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Endocrinology Vol. 138, No. 9 3735-3740
Copyright © 1997 by The Endocrine Society

ARTICLES

T-Type Calcium Channels Facilitate Insulin Secretion by Enhancing General Excitability in the Insulin-Secreting ß-Cell Line, INS-11

Arin Bhattacharjee, Richard M. Whitehurst, Jr., Min Zhang, Lin Wang and Ming Li

Department of Pharmacology (A.B., M.Z., L.W., M.L.) and Department of Pediatrics (R.M.W.), University of South Alabama College of Medicine, Mobile, Alabama 36688

Address all correspondence and requests for reprints to: Ming Li, Ph.D. Department of Pharmacology, University of South Alabama, College of Medicine Mobile, Alabama 36688. E-mail: mli{at}jaguar1.usouthal.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study addresses the function of T-type voltage-gated calcium channels in insulin-secreting cells. We used whole-cell voltage and current recordings, capacitance measurements, and RIA techniques to determine the contribution of T-type calcium channels in modulation of electrical activity and in stimulus-secretion coupling in a rat insulin secreting cell line, INS-1. By employing a double pulse protocol in the current-clamp mode, we found that activation of T-type calcium channels provided a low threshold depolarizing potential that decreased the latency of onset of action potentials and furthermore increased the frequency of action potentials, both of which are abolished by administration of nickel chloride (NiCl2), a selective T-type calcium channel blocker. Moreover application of high frequency stimulation, as compared with low frequency stimulation, caused a greater change in membrane capacitance ({Delta}Cm), suggesting higher insulin secretion. We demonstrated that glucose stimulated insulin secretion in INS-1 is reduced dose dependently by NiCl2. We conclude that T-type calcium channels facilitate insulin secretion by enhancing the general excitability of these cells. In light of the pathological effects of both hypo and hyperinsulinemia, the T-type calcium channel may be a therapeutic target.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOSE-INDUCED electrical activity in pancreatic ß-cells is thought to involve slow waves of membrane depolarization on which action potentials are superimposed (1). Plateau depolarization is initiated via blockade of the ATP-sensitive potassium channels causing trains of calcium-dependent, tetrodotoxin-insensitive action potentials (2). This has been the hallmark of electrical activity in mouse pancreatic ß-cells. The trains of action potentials represent the opening and closing of the voltage-dependent L-type calcium channels, major calcium signaling channels also found in neurons and muscle cells. The secretion of insulin has been shown to be dependent on increases in intracellular calcium concentration (3, 4, 5, 6), and it has been proposed that the metabolism of glucose will cause depolarization of the cell to membrane voltages that will activate the L-type calcium channels and, therefore, facilitate insulin secretion. Recently, however, it was demonstrated that the electrical activities in human pancreatic ß-cells are much more complex because of the prominence of additional inward voltage-dependent ionic currents, namely sodium and T-type calcium currents (7).

The roles of low voltage-activated T-type calcium channels in insulin stimulus-secretion coupling have not been properly assessed, in part because these channels are not normally expressed in the mouse model. A slow deactivating calcium current in rat pancreatic ß-cells was reported (8, 9) and more recently the presence of T-type calcium channels was demonstrated in human ß-cells (10, 11). The low voltage activation of these calcium channels suggests that they may play a pacemaker role (12). T-type calcium currents have also been shown to trigger burst firing and increase the number and/or frequency of action potentials in neurons (12, 13, 14).

In the present study we have investigated the roles of T-type calcium currents in stimulus-secretion coupling in INS-1 cells by using conventional whole-cell as well as perforated whole-cell, patch clamp techniques. INS-1 is a stable rat ß-cell line that responds to glucose at a physiological range of concentrations (15, 16, 17). INS-1 cells express a considerable level of L-type calcium current as well as T-type calcium current. Because human ß-cells demonstrate similar calcium current profiles, the INS-1 cell line then becomes a suitable model for ß-cell electrophysiology studies. In this communication, we tested the hypothesis that the T-type calcium current participates in the generation of rhythmic activity and facilitates bursting of action potentials in INS-1 and, therefore, the T-type calcium current is an enhancer of insulin secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
RIA
The assay is based on the competition between unlabeled insulin and a fixed amount of 125I-labeled insulin for a limited number of insulin-antibody (LINCO Research Inc., St. Charles, MO) binding sites (18). With fixed amounts of antibody and radioactive ligand, the amount of radioactive ligand bound by the antibody will be inversely proportional to the concentration of added nonradioactive ligand. The separation of antibody-insulin and free insulin was achieved with centrifugation (3000 rpm, 20 min) and decantation of the supernatant. Measurement of the radioactivity in the pellet enabled the amount of labeled insulin in the bound fraction to be calculated. The concentration of unlabeled insulin in the sample was then determined by interpolation from a standard curve.

Cell Culture
INS-1 cells were kindly provided by Dr. Barbara E. Corkey (Boston University). This cell line was cultured in RPMI 1640 medium containing 10% FBS, 25 U/ml penicillin, 25 mg/ml streptomycin and 50 µM mercaptoethanol (15) in an atmosphere of 5% CO2 in air, at 37 C, for 2–5 days before recording. HIT-T15 cells were cultured under identical conditions except media was not supplemented with mercaptoethanol.

Electrical recordings and data analysis
Whole-cell voltage-clamp, current-clamp and capacitance measurements were carried out by the standard giga-seal patch clamp method (19). The whole-cell recording pipettes were made of hemo capillary tubes (Fisher Scientific, Pittsburgh, PA), pulled by a two-stage puller (P-30, Sutter Instrument, Novato, CA) and heat-polished before use. Capacitance measurement recording pipettes were coated with Sylgard (Dow Corning, Midland, MI), with the dipping-in method as previously described (20). Pipette resistance was in the range of 2–5 M{Omega} in our internal solutions. The voltage-clamp and current-clamp recordings were performed at room temperature (22–25 C), while capacitance measurements were performed at more physiological temperatures (32–35 C). An EPC-9 patch-clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) filtered at 2.9 kHz was used, and data were acquired using Pulse/PulseFit software (HEKA). Voltage-dependent currents have been corrected for linear leak and residual capacitance by using an on-line P/n subtraction paradigm.

Solutions for recording
For whole-cell voltage-clamp recordings, the extracellular solution contained (in mM): 10 CaCl2, 110 tetraethylammonium Cl (TEA-Cl), 10 CsCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 40 sucrose, 0.5 3,4-diaminopyridine, at pH 7.3. For steady-state inactivation experiments, a bath solution containing (in mM) 2 CaCl2, 120 NaCl, 15 HEPES, 30 TEA-Cl, and 1 µM tetrodotoxin (TTX) was used. The intracellular solution contained (in mM): 130 N-methyl-D-glucamine, 20 EGTA (free acid), 5 bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetate (BAPTA), 10 HEPES, 6 MgCl2, 4 Ca(OH)2, with pH adjusted to 7.4 with methanesulfonate. A concentration of 2 mM ATP was included in the intracellular solution to prevent run-down of Ca2+ currents. For current-clamp recordings, the perforated-patch pipette solution contained (in mM): 140 KCl, 10 NaCl, 2 MgCl2, 5 HEPES, and 0.24 mg/ml amphotericin B, pH 7.4. The bath solution contained 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, and 30 sucrose, pH 7.3. For capacitance measurements, the pipette solution contained 130 Cs-aspartate, 30 HEPES, 2 MgCl2, 10 CsCl, pH 7.4. The bath solution contained 110 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 20 HEPES, and 40 sucrose, pH 7.4.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two types of voltage-dependent calcium currents have been recorded in INS-1 cells as shown in a representative family of calcium currents in an INS-1 cell (Fig. 1AGo). The cell was held at -80 mV and test potentials, with duration of 200 msec, were applied from -50 to 0 mV. The traces show the presence of both low voltage-activated (T-type) and high voltage-activated (L-type) calcium currents. INS-1 cells consistently exhibited T-type calcium current.



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  		Figure 1. A, Representative family of calcium currents in an INS-1 cell. B, Plot of steady-state inactivation of calcium currents from INS-1 cells. The obtained currents at this test pulse were then normalized to the maximal current and plotted. C, The inhibitory effect of NiCl2 on T-type calcium currents in INS-1 cells (n = 4). Concentration dependent block of T-type and L-type currents were normalized to the maximal currents. Error bars represent SEM.

 
As a low voltage-activated channel, the voltages that are required for both activation and inactivation of the T-type calcium channel are more negative than that of high voltage-activated calcium channels. To determine the possibility that T-channels are in the noninactivated state at cell resting membrane potentials, we characterized the steady-state inactivation of the T-type calcium currents in INS-1 cells (Fig. 1BGo). Representative cells (n = 3) were given varied prepulse potentials (-90 to -40 mV) of 1000 msec duration then immediately stepped to a test potential of -30 mV for 100 msec in bath solution containing 2 mM CaCl2 to negate surface potential effects. Currents obtained at this test pulse were then normalized and plotted. A test potential of -30 mV was chosen because the predominant calcium current is T-type. Note that at potentials negative to -65 mV, approximately 90% of the T-type calcium channels are activated whereas at -50 mV less than 40% are activated. This sharp and less negative steady-state inactivation resembles the inactivation properties of T-type calcium current in other endocrine cells (21, 22).

It has been shown that nickel blocks T-type calcium channels in pancreatic ß-cells and in other cell types (12, 23, 24, 25). The ability of NiCl2 to selectively antagonize T-type calcium currents vs. L-type calcium currents was also examined in INS-1 cells (Fig. 1CGo) in a dose-dependent fashion. Increasing concentrations of NiCl2 were perfused into the recording chamber and the current amplitudes were measured. For testing the effects of NiCl2 on T-type calcium current, the cells were held at -80 mV with a test pulse applied at -30 mV. To test the effects of NiCl2 on L-type current, the cells were held at -40 mV with a test pulse applied at +10 mV because the calcium current at this voltage comprises mainly of L-type calcium current. Nickel chloride is a very selective blocker of T-type calcium current (IC50 {approx} 20 µM) as much higher concentrations of NiCl2 are required to block L-type calcium current.

We determined the role of T-type calcium current on INS-1 cell electrical activity, using the perforated variant of whole cell current-clamp technique. By applying preceding hyperpolarizing current pulses, we obtained slow rising and falling triangle-like potentials, or low threshold spikes (LTS) (Fig. 2Go). The amplitude and rising rate of the LTS were increased as the preceding hyperpolarization was increased. In this set of experiments, the extracellular solution contained 2 µM tetradotoxin and 10 µM of nifedipine to block voltage-sensitive sodium channels and L-type calcium channel, respectively. The effective hyperpolarizing voltages ranged from -60 mV to -80 mV, corresponding to the steady-state inactivation potentials of T-type calcium channel; therefore, this LTS is most likely the result of the deinactivation of T-type calcium channel in the cells.



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  		Figure 2. The generation of LTS in INS-1 cells. Membrane potentials were recorded in current-clamp mode using the perforated-patch variant of whole-cell patch clamp. Preceding hyperpolarizing current pulses, with increasing magnitude (indicated by arrows) were applied resulting in slow rising and falling triangle-like potentials, or LTS. The extracellular solution contained 150 mM NaCl, 2 mM CaCl2, 2 µM tetradotoxin, and 10 µM nifedipine.

 
The function of the LTS may be to provide a pacemaker-like intermediate potential that triggers action potential generated by activating sodium channels and/or L-type calcium channels. To test the effects of the LTS on action potential generation, we used a double pulse protocol (Fig. 3Go). This protocol consists of two identical depolarizing test pulses with an intervening hyperpolarizing pulse, preceding the second test pulse. The first test pulse generates a control potential, and the second test pulse generates a similar control potential plus a new component of membrane potential attributed to T-type calcium channel deinactivation produced by the hyperpolarizing pulse. To eliminate the time-dependent variation of membrane potential during the current clamp recording, the double pulse protocol was employed to ensure an internal control for every voltage recording sweep. The current was clamped in the cell such that the membrane potential remained between -50 to -60 mV. At these potentials, a large proportion of T-type calcium channels should be inactivated. For example, a 1 pA test pulse is not enough to generate an action potential in pulse one, but when the cell is hyperpolarized, an equivalent 1 pA test pulse elicited an action potential in pulse two (Fig. 3Go). A hyperpolarization to a membrane potential near -80 mV should deactivate all the T-type calcium channels (Fig. 1BGo).



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  		Figure 3. Representative trace of the double pulse protocol in an INS-1 cell. The current was clamped such that the membrane potential remained between -55 and -60 mV. Both test pulses were of 1 pA magnitude.

 
The effect of the LTS on action potentials was examined directly with the double pulse protocol. This time a test pulse was administered sufficient to generate at least one action potential in the first pulse (Fig. 4AGo). The number of action potentials after hyperpolarization is greater in the second test pulse. The more depolarized membrane potential due to the LTS may facilitate the initiation of action potentials.



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  		Figure 4. The LTS increases the frequency of action potentials. A, A current (2 pA) was injected sufficient to generate at least one action potential in the test pulse one. B, Comparison of latencies of onset of action potentials between test pulse one and test pulse two with the double-pulse protocol (8 cells, n = 40 traces). Error bars represent SEM. The asterisk represents P <= 0.01 with Student’s t test.

 
It was also observed that the time it takes to generate the first action potential in the first test pulse was longer than that in the second test pulse. This time was defined as latency of onset of action potential and pooled data (n = 40) from eight cells determined that the latency was greater in test pulse one than test pulse two (Fig. 4BGo). These results demonstrate that T-type calcium current decreases the latency of onset of action potential and increases the number of action potentials.

To further establish that T-type calcium current enhances cell excitability, we administered 100 µM NiCl2 to effectively block T-type calcium channels. In contrast to the control experiments, NiCl2 caused a delay in the onset of an action potential in the second test pulse (Fig. 5AGo) and a decrease in number of action potentials during the second test pulse. The averaged difference between latencies of action potential onset in test pulse one and two, defined as {Delta} latency, was taken under conditions with and without application of NiCl2. NiCl2 caused an averaged 100 msec greater latency of action potential onset in test pulse two, indicated by a negative {Delta} latency (Fig. 5BGo).



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  		Figure 5. A, Representative trace where 100 µM nickel is applied to the bath. NiCl2 caused a delay in action potential onset in the second pulse. B, NiCl2 caused a longer latency of action potential onset in test pulse as determined by a negative {Delta}latency (five cells, n = 20 traces). Error bars are SEM The asterisk represents P <= 0.01 with Student’s t test.

 
The time it takes to charge the membrane capacitor after hyperpolarization may contribute to this negative {Delta} latency. Theoretically, the time course of the membrane capacitance discharge can be calculated by the following equation (26): E = E0e-t/RC, where the membrane potential (E) at any moment is inversely proportional to the time (t) of discharging with the time course ({tau} = RC). For the INS-1 cells, the membrane resistance (Rm) is approximately 3 giga-Ohm; the membrane capacitance (Cm) is averaged 20–25 pF, therefore the {tau} is estimated to be 60 msec in our experimental conditions. If we assume that the T-type calcium channel activation threshold is approximately -55 mV, the {Delta} latency for charging the membrane from first pulse (-60 mV) and the second pulse (-80 mV) can be estimated. The estimated {Delta} latency is between 96 and 120 msec, which fits roughly with our experimental data. The effect of T-type calcium current on latency (Fig. 4BGo) in our model then, is an underestimate value based on the inherent nature of membrane capacitance.

It has been demonstrated that a single action potential is sufficient to evoke a transient rise in [Ca2+]i, in mouse ß-cells (27). Higher frequency of action potentials has been demonstrated to cause a greater and longer rise in [Ca2+]i and increases in ß-cell exocytosis (28). In our system, we measured changes in membrane capacitance in response to two different stimulation frequencies, mimicking the electrical activity in these cells as an index of exocytosis. This uses the fact that exocytosis involves fusion of the secretory granules with the plasma membrane, resulting in an increase in the cell surface area. Cells were held at -60 mV and then repetitive depolarizations to 0 mV, each a duration of 10 msec were applied for 3 sec. The low frequency stimulation consisted of 10 pulses/sec, and the high frequency stimulation consisted of 20 pulses/sec and both stimulations were applied to the same cell (Fig. 6AGo). High frequency stimulation caused a greater increase in membrane capacitance compared with low frequency stimulation, suggesting that more secretory vesicles fused to the membrane with high frequency stimulation. Pooled data from 12 cells of approximate equal size demonstrated that high frequency stimulation induces a greater change in membrane capacitance than low frequency stimulation (Fig. 6BGo). This should be expected because high frequency stimulation will lead to higher cytosolic calcium concentrations and will likely facilitate more secretory vesicles fusing to the membrane.



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  		Figure 6. A, Representative trace of changes in membrane capacitance (one measurement of cell capacitance for every 5 sec) in an INS-1 cell when two different stimulations were applied to the same cell. Cells were held at -60 mV and then repetitive depolarizations to 0 mV, each a duration of 10 msec were applied for every 3 sec. The low frequency stimulation consisted of 10 pulses/sec, and the high frequency stimulation consisted of 20 pulses/sec. B, A high frequency stimulation induces a greater change in membrane capacitance than a low frequency stimulation (n = 12 cells). Data were collected with low frequency pulse applied first and also in the reverse order.Error bars are SEM. The asterisk represents P <= 0.01 with Student’s t test.

 
To directly demonstrate the role of T-type calcium current in glucose-stimulated insulin secretion, we incubated INS-1 cells with 11.1 mM glucose and 0, 3, 10, or 30 µM NiCl2 and measured the insulin release using a standard RIA (Fig. 7AGo). NiCl2 reduced insulin secretion in a dose-dependent manner. On the other hand, clonal hamster-secreting cells (HIT-T15), which do not consistently exhibit T-type calcium current (29), were not affected by 30 µM NiCl2 (Fig. 7BGo). This suggests that T-type calcium current in INS-1 cells significantly contributes to glucose-stimulated insulin secretion.



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  		Figure 7. A, The dose-dependent effect of nickel on insulin secretion in INS-1 cells. Cells were placed in a medium containing 11.1 mM glucose for 1 h with and without NiCl2. B, The effect of 30 µM nickel on HIT-15T cells. Insulin release was determined by a standard RIA. Numbers above bars represent the numbers of independent experiments performed. Error bars represent SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have determined a role for T-type calcium channels in stimulus-secretion coupling. The double pulse protocol we employed demonstrated the effects of T-type calcium current in altering the electrical activity of INS-1 cells. We believe that T-type calcium currents have a permissive role and contributes to the depolarization of the membrane, allowing for activation of the L-type calcium channels. The true contribution of T-type calcium current may be seen in the intermittent repolarizing phases of the glucose-induced electrical activity cycle. The low threshold spike may give rise to the plateau potential. Ultimately, T-type calcium channels may play a pacemaker role especially at postabsorptive glucose levels.

The resting membrane potential of INS-1 cells has been reported to be between -72 and -80 mV (17, 30) and 15 mM glucose depolarizes the cell to approximately -56 mV. Based upon the voltage-dependent inactivation curve obtained for T-type calcium channels in INS-1 cells, we can conclude that T-type calcium currents are a significant contributor to the underlying electrical activities of INS-1 cells in the physiological range of glucose concentrations. Indeed, the voltage-dependent inactivation profile for the T-type calcium current in INS-1 cells is very similar to other endocrine cell types, where a very large proportion of the channels are still available for conducting current at voltages negative to -55 mV.

T-type calcium current mediated LTS has been demonstrated to play a critical role in regulating the rhythmicity in the thalamic neuronal network (31). In that system, each action potential is followed by a marked after-hyperpolarization (A-HP), which provides a potential required for deinactivation of T-type calcium channels. In INS-1 cells, however, no such A-HP is seen during the repetitive firing. The function of T-type calcium current may be to provide a small but sustained depolarization which facilitates higher firing frequency.

It has been demonstrated that 100 µM nickel chloride reduces the frequency of action potentials in human ß-cells (7). In our experiments, we observed that low concentrations of NiCl2 reduced the number of action potentials in INS-1 cells. The underlying mechanism responsible for this effect is unclear. It is possible that the T-type calcium current in ß-cells undergoes voltage-dependent potentiation as is seen in other cell types (32, 33). Further T-type calcium current recruitment upon strong depolarization may facilitate the firing of subsequent action potentials and thus increase cell excitability.

We have only addressed the contribution of T-type calcium current in terms of electrical activity. The amount of calcium that enters via T-type calcium channels will also be significant for secretion, particularly to vesicles that are in close proximity to the membrane. For example, a calcitonin-secreting cell line has been shown to only possess T-type calcium channels and not L-type calcium channels (21). The T-type calcium channels in this cell type are the major Ca2+ signaling channels for secretion. It is conceivable that T-type calcium channels facilitates much of the calcium entry (unpublished observations) at lower glucose concentrations.

It has been shown that T-type calcium current are important for neuronal and cardiac electrical activities (34, 35, 36, 37, 38, 39, 40). The functions of T-type calcium channels in endocrine cell types have also been emerging (21, 22, 41, 42). The functions of T-type calcium channels in pancreatic ß-cells have largely been overlooked due in part to the choice of ß-cell models. It has been well documented that the mouse model does not exhibit T-type calcium current. The INS-1 cell line responds normally to glucose, and the presence of T-type calcium channels may indicate that this model is suitable for electrophysiology studies since T-type calcium channels are prominent in human pancreatic ß-cells.


   Footnotes
 
1 This work was supported by The American Diabetes Association and American Heart Association. Back

Received February 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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