The Journal of Neurophysiology Vol. 78 No. 2 August 1997, pp. 1161-1169
Copyright ©1997 by the American Physiological Society

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Calcineurin Modulates G Protein-Mediated Inhibition of N-Type Calcium Channels in Rat Sympathetic Neurons

Yu Zhu and Jerrel L. Yakel

Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Zhu, Yu and Jerrel L. Yakel. Calcineurin modulates G protein-mediated inhibition of N-type calcium channels in rat sympathetic neurons. J. Neurophysiol. 78: 1161-1169, 1997. The modulation of N-type voltage-gated calcium (Ca²⁺) channels by G protein-coupled receptors was investigated in sympathetic neurons of the male rat major pelvic ganglion (MPG) with the use of whole cell patch-clamp recording techniques from acutely dissociated neurons. By inhibiting calcineurin, a Ca²⁺/calmodulin-regulatedprotein phosphatase, the ₂ noradrenergic and somatostatin receptor-induced inhibition of these N-type Ca²⁺ channels was greatly reduced. Both of these receptor pathways utilize a pertussis toxin-sensitive G protein (G_PTX). The guanosine 5-o-(3-thiotriphosphate) (GTPS)-induced decrease in the amplitude and activation kinetics of Ca²⁺ currents, an effect that was similar to the activation of G_PTX-coupled receptors, also was reduced by the inhibition of calcineurin. Calcineurin does not regulate the muscarinic receptor-induced inhibition of the N-type Ca²⁺ channels, a pathway that utilizes a different G protein in the MPG neurons. Thus calcineurin appears to selectively regulate the coupling between the G_PTX and the Ca²⁺ channel.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Voltage-gated calcium (Ca²⁺) channels are modulated by neurotransmitters acting through various G protein-coupled receptor pathways. In sympathetic neurons, at least five different G protein pathways for modulating the N-type Ca²⁺ channels are known (see review by Hille 1994). One of these is a membrane-delimited pathway that involves a pertussis toxin (PTX)-sensitive heterotrimeric G protein (G_PTX) (Hille 1994) and is regulated by protein phosphorylation. In rat sympathetic neurons, the activation of protein kinase C (PKC) enhanced the amplitude and attenuated the inhibition of N-type Ca²⁺ channels by the activation of G_PTX-coupled receptors (Swartz 1993; Zhu and Ikeda 1994; Zhu and Yakel 1997). The action of PKC was potentiated by the inhibition of okadaic acid-sensitive phosphatases.

Calcineurin is a Ca²⁺/calmodulin-regulated protein phosphatase that has been shown to negatively regulate various ligand- and voltage-gated ion channels as well as neurotransmitter release (see review by Yakel 1997). For example, calcineurin enhances the inactivation of voltage-gated Ca²⁺ channels in molluscan neurons (Chad and Eckert 1986), and modulates glutamatergic synaptic transmission via a presynaptic mechanism of action in rat cortex (Nichols et al. 1994; Victor et al. 1995). The molecular details concerning how calcineurin regulates neurotransmitter release are currently unknown.

Here we report that by inhibiting calcineurin, the G_PTX-coupled receptor-mediated inhibition of the N-type Ca²⁺ channels in rat sympathetic neurons from the male rat major pelvic ganglion (MPG) was greatly reduced. The muscarinic receptor-induced inhibition of these channels, which does not involve the activation of a G_PTX-coupled and membrane-delimited pathway in MPG neurons (Zhu and Yakel 1997) as it does in superior cervical ganglion sympathetic neurons (Hille 1994), was unaffected by the inhibition of calcineurin. These data suggest that calcineurin may play an important role in regulating neurotransmitter release in sympathetic neurons by reducing presynaptic inhibition, and may explain why the immunosuppressant drug cyclosporin, a potent inhibitor of calcineurin, leads to neurotoxicity and neurogenic hypertension.

	METHODS

Abstract Introduction Methods Results Discussion References

Single neurons of the MPG were isolated from 12- to 16-wk-old male Wistar rats with the use of an enzymatic method as previously described (Zhu et al. 1995). Briefly, animals were killed with CO₂ and the MPGs were dissected, cleaned of connective tissue, and treated with enzymes. Single neurons were dispersed and then plated on glass coverslips coated with poly-D-lysine and short-term (<24 h) cultured in a humidified atmosphere containing 5% CO₂ in air at 37°C.

The whole cell patch-clamp recording technique was used to record calcium (Ca²⁺) currents (I_Ca) from single MPG neurons. Patch pipettes (resistances 0.8-2.5 M) were made from Corning 7052 glass; the series resistance (usually <5 M) and membrane capacitance were compensated (>80%) with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA). Voltage protocols and data analysis (including curve-fitting procedure) were performed with the use of the pCLAMP6 software (Axon Instruments). Traces were digitized and filtered at 5 kHz. Unless otherwise indicated, depolarizations were given every 10 s from a holding potential of 80 mV to +10 mV to elicit maximum currents. The amplitudes of I_Ca were determined isochronally 10 ms after depolarization. Data are expressed as means ± SE where appropriate. Student's t-test was used to determine the significance in differences. P < 0.05 was considered significant.

The pipette solution contained (in mM) 120 NMG-CH₃SO₃,20 tetraethylammonium (TEA) chloride, 11 ethylene glycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA), 1CaCl₂, 10 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 4 MgATP, 0.3 guanosine 5-triphosphate (GTP), and 14 creatine phosphate, pH 7.2. The external solution contained (in mM) 145 NMG-CH₃SO₃, 10 CaCl₂, 10 HEPES, 15 glucose, and 0.0001 tetrodotoxin, pH 7.4. All experiments were performed at room temperature (21-24°C). Drugs were applied to neurons through quartz tubing placed 300 µm away from the neuron; the application of drug was controlled by computer-driven valves (General Valve, Fairfield, NJ). Agonists were typically applied 8-10 min after establishment of the recording. Compounds used in this study were obtained from the following sources: UK14304, somatostatin-28 (SST-28), okadaic acid, muscarine, and PTX were from RBI (Natick, MA); calcineurin autoinhibitory fragment (CIF) was obtained from Bachem (Torrance, CA); cyclosporin A and cyclophilin were generous gifts from Sandoz (Basel); CaN₄₂₀ was a generous gift from Dr. Brian Perrino; and all other drugs were obtained from Sigma.

	RESULTS

Abstract Introduction Methods Results Discussion References

Calcineurin inhibition reduced ₂ receptor-induced I_Ca inhibition

In sympathetic adult male rat MPG neurons, whole cell I_Ca composed mostly of N-type Ca²⁺ channels (~70%) (Zhu et al. 1995) were elicited by depolarization from 80 to +10 mV; peak amplitudes averaged 4.3 ± 1.7 (SE) nA (40 cells, Fig. 1). The bath application of the selective ₂ noradrenergic receptor agonist UK14304 (5 µM) reduced the amplitude of I_Ca by 53 ± 4% (30 cells) and slowed the activation kinetics (Fig. 1A). This inhibition of I_Ca was voltage dependent in that it was mostly relieved by a large preceding depolarization to +80 mV (Fig. 1B). Under control conditions in the absence of agonist, the depolarization to +80 mV increased the amplitude of I_Ca (the post current) by 37 ± 3% (46 cells); this process is referred to as facilitation. After this +80-mV depolarization, UK14304 inhibited the post current by only 18 ± 3% (19 cells, Fig. 1A). Overnight treatment (12-14 h) with PTX (500 ng/ml) reduced the UK14304-induced inhibition of I_Ca by >80% (data not shown). Thus UK14304 appears to preferentially activate the PTX-sensitive pathway (Hille 1994).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Calcineurin inhibition blocks UK14304-induced inhibition of Ca2+ currents (ICa). A: superimposed ICa traces recorded with double pulse voltage protocol (top) in absence and presence of 5 µM UK14304. B: time course of UK14304-induced ICa inhibition. Current amplitude was determined isochronally 10 ms after each pulse for both pre and post currents and plotted as function of time. C: same as A, except neuron is dialyzed with calcineurin autoinhibitory fragment (CIF). D: summary of pre current inhibition induced by UK14304. Numbers in parentheses: number of neurons examined. Double asterisk: P < 0.01. CysA, cyclosporin A; cyph, cyclophilin.

Inhibiting calcineurin by dialyzing neurons with the calcineurin autoinhibitory peptide fragment (CIF, 100 µM) (Hashimoto et al. 1990) or cyclosporin A and cyclophilin (both at 500 nM) significantly reduced the UK14304-induced inhibition of I_Ca to 29 ± 3% (14 cells, Fig. 1C) and 25 ± 6% (6 cells, Fig. 1D), respectively. Inhibiting protein phosphatases 1 and 2A by dialyzing neurons with okadaic acid (1 µM) was without effect (Fig. 1D).

Dialysis with CIF did not significantly affect either the amplitude, the kinetics, or the extent of facilitation of I_Ca. To determine whether dialysis with CIF altered the amplitude of I_Ca, we calculated the percent change in the amplitude at the end of the recording versus at the beginning (this was defined as the amplitude ratio), and compared this value with that obtained for dialysis with the control solution on the same day. The rate of I_Ca activation was determined by fitting the rising phase of I_Ca to single-exponential curve fits, and the rate of inactivation was determined by measuring the amount of inactivation during the depolarizing pulse. These various values for CIF-dialyzed cells versus controls for the amplitude ratio, time constant of activation, percent inactivation, and extent of facilitation were, respectively, 104 ± 6% (17 cells) versus 105 ± 8% (15 cells), 1.2 ± 0.1 ms versus 1.4 ± 0.1 ms, 5.6 ± 1% versus 2.7 ± 1%, and 42 ± 7% versus 32 ± 3%.

Cyclosporin A/cyclophilin dialysis also did not significantly alter either the amplitude [the amplitude ratio was 94 ± 7% (6 cells) for cyclosporin A/cyclophilin-dialyzed cells vs. 107 ± 7 (10 cells) for controls] or the kinetics of activation of I_Ca (the activation time constant values were 1.1 ± 0.2 ms vs. 1.4 ± 0.1 ms, respectively). However, for dialysis with cyclosporin A/cyclophilin, the percent inactivation of I_Ca increased to 13 ± 1% from a control value of 2.1 ± 1%, and the extent of facilitation decreased to 6.5 ± 9% from a control value of 44 ± 7%. The significance of these differences between dialysis with CIF and cyclosporin A/cyclophilin on the properties of I_Ca is presently unclear.

Dialysis with a truncated and preactivated form of calcineurin (CaN₄₂₀, 112 nM) (Perrino et al. 1995), which no longer requires Ca²⁺ or calmodulin for full activity, did not significantly alter most properties of I_Ca (i.e., amplitude, kinetics of activation, or extent of facilitation) or the UK14304-induced inhibition of I_Ca (data not shown). The amplitude ratio for CaN₄₂₀ dialysis was 91 ± 9% (12 cells) versus a control value of 93 ± 7% (8 cells), the activation time constant was 1.6 ± 0.1 ms versus 1.6 ± 0.2 ms (control), and the extent of facilitation was 35 ± 7% versus 39 ± 8% (control). However, dialysis with CaN₄₂₀ significantly increased the percent inactivation of I_Ca to 11 ± 2% from a control value of 2.5 ± 1%.

Somatostatin- but not muscarinic-induced inhibition of I_Ca is also modulated by calcineurin

Somatostatin has previously been shown to utilize the same G_PTX-coupled receptor pathway as the ₂ receptors to inhibit the N-type Ca²⁺ channels in sympathetic neurons (Ikeda and Schofield 1989). SST-28 (100 nM) inhibited I_Ca by 45 ± 5% (5 cells) and slowed the activation kinetics (Fig. 2A). When the neurons were dialyzed with CIF, SST-28 inhibited I_Ca by only 25 ± 8% (5 cells, Fig. 2B).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Calcineurin inhibition blocks somatostatin-28 (SST-28)- but not muscarinic-inducedinhibition of ICa. A-D: superimposed ICa traces recorded with double pulse voltage protocol in absence and presence, respectively, of 100 nM SST-28 (A and B) or 10 µM muscarine (C and D) in neurons dialyzed either with control (A and C) or CIF (B and D). E: summary of pre current inhibition induced by SST-28 and muscarine. Asterisk, P < 0.05.

In rat sympathetic MPG neurons, muscarinic receptor activation does not activate a G_PTX-coupled and membrane-delimited pathway as it does in rat superior cervical ganglion (Zhu and Yakel 1997), but instead activates only a PTX-insensitive G protein pathway that is thought to involve a cytoplasmic messenger (i.e., not membrane delimited) (Hille 1994). Muscarine (10 µM) reversibly inhibited I_Ca by 29 ± 3% (14 cells) without slowing the activation kinetics (Fig. 2C). Dialysis with either CIF (Fig. 2D) or cyclosporin A/cyclophilin (Fig. 2E) did not significantly alter the muscarine-induced inhibition of I_Ca. This suggests that the modulatory effect of calcineurin may be selective for the particular G protein pathway utilized by both UK14304 and SST-28 (i.e., a G_PTX pathway) but not by muscarine.

Calcineurin modulates the G protein coupling to the Ca²⁺ channels

The site of calcineurin regulation is likely to be at the level of the G protein or downstream. To test this, neurons were dialyzed with guanosine 5-o-(3-thiotriphosphate) (GTPS; 0.5 mM), which decreased the amplitude of I_Ca and slowed the activation kinetics in a manner similar to UK14304 and SST-28 (Fig. 3A). The inhibition by GTPS was voltage dependent in that a large preceding depolarization to +80 mV relieved most of this inhibition; GTPS dialysis greatly increased the extent of facilitation to 138 ± 22% (4 cells, after 9 min of dialysis) as compared with neurons without GTPS dialysis (37%, Fig. 3A).

View larger version (13K): [in this window] [in a new window]   FIG. 3. Dialysis with guanosine 5-o-(3-thiotriphosphate) (GTPS) inhibits ICa in CIF-dependent manner. A and B: ICa traces recorded with double pulse voltage protocol in neurons dialyzed either with GTPS (A) or CIF + GTPS (B). C: summary of extent of facilitation in neurons dialyzed either with GTPS or CIF + GTPS. Double asterisk: P < 0.01.

Dialysis with CIF (in addition to GTPS) significantly reduced the extent of facilitation (61 ± 14%, 4 cells, Fig. 3C) as compared with GTPS alone, as well as reversing the GTPS-induced kinetic slowing of I_Ca (Fig. 3B). This provides further evidence that calcineurin is acting on the G protein itself or downstream of the G protein.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We have shown that in rat sympathetic neurons from MPG, the inhibition of N-type Ca²⁺ channels by the activation of ₂ noradrenergic and somatostatin receptors, which both utilize a G_PTX-coupled receptor pathway, is greatly reduced by inhibiting calcineurin. Calcineurin does not regulate the muscarinic receptor-induced inhibition of the N-type Ca²⁺ channels, a pathway that utilizes a different G protein. The GTPS-induced decrease in the amplitude and activation kinetics of I_Ca, effects that were similar to the activation of G_PTX-coupled receptors, also was reduced by the inhibition of calcineurin. Thus calcineurin appears to regulate the coupling between the G_PTX and the Ca²⁺ channel.

The inhibition of calcineurin with CIF did not have any significant affect on either the amplitude, the kinetics, or the extent of facilitation of I_Ca. However, the inhibition of calcineurin with cyclosporin A/cyclophilin, which also did not significantly affect either the amplitude or the kinetics of activation of I_Ca, did significantly increase the rate of inactivation as well as decrease the extent of facilitation of I_Ca. The different results obtained between CIF and cyclosporin A/cyclophilin could be related to their different mechanisms of inhibiting calcineurin (see review by Yakel 1997). From these data we propose that there is a calcineurin-regulated phosphorylation site on either on the G_PTX, the Ca²⁺ channel, or any putative intermediate molecule. When this site is dephosphorylated by calcineurin, the G_PTX-coupled receptor pathway inhibiting I_Ca can proceed. If this site is phosphorylated, however (e.g., by the inhibition of calcineurin), the ability of this G_PTX pathway to inhibit I_Ca is greatly attenuated.

Activating PKC, similar to inhibiting calcineurin, greatly attenuated the G_PTX-coupled receptor-induced inhibition of the N-type Ca²⁺ channels in rat sympathetic neurons (Swartz 1993; Zhu and Ikeda 1994; Zhu and Yakel 1997). This suggests that the kinase that phosphorylates the calcineurin-regulated site could be PKC. In addition, PKC activation also significantly increased the rate of inactivation and decreased the extent of facilitation of I_Ca, effects that were observed with cyclosporin A/cyclophilin but not with CIF. PKC activation also greatly increased the amplitude of I_Ca (>40%), an effect not observed with either CIF or cyclosporin A/cyclophilin. Therefore whether or not PKC is in fact the kinase that phosphorylates the calcineurin-regulated site is currently unknown. Recently Zamponi et al. (1997) have shown that PKC phosphorylates the ₁ subunit of the N-type Ca²⁺ channels (_1B) in the I-II cytoplasmic linker domain near one of the G subunit binding sites, and that the PKC-dependent phosphorylation of this site antagonizes the G-induced inhibition of expressed N-type Ca²⁺ channels. Whether or not this PKC site is dephosphorylated by calcineurin remains to be determined.

In rat sympathetic neurons, norepinephrine inhibits its own release by inhibiting N-type voltage-gated Ca²⁺ channels through activation of ₂ receptors (Hirning et al. 1988). Interfering with this presynaptic negative feedback inhibitory process (e.g., by inhibiting calcineurin) could lead to hyperexcitability of the sympathetic neurons and contribute to pathological conditions such as hypertension. In the brain, many presynaptic G protein-coupled receptors also inhibit neurotransmitter release. Should calcineurin be modulating the release of neurotransmitter via a mechanism similar to that described here (i.e., the G_PTX-induced inhibition of I_Ca), then the inhibition of calcineurin could have profound effects on the regulation of neurotransmitter release (see review by Yakel 1997). It has been shown that calcineurin negatively regulates glutamatergic transmitter release in the rat brain, although the mechanism whereby calcineurin exerts this effect is unknown (Nichols et al. 1994; Sihra et al. 1995; Victor et al. 1995). Calcineurin inhibition with the immunosuppressant drug cyclosporin leads to neurotoxicity (Hughes 1990) and neurogenic hypertension (Lyson et al. 1993). The possibility exists that this neurotoxic effect of cyclosporin could be due to relief of presynaptic inhibition by a mechanism involving regulation of voltage-gated Ca²⁺ channels leading to the enhanced release of glutamate.

	ACKNOWLEDGEMENTS

  We thank S. Jones and D. Armstrong for critical reading of this manuscript, B. Perrino for providing us with CaN₄₂₀, and H. Boddeke for providing us with cyclosporin A and cyclophilin.

	FOOTNOTES

  Address for reprint requests: J. L. Yakel, National Institute of Environmental Health Sciences, F2-08, PO Box 12233, 104 T. W. Alexander Drive, Research Triangle Park, NC 27709.

  Received 22 January 1997; accepted in final form 30 April 1997.

	REFERENCES

Abstract Introduction Methods Results Discussion References

• CHAD, J. E., ECKERT, R. An enzymatic mechanism for calcium current inactivation in dialysed Helix neurones. J. Physiol. (Lond.) 378: 31-51, 1986.[Abstract/Free Full Text]
• HASHIMOTO, Y., PERRINO, B. A., SODERLING, T. R. Identification of an autoinhibitory domain in calcineurin. J. Biol. Chem. 265: 1924-1927, 1990.[Abstract/Free Full Text]
• HILLE, B. Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci. 17: 531-536, 1994.[Medline]
• HIRNING, L. D., FOX, A. P., MCCLESKEY, E. W., OLIVERA, B. M., THAYER, S. A., MILLER, R. J., TSIEN, R. W. Dominant role of N-type Ca²⁺ channels in evoked release of norepinephrine from sympathetic neurons. Science 239: 57-61, 1988.[Abstract/Free Full Text]
• HUGHES, R. L. Cyclosporine-related central nervous system toxicity in cardiac transplantation. N. Engl. J. Med. 323: 420-421, 1990.[Medline]
• IKEDA, S. R., SCHOFIELD, G. G. Somatostatin blocks a calcium current in rat sympathetic ganglion neurones. J. Physiol. (Lond.) 409: 221-240, 1989.[Abstract/Free Full Text]
• LYSON, T., ERMEL, L. D., BELSHAW, P. J., ALBERG, D. G., SCHREIBER, S. L., VICTOR, R. G. Cyclosporine- and Fk506-induced sympathetic activation correlates with calcineurin-mediated inhibition of T-cell signaling. Circ. Res. 73: 596-602, 1993.[Abstract/Free Full Text]
• NICHOLS, R. A., SUPLICK, G. R., BROWN, J. M. Calcineurin-mediated protein dephosphorylation in brain nerve terminals regulates the release of glutamate. J. Biol. Chem. 269: 23817-23823, 1994.[Abstract/Free Full Text]
• PERRINO, B. A., NG, L. Y., SODERLING, T. R. Calcium regulation of calcineurin phosphatase activity by its B subunit and calmodulin. J. Biol. Chem. 270: 340-346, 1995.[Abstract/Free Full Text]
• SIHRA, T. S., NAIRN, A. C., KLOPPENBURG, P., LIN, Z., POUZAT, C. A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release. Biochem. Biophys. Res. Commun. 212: 609-616, 1995.[Medline]
• SWARTZ, K. J. Modulation of Ca²⁺ channels by protein kinase C in rat central and peripheral neurons: disruption of G protein-mediated inhibition. Neuron 11: 305-320, 1993.[Medline]
• VICTOR, R. G., THOMAS, G. D., MARBAN, E., O'ROURKE, B. Presynaptic modulation of cortical synaptic activity by calcineurin. Proc. Natl. Acad. Sci. USA 92: 6269-6273, 1995.[Abstract/Free Full Text]
• YAKEL, J. L. Calcineurin regulation of synaptic function: from ion channels to transmitter release and gene transcription. Trends Pharmacol. Sci. 18: 124-134, 1997.[Medline]
• ZAMPONI, G. W., BOURINET, E., NELSON, D., NARGEOT, J., SNUTCH, T. P. Crosstalk between G proteins and protein kinase C mediated by the calcium channel ₁ subunit. Nature 385: 442-446, 1997.[Medline]
• ZHU, Y., IKEDA, S. R. Modulation of Ca²⁺-channel currents by protein kinase C in adult rat sympathetic neurons. J. Neurophysiol. 72: 1549-1560, 1994.[Abstract/Free Full Text]
• ZHU, Y., YAKEL, J. L. Modulation of Ca²⁺ currents by various G protein-coupled receptors in sympathetic neurons of male rat pelvic ganglia. J. Neurophysiol. 78: 780-789, 1997.[Abstract/Free Full Text]
• ZHU, Y., ZBORAN, E. L., IKEDA, S. R. Phenotype-specific expression of T-type calcium channels in neurons of the major pelvic ganglion of the adult male rat. J. Physiol. (Lond.) 489: 363-375, 1995.[Abstract/Free Full Text]

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