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 alpha 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 (GPTX). The guanosine 5'-o-(3-thiotriphosphate) (GTPgamma S)-induced decrease in the amplitude and activation kinetics of Ca2+ currents, an effect that was similar to the activation of GPTX-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 GPTX 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 (GPTX) (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 GPTX-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 GPTX-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 GPTX-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 (ICa) from single MPG neurons. Patch pipettes (resistances 0.8-2.5 MOmega ) were made from Corning 7052 glass; the series resistance (usually <5 MOmega ) 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 ICa 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(beta -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 alpha 2 receptor-induced ICa inhibition

In sympathetic adult male rat MPG neurons, whole cell ICa 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 alpha 2 noradrenergic receptor agonist UK14304 (5 µM) reduced the amplitude of ICa by 53 ± 4% (30 cells) and slowed the activation kinetics (Fig. 1A). This inhibition of ICa 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 ICa (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 ICa by >80% (data not shown). Thus UK14304 appears to preferentially activate the PTX-sensitive pathway (Hille 1994).


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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 ICa 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 ICa. To determine whether dialysis with CIF altered the amplitude of ICa, 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 ICa activation was determined by fitting the rising phase of ICa 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 ICa (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 ICa 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 ICa 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 ICa (i.e., amplitude, kinetics of activation, or extent of facilitation) or the UK14304-induced inhibition of ICa (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 ICa to 11 ± 2% from a control value of 2.5 ± 1%.

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

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


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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 GPTX-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 ICa 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 ICa. 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 GPTX 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) (GTPgamma S; 0.5 mM), which decreased the amplitude of ICa and slowed the activation kinetics in a manner similar to UK14304 and SST-28 (Fig. 3A). The inhibition by GTPgamma S was voltage dependent in that a large preceding depolarization to +80 mV relieved most of this inhibition; GTPgamma S dialysis greatly increased the extent of facilitation to 138 ± 22% (4 cells, after 9 min of dialysis) as compared with neurons without GTPgamma S dialysis (37%, Fig. 3A).


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FIG. 3. Dialysis with guanosine 5'-o-(3-thiotriphosphate) (GTPgamma S) inhibits ICa in CIF-dependent manner. A and B: ICa traces recorded with double pulse voltage protocol in neurons dialyzed either with GTPgamma S (A) or CIF + GTPgamma S (B). C: summary of extent of facilitation in neurons dialyzed either with GTPgamma S or CIF + GTPgamma S. Double asterisk: P < 0.01.

Dialysis with CIF (in addition to GTPgamma S) significantly reduced the extent of facilitation (61 ± 14%, 4 cells, Fig. 3C) as compared with GTPgamma S alone, as well as reversing the GTPgamma S-induced kinetic slowing of ICa (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 alpha 2 noradrenergic and somatostatin receptors, which both utilize a GPTX-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 GTPgamma S-induced decrease in the amplitude and activation kinetics of ICa, effects that were similar to the activation of GPTX-coupled receptors, also was reduced by the inhibition of calcineurin. Thus calcineurin appears to regulate the coupling between the GPTX 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 ICa. However, the inhibition of calcineurin with cyclosporin A/cyclophilin, which also did not significantly affect either the amplitude or the kinetics of activation of ICa, did significantly increase the rate of inactivation as well as decrease the extent of facilitation of ICa. 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 GPTX, the Ca2+ channel, or any putative intermediate molecule. When this site is dephosphorylated by calcineurin, the GPTX-coupled receptor pathway inhibiting ICa can proceed. If this site is phosphorylated, however (e.g., by the inhibition of calcineurin), the ability of this GPTX pathway to inhibit ICa is greatly attenuated.

Activating PKC, similar to inhibiting calcineurin, greatly attenuated the GPTX-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 ICa, effects that were observed with cyclosporin A/cyclophilin but not with CIF. PKC activation also greatly increased the amplitude of ICa (>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 alpha 1 subunit of the N-type Ca2+ channels (alpha 1B) in the I-II cytoplasmic linker domain near one of the Gbeta gamma subunit binding sites, and that the PKC-dependent phosphorylation of this site antagonizes the Gbeta gamma -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 alpha 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 GPTX-induced inhibition of ICa), 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

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society