Correspondence to: Ann R. Rittenhouse, Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655. Fax:508-856-5997 E-mail:ann.rittenhouse{at}umassmed.edu.
Released online: 14 February 2000
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Abstract |
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N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming 1B subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-proteinmediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-
-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-
-Sinduced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-proteinmediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-proteinmediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potentialdependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-ß-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.
Key Words: G-protein, inactivation, L-type calcium channel, phorbol ester, phosphorylation
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INTRODUCTION |
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N-type voltage-gated calcium channel activity in rat superior cervical ganglion (SCG)1 neurons is modulated by a variety of mechanisms. G-proteincoupled, membrane-delimited pathways have been shown to decrease whole-cell barium current amplitude by selective inhibition of N-type current (
The rate of reinhibition of currents facilitated by prepulse application is related to the concentration of activated G-proteins. This was demonstrated with increasing concentrations of GTP--S (
(
Additional modulation of N-type calcium channel activity exists via phosphorylation by protein kinase C. Activation of PKC by phorbol esters leads to an enhancement of whole-cell current amplitude in sympathetic neurons, as well as an attenuation of subsequent transmitter-induced, membrane-delimited inhibition (1B subunit of the channel has been proposed as one possible site for convergence of these pathways (
Previous data (
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METHODS |
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Cell Preparation and Culture
SCG were dissected from 14 d old Sprague-Dawley rats (Charles River Laboratories) and dissociated by trituration (
Electrophysiology
Barium currents were recorded using the whole-cell configuration of an Axopatch 200B patch-clamp amplifier (Axon Instruments). Except where noted, voltage steps were applied every 4 s from a holding potential of -90 mV. When used, prepulses preceded the test pulse by 5 ms. Currents were recorded at 2024°C, passed through a four-pole low-pass Bessel filter at 1 kHz, and then digitized at 5 kHz with a 1401 plus interface (Cambridge Electronic Design), except the activation data, which were filtered at 5 kHz and digitized at 20 kHz. Data were collected using the Patch software suite, version 6.3 (Cambridge Electronic Design), and stored on a personal computer for off-line analysis. Capacitive currents were corrected online, and leak currents were subtracted using a scaled-up hyperpolarizing pulse. Pipettes were pulled (PB-7 puller; Narishige) from borosilicate capillary tubes (2-000-210; Drummond Scientific) and heat-polished just before use (MF-9 microforge; Narishige), leading to pipette tip resistances ranging from 2 to 2.5 M. For most recordings, pipette tips were coated with Sylgard (Dow Corning) to minimize capacitance. Drugs were applied via gravity-driven bath perfusion, with an estimated time to complete bath exchange of 510 s.
The control bath solution consisted of (mM): 125 NMDG-aspartate, 10 HEPES, 20 barium-acetate, 0.0005 tetrodotoxin, pH 7.5 (296 mOsm). The pipette solution contained (mM): 122 cesium-aspartate, 10 HEPES, 10 EGTA, 5 MgCl2, 4 ATP (disodium salt), 0.4 GTP (sodium salt), pH 7.5 (293 mOsm); where indicated, GTP was substituted with 0.1 mM of either GTP--S or GDP-ß-S (lithium salts).
Transmitters were excluded from the bath, and G-proteins were directly activated by including GTP or GTP--S in the pipette solution (
Pharmacology
Phorbol 12-myristate 13-acetate (PMA) and 4--phorbol 12-myristate 13-acetate (4-
-PMA) were obtained from Research Biochemicals, Inc. GTP-
-S and GDP-ß-S were obtained from either Research Biochemicals, Inc. or Sigma Chemical Co. The PKC inhibitor bisindolylmaleimide I (BLM) was obtained from Calbiochem Corp., and
-conotoxin GVIA (CTX) was from Bachem. All other chemicals and reagents were obtained from Sigma Chemical Co. Stock solutions of tetrodotoxin and CTX were prepared in double-distilled water; stock solutions of PMA, 4-
-PMA, and BLM were prepared in DMSO. Currents obtained in control bath solution containing the maximal final concentration of DMSO (0.124%) were indistinguishable from solutions lacking DMSO (not shown).
Data Analysis
Analysis software included Patch 6.3, Microsoft Excel 97, and Origin 5.0 (Microcal Software, Inc.). Current amplitude was measured isochronically for all recordings. Data are presented as mean ± SEM. Statistical significance was determined using a Student's two-tailed, paired t test or a two-way t test for two means; data were considered significantly different if P < 0.05. Sample size is given in parentheses within the figures, unless provided elsewhere. Fraction remaining was measured as the ratio of current amplitude at the end of the test pulse to the amplitude at the onset of the test pulse (see Fig 3 A); this method of measuring fast inactivation has been described previously as residual fraction of peak current (
where I/Imax is normalized current, V is voltage in millivolts, Vh is the voltage at half-maximal current, k is the slope of activation in millivolts per e-fold change in current, and I1 and I2 are the minimum and maximum values of I, respectively.
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RESULTS |
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Neonatal Rat SCG Neurons Exhibit Tonic G-Proteinmediated Inhibition
To examine the effects of PKC on whole-cell currents, it was necessary to first confirm that, under our recording conditions, tonic inhibition of whole-cell currents by G-proteins could be observed, and that activating PKC could block this inhibition. Tonic inhibition was observed in neonatal rat SCG neurons in the absence of G-proteincoupled receptor activation, when GTP was included in the pipette solution. Stepping from -90 to +10 mV (Fig 1 A) elicited an inward current whose amplitude was greatly facilitated if this step was preceded by a pulse to +80 mV (Fig 1 B, left, and Fig 2). 4 s later, current amplitude had returned to baseline, consistent with time-dependent recovery of tonic G-proteinmediated inhibition (
The increase in current amplitude following a prepulse was voltage dependent, as shown by the currentvoltage analysis (Fig 1 B, right). Maximal facilitation was observed from 0 to +10 mV, and dropped to undetectable levels as the test pulse became more positive, consistent with voltage-dependent relief of G-proteinmediated inhibition (
Tonic inhibition of whole-cell currents displayed additional characteristics that have been described previously. In addition to decreased current amplitude, G-proteininhibited whole-cell currents also exhibited slowed activation kinetics and increased facilitation; these effects were more pronounced when GTP--S was substituted for GTP in the pipette solution (Fig 1 C and 2). In contrast, G-proteinmediated inhibition was minimized by dialysis of the cell with GDP-ß-S (Fig 1 D). This was reflected by a loss of prepulse facilitation (Fig 2).
In addition to slowing voltage-dependent activation, modulation by G-proteins has also been shown to decrease voltage-dependent fast inactivation (-S greatly decreased inactivation, although application of a prepulse was sufficient to increase fast inactivation to the same level as with GDP-ß-S.
Taken together, these results verify that, under the conditions used in this study, inhibition of whole-cell currents by G-proteins is readily observable. Moreover, the effects of this inhibition on current amplitude, facilitation, and kinetics can be completely reversed either by applying prepulses or by including GDP-ß-S in the pipette solution.
PMA Enhances Whole-Cell N-Type Currents by Preventing Tonic G-Proteinmediated Inhibition
PKC activation has been shown previously to enhance whole-cell currents and lead to a reduction in G-proteinmediated inhibition in SCG neurons (
If PKC activation is sufficient to account for the increased current amplitude, decreased facilitation, and altered kinetics observed in cells that show tonic inhibition, then we would predict that these three parameters should change along a similar time course. This was addressed by measuring each of these parameters in a single recording (Fig 5). Indeed, as expected, after application of PMA to the bath, the changes observed in facilitation and fraction remaining closely paralleled the change in unfacilitated current amplitude.
To confirm that the effects observed after PMA application were due to activation of PKC, we conducted two control experiments (Fig 2 Fig 3 Fig 4). First, the inactive PMA analogue 4--PMA (
-PMA was without effect on facilitation, current amplitude, or fast inactivation. Second, to determine whether PMA's effects were due to selective activation of PKC, the PKC-specific inhibitor BLM (
Because neonatal rat SCG neurons contain additional types of calcium currents, the changes in whole-cell currents observed after PMA application could be due to actions on currents other than N-type. Therefore, to examine the effects of PMA on nonN-type currents, cells were incubated in a solution containing the N-type calcium channel blocker CTX for 10 min before patch-clamp recording (Fig 2 and Fig 4). CTX was excluded from the recording solutions to allow washout of any reversible block of nonN-type calcium channels (
G-Proteinmediated Inhibition Blocks PKC's Effect on Whole-Cell Currents
We observed that PMA only affected whole-cell currents that demonstrated G-proteinmediated inhibition, causing a relief of that inhibition (Fig 1 and Fig 4). Moreover, PMA appeared to act faster when prepulses were applied during the recording. This is consistent with the previous observation that relatively long prepulses were required to demonstrate PMA's effect on G-proteinmediated inhibition (-S was included in the pipette solution.
After membrane breakthrough, current amplitude rapidly decreased (Fig 6 A). This decrease was due to influx of GTP--S and subsequent activation of G-proteins, as application of a prepulse was sufficient to restore current amplitude. Currents were then elicited without prepulses, to minimize dissociation of G-proteinchannel interactions. In the absence of prepulses, PMA was essentially without effect on whole-cell current amplitude (Fig 6 C). Even after 8 min of stimulation with PMA, considerable G-proteinmediated inhibition remained, as prepulses could still facilitate current amplitude (Fig 6 A).
In separate experiments, currents were recorded in which every other test pulse to +10 mV was preceded by a pulse to +80 mV (Fig 6 B). Under these conditions, application of PMA was sufficient to enhance whole-cell current amplitude. Because GTP--S increases the level of available Gß
subunits in the cytoplasm, we would expect PMA to have a slower effect when recording with GTP-
-S than with GTP. We therefore measured the time constants of PMA's effect when prepulses were applied under both conditions. The time constant observed was 3.85 ± 0.69 min (n = 13) with GTP, and 5.18 ± 0.68 min (n = 3) with GTP-
-S. In addition, because GTP-
-S leads to a greater inhibition of current than GTP (Fig 1 and Fig 2), we would expect PMA to cause a greater enhancement of whole-cell currents after dialysis of GTP-
-S. As predicted, when prepulses were applied throughout the recording, PMA increased current amplitude approximately twofold (Fig 6 C), compared with 1.3-fold with GTP (Fig 4). These results indicate that phosphorylation occurred only after G-proteins were displaced from the channel. Together with the finding that phosphorylation by PKC prevents G-proteinmediated inhibition, these findings support mutual exclusivity between G-protein binding and phosphorylation.
GDP-ß-S Precludes PKC-mediated Enhancement of Current Amplitude
The above data indicate that PKC activation prevents G-proteinmediated inhibition. However, it is unclear whether PKC affects whole-cell currents in additional ways. Therefore, we next examined whether PKC modulates whole-cell currents in the absence of G-protein activity. When GDP-ß-S was included in the pipette, we observed an increase in current amplitude after membrane breakthrough (not shown), consistent with previously published results (-S was included in the pipette, PMA had no significant effect on current amplitude when GDP-ß-S was used (Fig 4).
Holding Potentialinduced Inactivation of Whole-Cell Currents Is Not Affected by PKC Activation
Although it did not affect current amplitude, PKC activation might affect other properties of the current. Thus, we next examined whether phosphorylation has an effect on holding potentialdependent inactivation. Data were collected from 100-ms test pulses to +10 mV, preceded by 2.2-s prepulses to varying potentials. For these experiments, no attempt was made to isolate fast inactivation from steady state inactivation; hence, all inactivation measured with this protocol was defined as holding potentialinduced inactivation (
Changes in holding potential can also affect fast inactivation (
Stimulation of PKC Does Not Affect Voltage-dependent Activation of Whole-Cell Currents
Our data suggest that G-proteinmediated inhibition shifts the voltage dependence of current activation to more positive voltages (Fig 1). Moreover, previous data (-S (Fig 8 B). Consistent with relief of G-proteinmediated inhibition, prepulses significantly facilitated voltage-dependent activation without shifting the threshold of activation. Cells dialyzed with GDP-ß-S (Fig 8 C) displayed voltage-dependent activation that was similar to cells recorded with prepulses after GTP-
-S dialysis (Table 1). This is consistent with a loss of G-proteinmediated inhibition. Subsequent application of PMA had no effect on voltage-dependent activation, supporting the hypothesis that
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DISCUSSION |
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In this study, we sought to determine what effect, if any, activation of PKC by PMA has on whole-cell barium currents in neonatal rat SCG neurons, in the absence of G-protein modulation. Before proceeding with this analysis, however, we established that, under our recording conditions, whole-cell currents can undergo voltage-dependent G-proteinmediated inhibition, and that this inhibition is restricted to N-type currents (
Having confirmed the presence of tonic G-proteinmediated inhibition, and its modulation by PKC, we next examined whether these two mechanisms can preclude one another. By examining the effect of PMA on current amplitude in the absence of prepulses, we demonstrated that G-proteinmediated inhibition is sufficient to block the effects of PKC activity. Moreover, our findings are consistent with the hypothesis that activation of PKC is sufficient to block G-proteinmediated inhibition. These results support a model of mutual exclusivity between phosphorylation and G-proteinmediated inhibition, consistent with previously published results (
Lastly, we examined whether PKC activation in the absence of G-proteinmediated inhibition causes additional modulation of whole-cell currents. When inhibition was first minimized by including GDP-ß-S in the pipette solution, bath application of PMA was without significant effect on current amplitude, fast and holding potentialdependent inactivation, or voltage-dependent activation, suggesting that PKC's only role in modulating N-type currents is to block G-proteinmediated inhibition. These results are somewhat surprising since multiple putative PKC consensus sites are present on the pore-forming 1B subunit (
1B subunit can account for the loss of inhibition by G-proteins (
N-type calcium channels inhibited by G-proteins have been termed "reluctant" by
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In contrast, channels not inhibited by G-proteins have been called "willing," indicating a more rapid response to changes in membrane potential (
In conclusion, we have confirmed that N-type calcium channel activity in neonatal rat SCG neurons undergoes voltage-dependent G-proteinmediated inhibition. In addition, stimulation of PKC enhances whole-cell barium currents by blocking this inhibition. Moreover, when G-proteins are activated with GTP--S, enhancement by PMA only occurs after prepulses, indicating that G-proteins must dissociate from the channel to observe the effects of phosphorylation by PKC. Finally, we have demonstrated that, under our recording conditions, there appears to be no functional effect of phosphorylation by PKC on N-type calcium channel activity beyond causing a long-term block of G-proteinmediated inhibition. Because of the existence of many PKC consensus sites on the N-type calcium channel (
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Footnotes |
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Portions of this work were previously published in abstract form (Barrett, C.F., and A.R. Rittenhouse. 1998. J. Gen. Physiol. 112:18a).
1 Abbreviations used in this paper: BLM, bisindolylmaleimide I; CTX, -conotoxin GVIA; SCG, superior cervical ganglion.
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Acknowledgements |
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We thank Drs. Alex Dopico and Liwang Liu for critiquing various versions of this manuscript.
This publication was made possible by support from the National Institutes of Health (grant NS34195) and its contents are solely the responsibility of the authors and do not necessarily reflect the official view of these granting agencies. A.R. Rittenhouse is the recipient of an Established Investigator Award from the American Heart Association (grant 9940225).
Submitted: 24 November 1999
Revised: 13 January 2000
Accepted: 14 January 2000
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