cAMP to determine whether these currents are likely to be carried through the same channels.

MATERIALS AND METHODS

HIT-T15 cells were obtained from the American Type Culture Collection. TC6 cells were obtained from Dr. Shimon Efrat (Albert Einstein College of Medicine, New York, NY). HIT-T15 cells (passages 67-75) were maintained in Ham's F-12 medium containing 10 mM glucose, 10% heat-inactivated horse serum, and 2.5% fetal bovine serum. TC6 cells (passages 24-59) were maintained in Dulbecco's modified Eagle's medium containing 25 mM glucose, 15% horse serum, and 2.5% fetal bovine serum. Culture media also contained 100 units/ml penicillin G and 100 µg/ml streptomycin. Cells were plated onto glass coverslips coated with 1 mg/ml of type V concanavalin A (Sigma), which facilitates their adherence to glass. Cultures were maintained at 37 °C in a 5% CO₂ atmosphere incubator, and experiments were conducted 1-5 days post-plating.

Cells were bathed in a standard extracellular solution (SES) containing: 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES (295 mosM; pH adjusted to 7.4 with NaOH, approximately 4 mM), and 0.8 mM D-glucose unless indicated as being different in the text. Na⁺-free, N-methyl-D-glucamine (NMG) solutions were prepared using 138 mM NMG substituted for NaCl and adjusted to pH 7.4 with HCl. A Na⁺-free, 100 Ca solution was also prepared containing: 100 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES (282 mosM; pH adjusted to 7.4 with KOH, approximately 6 mM) Other test solutions were prepared by substitution of NaCl with 140 mM KCl, CsCl, LiCl, or choline chloride. Ca²⁺-free solutions were prepared by substituting MgCl₂ or MnCl₂ for CaCl₂. Low [Cl]_o solution was prepared by substituting NaCl with Na-aspartate.

Test solutions containing MTX were applied to individual cells by focal application from micropipettes using a PicoSpritzer II pressure ejection system (General Valve, Fairfield, NJ). A gravity-fed bath superfusion system was used to exchange and refresh bath solutions. Maitotoxin, econazole, staurosporine, nimodipine, and glyburide were obtained from Sigma. Tetrodotoxin, SKF 96365, genistein, GTPS, ryanodine, and heparin were obtained from Calbiochem. Tetraethylammonium chloride (TEA) and 4-aminopyridine (4AP) were obtained from Aldrich.

Cells were prepared for measurement of [Ca²⁺]_i by incubation in fura-2 acetoxymethyl ester (fura-2 AM; Molecular Probes, Inc., Eugene, OR). Cells were loaded in SES supplemented with 2% fetal bovine serum, 0.03% pluronic F-127, and 1 µM fura-2 AM for 15 min at room temperature (20-22 °C). Coverslips with fura-loaded adherent cells formed the base of a recording chamber mounted on a temperature-controlled stage (Micro Devices, Jenkintown, PA). Cells were visualized using a Zeiss IM35 microscope equipped with a Nikon UVF100 100× objective. Measurements of [Ca²⁺]_i were performed at 1-s intervals from the average of 10 video frames using a dual excitation wavelength video imaging system (IonOptix Corp., Milton, MA). Experiments were conducted at 32 °C. [Ca²⁺]_i was estimated from the ratio of 510 nm emission fluorescences due to excitation by 350 nm and 380 nm wavelength light from the following equation (18).

(Eq. 1)

K_d is the dissociation constant of fura-2 (225 nM),  is the ratio of 380 nm induced fluorescences of free/bound fura-2, R is the measured ratio of 350 nm/380 nm fluorescences and R_min and R_max are 350 nm/380 nm fluorescence ratios in zero [Ca²⁺] and saturating [Ca²⁺], respectively. Values of , R_min, and R_max were determined using fura-2 pentapotassium salt and calibration solutions from Molecular Probes, Inc.

Cell resting potentials and membrane currents were measured under current clamp or voltage clamp using either whole-cell or perforated patch configurations (19, 20). Patch pipettes pulled from borosilicate glass (Kimax-51, tip resistance 2-4 megohms) were fire polished and tip-dipped in K- or Cs-pipette solution containing: 95 mM K₂SO₄ (or Cs₂SO₄), 7 mM MgCl₂, 5 mM HEPES (pH adjusted to 7.4 with NaOH; final concentration of Na⁺ approximately 2 mM). Pipettes were then back-filled with the same solution, to which nystatin (240 µg/ml) was added for perforated patch recording.

The patch pipette was connected to an Heka Electronik EPC-9 patch clamp amplifier (Instrutech Corp., Mineola, NY) interfaced with a Macintosh Quadra 840AV computer running Pulse version 8.0 software (Instrutech Corp.). The series resistance (R_s) and cell capacitance (C_m) were monitored following seal formation, and experiments were conducted when R_s declined to <35 megohms. In voltage clamp experiments, R_s was compensated for by 60%.

Values are given as mean ± S.E. Statistical significance was determined using Student's t test computed using Lotus 1-2-3 spreadsheets.

RESULTS

MTX activates a non-selective cation current in TC6 cells (Fig. 1) that has a reversal potential of -7.8 ± 1.0 mV (n = 72) in SES (142 mM [Na⁺]_o). This current was observed in all cells tested, and its reversal potential becomes more negative as [Na⁺]_o is reduced (Fig. 1, Table I), although the shift is less than predicted from the Nernst potential for Na⁺. I_Ca-NS is also observed in solutions where extracellular Na⁺ is replaced by Cs⁺, K⁺, Li⁺, choline⁺, or NMG⁺ and in cells dialyzed with K⁺, Cs⁺, Na⁺, choline⁺, or NMG⁺ with Na⁺ in the bathing solution (Table II), confirming the non-selective nature of this current. Changing from normal [Cl]_o to low [Cl]_o extracellular solution did not affect the amplitude or reversal potential of the current (data not shown), confirming the cation selectivity of I_Ca-NS.

Table I. Sodium dependence of ICa-NS reversal potential (mV) Table shows the effect of changing [Na+]o on the reversal potential (mV) of the MTX-activated current (ICa-NS) in TC6 cells. The reversal potential shifted by less than would be predicted from the Nernst potential for Na+ suggesting that the channels are non-selective. Other values given in this table are control values obtained from cells subsequently transferred to different [Na+]o solutions. The number of cells recorded is indicated in parentheses. 142 Na 70 Na/70 NMG 70 Na/50 Ca 14 Na/NMG Na-free/NMG Na-free/100 Ca  7.8  ± 1.0  (72)  6.0  ± 3.7  (5)  21.6  ± 2.1  4.6  ± 2.1  (5)  30.6  ± 2.9  3.4  ± 1.2  (5)  38.4  ± 2.5  13.6  ± 3.3  (6)  39.7  ± 1.8  1.4  ± 0.5  (9)  25.0  ± 1.4

Table II. Ionic dependence of reversal potentials (mV) for MTX-induced currents Table shows the effect of extracellular (bath) and intracellular (pipette) ion substitutions on the reversal potential of ICa-NS. The reversal potential with SES (Na+) bath solution and Cs+-pipette solution is taken to be the reference value and ion substitutions that produced a significant change in the reversal potential are indicated. These data confirm the non-selective nature of the MTX-sensitive channels. *, p < 0.001; **, p = 0.005; ***, p = 0.014.  Cytosol Bath Na K Cs Li Choline Cs  7.8  ± 1.0 (72)  2.6  ± 0.7 (5) +2.7  ± 1.7 (6)**  3.9  ± 0.6 (7)  22.8  ± 1.0 (6)* Na  2.7  ± 1.2 (6) Choline +1.8  ± 3.3 (7)*** NMG +10.0  ± 4.9 (9)*

It is reported that Ca²⁺ is impermeant through MTX-activated channels in mouse -cells (10) in contrast with a report that Ca²⁺ is permeant through such channels in mouse L-cells (21). We therefore decided to re-examine the Ca²⁺ permeability of I_Ca-NS. Ca²⁺ influx through MTX-sensitive channels in HIT-T15 cells is suggested by an increase of [Ca²⁺]_i when hyperpolarizing voltage steps are applied following activation of the MTX-sensitive current and by the rapid and reversible fall of [Ca²⁺]_i when extracellular Ca²⁺ is removed (Figs. 2 and 3A). Fig. 2 shows that a hyperpolarizing voltage clamp step from 70 mV to 100 mV had no effect on [Ca²⁺]_i in a HIT-T15 cell before stimulation with MTX, but that similar voltage steps applied after activation of I_Ca-NS resulted in a pronounced increase of [Ca²⁺]_i that was reversibly abolished by removal of extracellular Ca²⁺. Hyperpolarizing pulses applied following activation of non-selective cation currents by GLP-1 (22) or 8-bromo-cAMP (7) also increased [Ca²⁺]_i.

We further tested the effects of removal of extracellular Ca²⁺ to confirm the activation of MTX-sensitive currents by Ca²⁺-free solutions (10) and to determine the effects of activation of the current on [Ca²⁺]_i in the absence of extracellular Ca²⁺. In 15/18 cells tested, bath perfusion with a Ca²⁺-free solution containing 50 µM EGTA (the Ca²⁺ concentration in this solution was measured (using K₅ fura-2) to be 150 nM) before stimulation with MTX caused a small, reversible increase of the membrane current and a fall of [Ca²⁺]_i (Fig. 3A). Holding currents at 70 mV increased from 0.96 ± 0.16 pA/pF in SES to 1.64 ± 0.30 pA/pF (p = 0.005) and [Ca²⁺]_i decreased from 117 ± 15 nM to 99 ± 15 nM on removal of extracellular Ca²⁺ (not statistically significant, p = 0.4). In 3/18 cells the holding current showed a much larger increase on transfer to Ca²⁺-free solution (Fig. 3B). The holding current in these cells increased by 21.8 ± 4.3 pA/pF and [Ca²⁺]_i reduced from 75 ± 15 nM to 37 ± 4 nM (p = 0.1). This current inactivates rapidly, and [Ca²⁺]_i is transiently elevated when SES (2.6 mM Ca²⁺) is reintroduced to the bath (Fig. 3B). Elevation of [Ca²⁺]_i and activation of a non-selective cation current were also observed in a similar subset of cells following stimulation with thapsigargin (2-10 µM, data not shown), as reported previously (10).

Bath perfusion with Ca²⁺-free solution after the activation of I_Ca-NS results in a rapid, reversible fall of [Ca²⁺]_i but has only a small effect on the current amplitude (Figs. 2 and 3A). The reversal potential of the MTX-activated current in Ca²⁺-free solution is 10.1 ± 2.8 mV (n = 12), not significantly different (p = 0.43) from the reversal potential measured in SES. These data suggest that Ca²⁺-influx carries only a small proportion of the current through MTX-sensitive channels in the presence of Na⁺. However, the rapid and reversible effects of removing extracellular Ca²⁺ on [Ca²⁺]_i suggests that I_Ca-NS does permit Ca²⁺ entry.

Further evidence for the influx of divalent cations through MTX-sensitive channels is suggested by increased Mn²⁺ quenching of intracellular fura-2 fluorescence following MTX-stimulation (Fig. 3C). Fig. 3C (i and ii) shows the raw fura-2 fluorescence emission values, application of a 60-s pulse of Ca²⁺-free, Mn²⁺-substituted solution before stimulation with MTX produces a gradual quenching of the fluorescence signals, a small increase in the holding current (Fig. 3C, iii), and little or no change in the 350 nm/380 nm fluorescence ratio (Fig. 3C, iv). Following activation of the inward current and rise in [Ca²⁺]_i, a pulse of Ca²⁺-free, Mn²⁺-substituted solution produces rapid quenching of fura-2 fluorescence (Fig. 3C, i and ii) with little or no effect on membrane current (Fig. 3C, iii) and reduces the fluorescence ratio (Fig. 3C, iv), consistent with a decrease in [Ca²⁺]_i. Similar activation of Mn²⁺ quenching has been observed following stimulation of insulinoma cells with PACAP (6), cAMP (22), and thapsigargin (23, 24).

A role for Ca²⁺ influx through MTX-sensitive channels is further supported by the effects of applying high [Ca²⁺]_o solutions. A Na⁺-free test solution containing 100 mM Ca²⁺ was applied to a TC6 cell prior to stimulation with MTX and produces a small increase of [Ca²⁺]_i (Fig. 4), similar to the effects of applying elevated [Ca²⁺]_o solutions to mouse islets (25). Application of 100 mM [Ca²⁺]_o solution following stimulation with MTX produces a pronounced inhibition of I_Ca-NS (Fig. 4) and a negative shift in the reversal potential of the current (Table I). This inhibition of I_Ca-NS by the 100 mM Ca²⁺ solution is accompanied by a rise of [Ca²⁺]_i (Fig. 4). These data could be explained by Ca²⁺ influx through a single class of non-selective cation channel with a lower permeability to Ca²⁺ than to Na⁺ under these experimental conditions. Alternatively, I_Ca-NS may have two (or more) components, one component being monovalent cation selective, and the other, smaller, component being selective for divalent cations.

The non-selective cation current activated by 8-bromo-cAMP in HIT-T15 cells is inhibited by whole cell dialysis with Ca²⁺-free, EGTA-buffered solutions or by loading the cells with BAPTA (1,2-bis(2-aminophenoxy)ethane N,N,N,N-tetraacetic acid), a Ca²⁺ chelator (7) leading us to test the dependence of I_Ca-NS activation on [Ca²⁺]_i (Fig. 5). Whole cell recordings from HIT-T15 cells were performed with either normal K-pipette solution (nominally Ca²⁺-free) or the same solution with 5 mM EGTA added (Ca²⁺-free). Whole cell dialysis with Ca²⁺-free intracellular solution inhibited activation of I_Ca-NS compared with control cells from the same platings dialyzed with nominally Ca²⁺-free solution or compared with cells in perforated patch voltage clamp (Fig. 5). These observations suggest that physiological [Ca²⁺]_i levels are required for activation of the current. It is notable that dialysis of the cells with Ca²⁺-free solution does not induce activation of the current alone, whereas dialysis with this solution might be expected to deplete intracellular Ca²⁺ stores and thus activate store-operated currents.

Ca²⁺-activated non-selective cation (Ca-NS) channels are expressed in -cells that are activated at cytosolic [Ca²⁺] > 10⁴ M and are blocked by 1 mM ATP and also by 10 mM 4AP when applied to the cytosolic face of isolated, inside-out patches (26, 27). Activation of I_Ca-NS is observed with physiological [Ca²⁺]_i levels but inhibition of the current by dialysis of cells with Ca²⁺-free solutions suggests that it may be carried through Ca-NS channels. To test this possibility, TC6 cells were bathed in glucose-free SES with 5 mM TEA and 10 nM glyburide added (to block Ca²⁺-activated K⁺ channels and K⁺_ATP channels) and dialyzed in the whole cell recording mode with K-pipette solution and 10 mM 4AP either with or without 2 mM ATP. MTX-induced currents in cells dialyzed without ATP had a mean peak amplitude of 23.6 ± 7.9 pA/pF (n = 6), and cells from the same platings dialyzed with 2 mM ATP had peak amplitudes of 22.5 ± 7.5 pA/pF (n = 6, not significantly different, p = 0.9). These data indicate that activation of I_Ca-NS is not glucose-dependent, consistent with previous reports of MTX-stimulated, glucose-independent insulin secretion (28). These differences in the sensitivity of I_Ca-NS to [Ca²⁺]_i and to block by ATP and 4AP in whole cell recordings compared with inside-out patches may indicate that I_Ca-NS is not carried through the Ca-NS channels reported previously (26, 27) or may reflect the different recording configurations.

The non-selective cation current activated by PACAP in TC6 cells is inhibited by SKF 96365 (22), a blocker of depletion-activated currents (29) that inhibits MTX-induced Ca²⁺ influx and insulin secretion (30). Fig. 6 shows that application of 50 µM SKF 96365 reversibly inhibits I_Ca-NS in TC6 cells, similar to its effect on the PACAP-induced current in these cells (22), further supporting the suggestion that these currents are carried by the same channel type.

The activation of Mn²⁺ quenching of intracellular fura-2 fluorescence by MTX (Fig. 3C) is similar to that observed following thapsigargin treatment of insulinoma cells (23, 24). Thapsigargin-sensitive Ca²⁺ pools can also be depleted by econazole, which inhibits Ca²⁺-ATPases (31) and thereby elevates [Ca²⁺]_i and also inhibits Mn²⁺ quenching in HIT cells (23). We therefore tested the effect of econazole on the activation of I_Ca-NS. Fig. 7A shows that 10 µM econazole significantly reduces the amplitude of I_Ca-NS compared with control cells from the same platings. The basal (pre-MTX) [Ca²⁺]_i rose from 52 ± 7 nM to 182 ± 41 nM (n = 5, p = 0.01) in the presence of econazole and the peak amplitude of I_Ca-NS decreased from 12.6 ± 2.3 pA/pF to 3.6 ± 1.1 pA/pF (n = 5, p = 0.01). The rise of [Ca²⁺]_i above basal during the MTX response was reduced from 449 ± 206 nM to 49 ± 14 nM (p = 0.09) by econazole.

The effect of genistein, a tyrosine kinase inhibitor, on the activation of I_Ca-NS was tested as capacitative Ca²⁺ influx activated by thapsigargin can also be inhibited by genistein (Ref. 32, Fig. 7B). The peak amplitude of I_Ca-NS reduces from 20.2 ± 3.3 pA/pF (n = 6) in control cells to 8.2 ± 2.6 pA/pF (n = 8, p = 0.01) following exposure of cells from the same platings to 100 µM genistein. Basal [Ca²⁺]_i (pre-MTX) is not significantly affected by genistein (88 ± 12 nM in control cells, 118 ± 19 nM in genistein, p = 0.2), while the peak rise of [Ca²⁺]_i during MTX responses reduced from 1026 ± 388 nM (control) to 313 ± 92 nM (genistein, p = 0.06).

Protein kinase C (PKC) activates capacitative Ca²⁺ entry in rat insulinoma (RINm5F) cells, and this activation of Ca²⁺ entry can be blocked by 1 µM staurosporine (24). We therefore tested the effects of 1 µM staurosporine on membrane currents and on the rise of [Ca²⁺]_i following MTX stimulation of TC6 cells (Fig. 7C). Staurosporine had no significant effect on I_Ca-NS; the mean peak amplitude of control currents was 54.2 ± 19.5 pA/pF (n = 5) compared with 51.1 ± 7.7 pA/pF (n = 6, p = 0.9) in cells from the same platings after addition of 1 µM staurosporine to the bathing solution. The pre-MTX basal [Ca²⁺]_i in control cells was 61 ± 11 nM and 96 ± 15 nM in cells bathed in staurosporine (p = 0.1), whereas the peak rise (increase above basal levels) of [Ca²⁺]_i was reduced from 1653 ± 252 nM to 142 ± 28 nM (p < 0.001) by staurosporine.

Activation of some types of non-selective cation current has been shown to be mediated by GTP-binding proteins (33). We therefore examined the potential role of GTP-binding proteins in the activation of I_Ca-NS by dialysis of TC6 cells with Cs-pipette solution supplemented with 10 mM 4AP, 2 mM Na₂ATP, and either 10 mM KF, 10 mM KF + 100 µM AlF₃, or 100 µM GTPS. The bath contained SES with 5 mM TEA and 1 µM TTX. Whole cell dialysis of cells for 15-20 min with KF (n = 4), KF + AlF₃ (n = 5), or GTPS (n = 5) failed to activate inward currents in cells that all subsequently responded to stimulation with MTX (data not shown).

MTX stimulates an increase in Ins(1,4,5)P₃ levels in -cell lines (8), and this increase might activate I_Ca-NS through an intracellular Ca²⁺ release mechanism. Heparin is a specific blocker of Ins(1,4,5)P₃ receptors that should inhibit activation of I_Ca-NS if Ins(1,4,5)P₃-gated Ca²⁺ stores play an important role. TC6 cells were dialyzed in whole cell recordings with Cs-pipette solution plus 10 mM 4AP, 2 mM Na₂ATP, and 0.5 mg/ml heparin for 3-4 min before stimulation with MTX. Activation of I_Ca-NS was not inhibited in cells dialyzed with heparin. The amplitude of the currents was not significantly different from that in control cells from the same platings, and the reversal potential of the currents was 7.0 ± 2.0 mV (n = 9), not significantly different from control values (p = 0.96).

GLP-1 enhances intracellular Ca²⁺ mobilization from ryanodine-sensitive stores in TC3 cells, and ryanodine reduces the amplitude of [Ca²⁺]_i spikes produced by depolarizing voltage clamp steps within 1 or 2 min (11). We introduced 100 µM ryanodine into TC6 cells by whole cell dialysis in the Cs/4AP/ATP-pipette solution (as above) and allowed 3-4 min dialysis before stimulation with MTX. Ryanodine failed to prevent activation of I_Ca-NS under these conditions, and the current amplitude and reversal potential (-8.0 ± 1.9 mV, n = 5) are not significantly different from control cells (p = 0.97).

DISCUSSION

We propose that MTX activates the same non-selective cation current as stimulation with the peptide hormones GLP-1 and PACAP (6, 7, 22). These hormones couple through GTP-binding proteins (G-proteins) to activate adenylyl cyclase and elevate intracellular cAMP in -cells (3-5), and cAMP analogs can also activate these non-selective cation currents. However, activation of G-proteins, by dialysis of cells with KF, KF + AlF₃, or GTPS (compounds that stimulate G-protein mediated activation of non-selective cation currents in epithelial cells (33) and activate K⁺_ATP channels in RINm5F and HIT-T15 insulinoma cells (34, 35)), neither activated nor inhibited the MTX-sensitive current.

Depletion of intracellular Ca²⁺ stores activates a MTX-sensitive current in mouse -cells (10), and the stimulation of Ins(1,4,5)P₃ production by MTX (8) raises the possibility that Ins(1,4,5)P₃-gated stores may play a role in the activation of this current. Parasympathetic, cholinergic stimulation of -cells stimulates Ins(1,4,5)P₃ production, potentiates glucose-induced insulin secretion (36), and also activates a TTX-insensitive Na⁺-dependent depolarizing current (37) that may also be carried through Ca²⁺ release-activated non-selective cation channels (15). The role of intracellular Ca²⁺ release in the activation of this current has, however, been disputed, and cholinergic activation of this Na⁺ current is reported to be mediated by M₃-type muscarinic receptors being coupled to Na⁺ channels (38). We observed that dialysis with heparin, a blocker of Ins(1,4,5)P₃ receptors, failed to inhibit activation of I_Ca-NS, suggesting that Ca²⁺ release from these stores may not be critical.

The presence of a Ca²⁺ store depletion-activated current in pancreatic -cells was proposed from studies showing: 1) that the state of filling of endoplasmic reticulum stores could regulate the membrane potential in mouse -cells (39), 2) that thapsigargin can activate Mn²⁺ quenching of intracellular fura-2 in RINm5F cells (24), and 3) that the Mn²⁺ quenching pathway is inhibited by econazole in HIT-T15 cells (23). Activation of I_Ca-NS by thapsigargin and its inhibition by both econazole and SKF 96365 are consistent with the suggestion that I_Ca-NS may represent a Ca²⁺ release-activated current (10, 31).

A role for PKC in the activation of store-operated Ca²⁺ entry in RINm5F cells was suggested from observations that the sustained [Ca²⁺]_i rise in response to combined stimulation with PKC-activating phorbol esters and thapsigargin is inhibited by staurosporine (24). We observed that staurosporine reduced the MTX-induced [Ca²⁺]_i rise but did not significantly inhibit the amplitude of I_Ca-NS, suggesting that PKC may not play a direct role in the activation of I_Ca-NS but does regulate [Ca²⁺]_i responses. Such effects could be mediated through inhibition of Ca²⁺ release from intracellular Ca²⁺ stores, or could reflect the inhibition of a small, divalent cation selective component of I_Ca-NS carried through a distinct set of channels other than the non-selective cation channels. The high concentration of staurosporine (1 µM) used in these experiments would also be expected to inhibit cAMP-dependent protein kinase, and further studies are required to elucidate the role(s) of PKC and cAMP-dependent protein kinase in the pathway(s) leading to the activation of I_Ca-NS and regulation of [Ca²⁺]_i changes.

Inhibition of both I_Ca-NS and the MTX-induced [Ca²⁺]_i rise by genistein, an inhibitor of tyrosine kinases (40) that blocks thapsigargin- and carbachol-induced Ca²⁺ entry (32, 41), suggests a role for tyrosine phosphorylation in the activation pathway of I_Ca-NS. However, the role of tyrosine phosphorylation remains ambiguous as only certain tyrosine kinase inhibitors (including genistein) effectively block capacitative Ca²⁺ entry (42). Thapsigargin-induced Ca²⁺ entry can also occur in the absence of detectable tyrosine phosphorylation but is still inhibited by tyrosine kinase inhibitors (43), and, therefore, the role of tyrosine kinases in activation of I_Ca-NS remains to be clarified.

Ca-NS channels are expressed in -cells that are activated at cytosolic [Ca²⁺] > 10⁴ M (26, 27). Activation of I_Ca-NS is observed at physiological [Ca²⁺]_i (approximately 100 nM) and is inhibited by dialysis of cells with Ca²⁺-free solution, similar to the inhibition of cAMP-activated currents (7) and suggesting a Ca²⁺-dependent step in the activation pathway of these channels. Ca-NS channels are also expressed in pancreatic acinar cells, and these channels are activated at much lower cytosolic Ca²⁺ concentrations in whole cell records than in isolated patches (44); a similar difference in Ca²⁺ sensitivity seems likely to occur for Ca-NS channels in -cells. Ca-NS channels are blocked by 1 mM ATP and by 10 mM 4AP when applied to the intracellular face of isolated membrane patches (27); however, these two compounds did not inhibit I_Ca-NS when introduced into the cytosol by whole cell dialysis (at 2 mM and 10 mM, respectively). It remains to be determined whether these differences in sensitivity to [Ca²⁺]_i, 4AP, and ATP are a consequence of the different recording conditions or suggest that Ca-NS channels do not carry I_Ca-NS.

Reducing extracellular [Cl] has no effect on I_Ca-NS amplitude or on its reversal potential, confirming the cation selectivity of the channel. Depletion of intracellular ATP levels through bathing cells in glucose-free media and dialyzing with ATP-free solutions or dialysis of cells with 2 mM ATP has no effect on I_Ca-NS amplitudes. This further distinguishes the MTX-induced current from the non-selective anion current described in insulin-secreting cells that increases in amplitude upon dialysis with 2 mM ATP (45).

Elevation of [Ca²⁺]_i is observed following the activation of non-selective cation currents by MTX, GLP-1, or PACAP in voltage clamped cells where activation of voltage-dependent Ca²⁺ channels is prevented (6, 7, 22). This rise of [Ca²⁺]_i is reversed by removal of extracellular Ca²⁺, suggesting that Ca²⁺ influx is associated with the non-selective current, although it remains to be determined whether a single class of channel is permeant to both monovalent and divalent cations, or if two (or more) distinct conductances are involved. The physiological role of Ca²⁺ influx associated with I_Ca-NS in the stimulation of insulin secretion remains to be determined. It has been reported that sustained Ca²⁺ influx through L-type VDCCs is strongly coupled to insulin secretion from HIT-T15 cells, whereas more transient Ca²⁺ influx through N-type Ca²⁺ channels is only weakly coupled (46). The Ca²⁺ influx associated with I_Ca-NS is prolonged and may, therefore, be able to contribute to the sustained [Ca²⁺]_i elevation that triggers secretion. However, the magnitude of this Ca²⁺ influx is likely to be small compared with that through L-type channels as, under physiological conditions, the cells will depolarize to a value close to that for the reversal potential of I_Ca-NS, and also influx through L-type VDCCs raises [Ca²⁺]_i very rapidly, whereas the [Ca²⁺]_i increase associated with I_Ca-NS develops much more slowly. This slow time course of the rise in [Ca²⁺]_i is consistent with a small amplitude Ca²⁺ influx and would explain why it is difficult to resolve a decrease in I_Ca-NS amplitude on changing to Ca²⁺-free solution with normal extracellular Na⁺ concentrations. It therefore seems that the main physiological role for these non-selective cation currents in the control of insulin secretion will be to depolarize the membrane potential and activate VDCCs.

The currents activated by GLP-1, PACAP, cAMP analogs, and MTX are Ca²⁺-dependent non-selective cation currents that activate over tens of seconds and persist for extended periods following removal of the stimulus. These currents are all insensitive to TTX, L-type Ca²⁺ channel blockers, TEA, and ryanodine but are inhibited by NMG, SKF96365, and La³⁺. Based upon these similarities between the temporal properties of the currents, and their associated [Ca²⁺]_i changes, and the pharmacology of the currents, we propose that these agents activate the same non-selective cation channels. The precise mechanism(s) controlling the activation of these non-selective cation channels remains to be determined, but a role for tyrosine kinase-induced phosphorylation is suggested by the effects of genistein. Activation of I_Ca-NS may also be controlled by the state of filling of intracellular Ca²⁺ stores, or may be partly due to Ca²⁺ release from intracellular stores raising cytosolic Ca²⁺ levels. We propose that activation of MTX-sensitive non-selective cation channels may play an important role in depolarizing -cells in response to stimulation by GLP-1 and PACAP during feeding to initiate insulin secretion without large elevations of blood glucose. We also suggest that Ca²⁺ is permeant through the MTX-sensitive channels and suggest that spontaneous activity of these channels may form the depolarizing, non-selective background conductance that permits Ca²⁺ influx (25) and opposes the activity of ATP-sensitive K⁺ channels in regulating the resting potential of -cells under both basal conditions and in response to hormonal stimulation.