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Open Access

Peer-reviewed

Research Article

Complex regulation of Cav2.2 N-type Ca2+ channels by Ca2+ and G-proteins

  • Jessica R. Thomas,

    Roles Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing

    Affiliation Dept. of Biomedical Sciences, Meharry Medical College, Nashville, TN, United States of America

    ⨯
  • Jinglang Sun,

    Roles Formal analysis, Investigation, Writing – review & editing

    Affiliation Dept. of Neuroscience and Center for Learning and Memory, University of Texas-Austin, Austin, TX, United States of America

    ⨯
  • Juan De la Rosa Vazquez,

    Roles Formal analysis, Investigation, Writing – review & editing

    Affiliation Dept. of Neuroscience and Center for Learning and Memory, University of Texas-Austin, Austin, TX, United States of America

    ⨯
  • Amy Lee

    Roles Conceptualization, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing

    amy.lee1@austin.utexas.edu

    Affiliation Dept. of Neuroscience and Center for Learning and Memory, University of Texas-Austin, Austin, TX, United States of America

    ⨯

Abstract

G-protein coupled receptors inhibit Cav2.2 N-type Ca2+ channels by a fast, voltage-dependent pathway mediated by Gαi/Gβγ and a slow, voltage-independent pathway mediated by Gαq-dependent reductions in phosphatidylinositol 4,5-bisphosphate (PIP2) or increases in arachidonic acid. Studies of these forms of regulation generally employ Ba2+ as the permeant ion, despite that Ca2+ -dependent pathways may impinge upon G-protein modulation. To address this possibility, we compared tonic G-protein inhibition of currents carried by Ba2+ (IBa) and Ca2+ (ICa) in HEK293T cells transfected with Cav2.2. Both IBa and ICa exhibited voltage-dependent facilitation (VDF), consistent with Gβγ unbinding from the channel. Compared to that for IBa, VDF of ICa was less sensitive to an inhibitor of Gα proteins (GDP-β-S) and an inhibitor of Gβγ (C-terminal construct of G-protein coupled receptor kinase 2). While insensitive to high intracellular Ca2+ buffering, VDF of ICa that remained in GDP-β-S was blunted by reductions in PIP2. We propose that when G-proteins are inhibited, Ca2+ influx through Cav2.2 promotes a form of VDF that involves PIP2. Our results highlight the complexity whereby Cav2.2 channels integrate G-protein signaling pathways, which may enrich the information encoding potential of chemical synapses in the nervous system.

Citation: Thomas JR, Sun J, De la Rosa Vazquez J, Lee A (2025) Complex regulation of Cav2.2 N-type Ca2+ channels by Ca2+ and G-proteins. PLoS ONE 20(2): e0314839. https://doi.org/10.1371/journal.pone.0314839

Editor: Steven Barnes, Doheny Eye Institute, UNITED STATES OF AMERICA

Received: November 18, 2024; Accepted: January 20, 2025; Published: February 7, 2025

Copyright: © 2025 Thomas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are contained within the manuscript and its Supporting Information files.

Funding: This work was supported by the National Institutes of Health (https://www.nih.gov) under grants R01 EY026817 and R03TR005086 and startup funds from The University of Texas at Austin (to A.L.). The sponsors did not play a role n the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

In nerve terminals, voltage-gated Cav2 channels are prominent mediators of Ca2+ influx which triggers the exocytotic release of neurotransmitters into the synaptic cleft. The inhibition of presynaptic Cav2 channels by neurochemicals such as GABA and norepinephrine potently suppresses neurotransmission via receptors coupled to heterotrimeric G-proteins (GPCRs) [1]. This inhibition can occur through a voltage-dependent, membrane-delimited pathway involving the Gαi/o class of G-proteins and the binding of Gβγ to the channel [2–6]. GPCRs coupled to the Gαq class of G-proteins also inhibit Cav2 channels through a slower, voltage-independent pathway [7, 8]. Mechanisms for this form of Cav2 channel modulation include enzymatic depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) [9, 10], which normally enhances the activity of Cav channels [11].

Among the Cav2 subtypes (Cav2.1, Cav2.2, Cav2.3), Cav2.2 channels exhibit particularly strong voltage-dependent inhibition by G-proteins [12, 13] which can be tempered by other signal mediators. For example, GPCRs that activate protein kinase C (PKC) diminish the impact of Gβγ on Cav2.2 [14–16]. PKC phosphorylates a threonine in the cytoplasmic linker between domains I and II, which prevents the interaction with Gβγ [17, 18]. Conversely, several proteins involved with synaptic release, such as syntaxin 1A and cysteine string proteins, enhance G-protein inhibition of Cav2.2 through interactions with both Gβγ and the channel [19–21]. Thus, the impact of GPCRs on neuronal Cav2.2 channels may vary with patterns of neuronal activity, exposure to various neuromodulators, and interactions with proteins in specific subcellular compartments.

Cav2.2 undergoes some voltage-dependent inhibition by G-proteins even without exogenous application of GPCR agonists, which could result from an excess of free Gβγ and/or activation of autoreceptor GPCRs [22–27]. In these studies, Ba2+ was often chosen as the permeant ion since Ba2+ currents (IBa) are larger in amplitude than Ca2+ currents (ICa) [28]. However, this approach can mask physiologically relevant forms of Cav2.2 modulation that rely on Ca2+ influx [29] and could affect the impact of G-proteins. To address this possibility, we compared tonic G-protein modulation of IBa and ICa in HEK293T cells transfected with Cav2.2. Our results indicate that tonic inhibition by Gβγ is stronger for IBa than for ICa and implicate PIP2 in modulation of ICa and not IBa when Gβγ-mediated inhibition is suppressed. Our findings add to the diverse modes by which Cav channels are regulated, some of which depend critically on the nature of the permeating cation.

Materials and methods

cDNAs. The following cDNAs were used: Cav2.2 e37b (Genbank # AF055477), β2a (Genbank # NM_053851), α2δ-1 (Genbank # NM_000722.3), pEGFP (Addgene). The C-terminal construct corresponding to G-protein coupled receptor kinase containing a myristic acid attachment signal (MAS-GRK2-ct) and zebrafish voltage-sensitive phosphatase (Dr-VSP) were described previously [8, 11].

Cell culture and transfection

Human embryonic kidney 293 cells transformed with the SV40 T-antigen (HEK 293T, American Type Culture Collection Cat# CRL-3216, RRID:CVCL_0063) were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO2. Cells were grown to 80% confluence and transfected using Fugene 6 (Promega) according to the manufacturer’s protocol. Cells were plated in 35 mm dishes and transfected with cDNAs encoding Cav channel subunits (Cav2.2, 1.8 μg; β2a, 0.6 μg; and α2δ-1, 0.6μg). In some experiments, 0.5 μg of MAS-GRK2-ct or Dr-VSP was co-transfected to buffer Gβγ or deplete PIP2, respectively. Cotransfection with cDNA encoding enhanced green fluorescent protein (pEGFP, 50 ng) allowed visualization of transfected cells.

Electrophysiological recordings

Whole-cell patch recordings were performed 24–72 hours after transfection with a EPC-8patch clamp amplifier and Patch master software (HEKA Elektronik). External recoding solution contained (in mM): 150 Tris, 1 MgCl2, and 5 CaCl2 or BaCl2. Intracellular solution contained (in mM): 140 N-methyl-D-glucamine 10 HEPES, 10 EGTA, 2 MgCl2, and 2 Mg-ATP. The pH of both solutions was adjusted to 7.3 using methanesulfonic acid. In some experiments BAPTA or Guanosine5′-[β-thio]diphosphate trilithium salt (GDPβS) was added to the intracellular solution to either buffer Ca2+ or block G proteins, respectively. Electrode resistances were 4–6 MΩ in the bath solution. Series resistance was compensated 60–70%. Leak currents were subtracted using a P/-4 protocol. Data were analyzed using Igor Pro software (Wavemetrics). Averaged data represent mean ± S.E., and result from at least 3 independent transfections.

Data presentation and statistical analysis

Data were incorporated into figures using Graph-Pad Prism software and Adobe Illustrator software. Statistical analysis was performed with Graph-Pad Prism software. The data were first analyzed for normality using the Shapiro–Wilk test. For parametric data, significant differences were determined by Student’s t test or ANOVA with post hoc Dunnett or Tukey test. For nonparametric data, the Mann-Whitney, Kruskal–Wallis, or Wilcoxon tests were used as well as post hoc Dunn’s test.

Results

In electrophysiological recordings of Cav channels, inhibition by G-proteins can be studied by evoking current-voltage (I-V) relationships before (P1) and after (P2) a depolarizing prepulse [23]. With this protocol, current amplitudes after the prepulse should be larger due to Gβγ unbinding from the channel [6]. We used this voltage protocol to test whether the tonic Cav2.2 modulation by G-proteins might differ for IBa and ICa in transfected HEK293T cells. In our experiments, we used the Cav2.2 splice variant containing exon 37b which lacks the voltage-independent, tyrosine kinase-dependent form of G-protein modulation seen for variants containing exon 37a [30]. We cotransfected cells with the auxiliary α2δ-1 subunit and β2a subunit, which produces stronger tonic G-protein modulation than channels containing the β1b subunit [23]. To account for differences in current amplitudes between cells due to variable levels of channel expression, we plotted I-V data normalized to the maximal current evoked by P2 (Inorm). As expected, the amplitudes of the normalized peak Ba2+ current (Inorm, at test pulse = 0 mV) were significantly higher after (median = -0.84) than before a +60-mV prepulse (median = -0.33, W = -28, p = 0.02 by Wilcoxon matched-pairs test; Fig 1A–1C). To verify the involvement of G-proteins, we used the guanosine diphosphate analog GDP-β-S which should limit the availability of Gβγ by stabilizing its association with Gα [31]. When GDP-β-S was included in the intracellular recording solution, the amplitude of peak Inorm was still higher after (median = -0.58) than before the prepulse (median = -0.41, W = -28, p = 0.02 by Wilcoxon matched-pairs test; Fig 1D–1F). However, the extent of the prepulse-induced increase in peak Inorm was 10-fold lower with GDP-β-S (Fractional facilitation (FF) = 1.05 ± 0.27 for control vs. 0.2 ± 0.04 for +GDP-β-S, t = 3.081, df = 12, p = 0.01 by unpaired t-test; Fig 1G). These results show that Cav2.2 undergoes tonic, voltage-dependent inhibition of Cav2.2 by G-proteins in HEK293T cells, as described previously for this channel in other cell-types [23, 26].

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Fig 1. VDF of IBa for Cav2.2 is blunted by GDPβS.

(A) Representative current traces and voltage protocol. IBa was evoked by a 10-ms test pulse from a holding voltage of -80 mV to the indicated voltages 10 s before (P1) and 5 ms after (P2) a 50-ms conditioning pre-pulse to +60 mV. The test pulses were followed by a 2-ms step to -60 mV prior to repolarizing to -80 mV. (B) Representative I-V plot of P1 and P2 currents for a single cell. Inorm represents the amplitude of the steady state current near the end of P1 or P2 pulse normalized to the maximal current evoked by the P2 voltage. Smooth line represents Boltzmann fits. (C) IBa for P1 and P2 pulses (both 0 mV) for each cell. (D-F) Same as in A-C but for cells where GDPβS (0.3 mM) was included in the intracellular recording solution. (G) Plot comparing fractional facilitations, (P2-P1)/P1, for IBa evoked by 0 mV test pulse between cells with and without intracellular GDPβS. Bar represents mean. p-value was determined by Wilcoxon test (C, F) or unpaired t-test (G).

https://doi.org/10.1371/journal.pone.0314839.g001

Like IBa, ICa also was increased by the prepulse under control conditions (mean = -0.56 before vs. mean = -0.74 after, t = 6.348, df = 6, p = 0.001; Fig 2A–2C) and with GDP-β-S (median = -0.68 before vs. median = -0.82 after, W = -66, p = 0.001 by Wilcoxon matched-pairs test; Fig 2D–2F). However, the depolarizing prepulse caused a significantly smaller increase in ICa than IBa under control conditions (FF = 0.35 ± 0.03 for ICa vs. 1.05 ± 0.27 for IBa, t = 2.549, df = 12, p = 0.02 by unpaired t-test). Moreover, facilitation caused by the prepulse did not significantly differ under control conditions and with GDP-β-S (FF = 0.25 ± 0.03, t = 2.007, df = 16, p = 0.06 compared to control by unpaired t-test; Fig 2G). Thus, tonic voltage-dependent inhibition of Cav2.2 by G-proteins is weaker and less sensitive to GDP-β-S for ICa than IBa.

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Fig 2. VDF of ICa for Cav2.2 is unaffected by GDPβS.

(A-G) Same voltage protocol and analysis as in Fig 1A and 1B) Representative current traces and voltage protocol (A) and I-V plot (B) for a single cell. Smooth line represents Boltzmann fits. (C) ICa for P1 and P2 pulses (both at 0 mV) for each cell. p-value was determined by paired t-test. (D-F) Same as in A-C but for cells where GDPβS (0.3 mM) was included in the intracellular recording solution. (G) Plot comparing fractional facilitations, (P2-P1)/P1, for ICa evoked by 0 mV test pulse between cells with and without intracellular GDPβS. Bar represents mean. p-value was determined by paired t-test (C), Wilcoxon test (F) or unpaired t-test (G).

https://doi.org/10.1371/journal.pone.0314839.g002

To further investigate this difference in G-protein regulation of ICa and IBa, we used a double pulse protocol where the effect of varying the voltage of the prepulse is measured on a test current evoked before (P1) and after (P2) the prepulse (Fig 3A–3F). With this protocol, VDF is evident as a progressive increase in the P2 vs P1 current amplitude with prepulse voltage [32]. For IBa, VDF was robust under control conditions and was reduced by GDP-β-S (Fig 3B and 3D). The amount of VDF was measured as the difference in the P2 and P1 currents with a prepulse to +80 mV (Fractional facilitation, FF80) and was significantly lower with GDP-β-S (mean FF80 = 0.13) compared to control conditions (mean FF80 = 0.48, t = 3.748, df = 14, p = 0.0022 by unpaired t-test; Fig 3F). VDF of ICa was also strong and showed a similar dependence on prepulse voltage as IBa. However, unlike IBa, VDF was not significantly different under control conditions (mean FF80 = 0.52) and with GDP-β-S (mean FF80 = 0.39, t = 2.076, df = 15, p = 0.056 by unpaired t-test; Fig 3C, 3E and 3F).

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Fig 3. Decline of VDF of IBa for Cav2.2 caused by GDPβS.

(A) Voltage protocol. ICa (or IBa) was evoked by a 10-ms test pulse from a holding voltage of -80 mV to -5 mV (-10 mV for IBa) 10-s before (P1) and 5-ms after (P2) a 50-ms conditioning pre-pulse to indicated voltages. The test pulses were followed by a 2-ms step to -60 mV prior to repolarizing to -80 mV to facilitate measurements of tail currents. (B, C) Tail currents for ICa or IBa evoked by P1 or P2 test pulses were normalized to that for the P1 pulse prior to the +80 mV prepulse (Inorm) and plotted against the prepulse voltage. (D, E) Same as in B-C but for cells where GDPβS (0.3 mM) was included in the intracellular recording solution. (F) Plot comparing fractional facilitations, (P2-P1)/P1, for ICa and IBa evoked before and after a +80 mV conditioning prepulse in cells with and without intracellular GDPβS. Bars represent mean. p-values were determined by unpaired t-test.

https://doi.org/10.1371/journal.pone.0314839.g003

A possible explanation for our results thus far was that VDF of ICa could involve an additional pathway that is recruited even when Gβγ is inhibited. To test this, we coexpressed Cav2.2 with a C-terminal construct of GPCR kinase 2 (GRK) which has no kinase activity but acts to sequester Gβγ [8]. With the I-V protocol to measure VDF, the peak current amplitude for both ICa and IBa was still increased by the +60 mV conditioning pulse in the presence of GRK (Fig 4A–4F). As expected, VDF for IBa was significantly weaker with GRK (FF = 0.19 ± 0.04, n = 7) than under control conditions (FF = 1.41 ± 0.27, n = 7; t = 3.102, df = 12, p = 0.009 by unpaired t-test; Fig 4G). In contrast, there was no significant difference in VDF for ICa with GRK (FF = 0.32 ± 0.07, n = 5) than under control conditions (FF = 0.34 ± 0.03, n = 7; t = 0.314, df = 10, p = 0.759 by unpaired t-test; Fig 4G). Similar results were obtained with the double pulse protocol (Fig 5A–5D). Compared to control conditions, GRK expression caused a significant reduction in VDF for IBa (58%, Fig 5B and 5D) but a non-significant slight increase in VDF for ICa (Fig 5C and 5D). These results agree with our hypothesis that VDF of ICa could proceed even when Gβγ is inhibited.

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Fig 4. VDF of IBa for Cav2.2 is suppressed by GRK2.

(A) Representative current traces and voltage protocol. ICa was evoked by the same voltage protocol as in Fig 1. (B) Representative I-V plot of P1 and P2 currents for a single cell co-transfected with GRK2. Smooth line represents Boltzmann fits. (C) ICa for P1 and P2 pulses for each cell. p-value was determined by paired t-test. (D-F) Same as in A-C but for cells recorded in Ba2⁺ bath solution. (G) Plots comparing fractional facilitations, (P2-P1)/P1, for ICa and IBa evoked by 0 mV test pulse. Bars represent mean. P-values were determined by unpaired t-test.

https://doi.org/10.1371/journal.pone.0314839.g004

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Fig 5. Reduced VDF of IBa for Cav2.2 caused by GRK2.

(A) Voltage protocol (same as in Fig 3). (B-C) Tail currents for ICa or IBa evoked by P1 or P2 test pulses were normalized to that for the P1 pulse prior to the +80 mV prepulse (Inorm) and plotted against the prepulse voltage. Cells were co-transfected with GRK2. (D) Plot comparing fractional facilitations, (P2-P1)/P1, for ICa and IBa evoked before and after an 80 mV conditioning pre pulse in cells with and without GRK2 transfection. Bars represent mean. p-values were determined by unpaired t-test.

https://doi.org/10.1371/journal.pone.0314839.g005

The apparent absence of an effect of GDPβS on VDF of ICa (Figs 2 and 3) could signify opposing regulation of Cav2.2 by another G-protein signaling pathway that is recruited when Ca2+ ions permeate the channel. One possibility was that GDP-β-S enabled a form of Ca2+-dependent facilitation (CDF) similar to that for Cav2.1 channels that is mediated by calmodulin (CaM) binding to the Cav2.1 C-terminal domain [33, 34]. This seemed unlikely since the VDF exhibited by Cav2.2 ICa in the double pulse protocol did not resemble CaM-dependent CDF of Cav2.1, which shows a U-shaped dependence on prepulse voltage that reflects the amount of Ca2+ influx during the prepulse [35, 36]. Moreover, Cav2.2 lacks key domains present in Cav2.1 that are required for CaM-dependent CDF [37]. In primary sensory neurons, Cav2.2 undergoes CDF that is mediated by CaM dependent protein kinase II (CaMKII), which requires cytoplasmic accumulation of Ca2+ [38]. In the voltage protocols for measuring VDF, the interval between the P1 and conditioning pulses is 10 s, which may allow for sufficient Ca2+ influx during the P1 pulse to activate Ca2+-dependent pathways such as those involving CaMKII. However, VDF of ICa with strong Ca2+ buffering with 10 mM BAPTA (FF = 0.39 ± 0.04, Fig 6A and 6B) or the 0.3 mM of the CaMKII inhibitor KN93 (FF = 0.34 ± 0.10, Fig 6C) was similar to that under control conditions with (FF = 0.39 ± 0.05) or without GDP-β-S (FF = 0.53 ± 0.06; F(3, 17) = 1.781, p = 0.189 by One-Way ANOVA; Fig 6D). These results argue against a role for CaMKII in VDF of Cav2.2 ICa.

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Fig 6. VDF of ICa for Cav2.2 is not mediated by CaMKII.

(A) Voltage protocol (same as in Fig 3). (B) Tail currents for ICa evoked by P1 or P2 test pulses were normalized to that for the P1 pulse prior to the +80 mV prepulse (Inorm) and plotted against the prepulse voltage. GDPβS (0.3 mM) and BAPTA (10 mM) was included in the intracellular recording solution. (C) Same as in B but for cells where GDPβS (0.3 mM) and KN93 was included in the intracellular recording solution. (D) Plot comparing fractional facilitations, (P2-P1)/P1, for ICa evoked before and after a +80 mV conditioning prepulse in cells with various intracellular conditions. Bars represent mean. p-value was determined by One-Way ANOVA.

https://doi.org/10.1371/journal.pone.0314839.g006

An alternative mechanism involves Gαq-dependent activation of phospholipase C (PLC), which causes the hydrolysis of PIP2 into inositol 1,4,5-trisphosphate and diacylglycerol, or increased liberation of arachidonic acid by phospholipase A2 [39]. It is well-established that PIP2 supports the function of Cav channels, and that GPCRs linked to Gαq cause a decline in PIP2 that lowers activity of Cav channels [9, 11, 40, 41]. GDP-β-S might suppress this Gαq signaling pathway, thus increasing Cav channel activity by reducing PIP2 hydrolysis. If selective for ICa, this effect of GDP-β-S on Gαq might mask the effect of GDP-β-S on the Gαi/o/ Gβγ-mediated pathway, leaving net VDF unchanged. To test this, we utilized a voltage-sensitive phosphatase (VSP) from zebrafish which enables the depletion of PIP2 in living cells following a strong depolarizing voltage step (i.e., +120 mV). This approach has been used previously to blunt Gαq-dependent inhibition of Cav channels [11].

To enable VSP activation, we modified our voltage protocol to include a +120-mV VSP-activating pulse prior to the P1 test pulse and VDF was measured as the ratio of the P2/P1 pulses with an intervening +20 mV prepulse (Fig 7A). A more modest depolarizing prepulse was used in these experiments to avoid additional activation of the VSP. GDPβS was included in the intracellular recording solution to replicate conditions that led to distinctions in VDF of ICa and IBa in Fig 3. When the double pulse protocol was given without the +120-mV pulse, P2/P1 for ICa did not differ in cells with (median = 1.371) and without VSP (median = 1.471; t = 1.612, df = 12, p = 0.133 by unpaired t-test) indicating that VSP did not affect VDF when not activated. In cells transfected with VSP, P2/P1 for ICa measured with the +120 mV pulse (mean = 1.016 ± 0.04) was significantly lower than when measured without the +120 mV pulse (mean = 1.39 ± 0.046; t = 8.264, df = 7, p < 0.0001 by paired t-test; Fig 7B and 7F). This result demonstrates that PIP2 enhances VDF of ICa. As in control cells transfected with Cav2.2 alone (i.e., -VSP), P2/P1 for IBa (+VSP) was not significantly different with (median = 1.184) or without the +120 mV pulse (median = 1.344, W = -15, p = 0.426 by Wilcoxon matched pair signed rank test; Fig 7D and 7F). Therefore, alterations in PIP2 do not affect VDF for IBa. Taken together, our results suggest that PIP2 enhances VDF of ICa but not IBa when G-proteins are inhibited.

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Fig 7. VDF of ICa for Cav2.2 is abolished after PIP2 depletion via VSP activation.

(A) Voltage protocol. An optional 1-s long +120 mV VSP activating pulse from a holding voltage of -80 mV is applied 10-s prior to the first test pulse. ICa or IBa was evoked by a 10-ms test pulse from a holding voltage of -80 mV to the indicated voltages (-5 mV for ICa or -10 mV for IBa) 10-s before (P1) and 5-ms after (P2) a 50-ms conditioning pre-pulse to +20 mV. The test pulses were followed by a 2-ms step to -60 mV prior to repolarizing to -80 mV to facilitate measurement of the tail current. (B) Paired representative ICa traces reflecting VDF from VSP and non-VSP transfected cells, with each cell tested with and without a +120 mV VSP activating pulse. (C) P1 and P2 evoked ICa comparison, obtained in conditions as dscribed in B. p-values determined by Wilcoxon test.(D, E) Same as in B-C but for cells recorded in Ba2⁺ bath solution. p-values determined by paired t-test (-VSP) and Wilcoxon test (+VSP). (F) P2/P1 evoked ICa and IBa ratio for each cell. p-values determined by paired t-test (-VSP) and Wilcoxon test (+VSP).

https://doi.org/10.1371/journal.pone.0314839.g007

Discussion

Our study reveals an unusual feature of G-protein modulation of Cav2.2 that requires the influx of Ca2+ through the channel. For IBa, VDF depends mainly on Gαi/o/ Gβγ which is blunted by GDPβS (Figs 1, 3F and 8A) and GRK (Figs 4 and 5). For ICa, VDF that remains in the presence of GDPβS (Figs 2 and 3) requires PIP2 since it is suppressed by Dr-VSP (Fig 7A–7C). We propose that when Ca2+ permeates the channel, GDPβS inhibits not only Gαi/o/ Gβγ but also Gαq/ Gβγ. The latter pathway promotes a decline in PIP2 since both Gαq and Gβγ can activate PLC [42]. Despite the competing effects of blunting the Gαi/o/ Gβγ pathway, GDPβS strengthens VDF of ICa by limiting Gαq-dependent reductions in PIP2 (Fig 8B).

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Fig 8. Model for distinct G-protein modulation of IBa and ICa mediated by Cav2.2.

(A, B) Cav2.2 channels are potentiated by PIP2 binding and inhibited by Gβγ binding to the channel. Gαq-mediated decreases in PIP2 would be expected to inhibit IBa and ICa, whereas Gβγ would be expected to promote VDF. For IBa, the main effect of GDP-β-S is to suppress VDF by inhibiting liberation of Gβγ from Gαi/o (A). For ICa, the main effect of GDP-β-S is to increase VDF by inhibiting Gαq-mediated decreases in PIP2 (B).

https://doi.org/10.1371/journal.pone.0314839.g008

In studies of Cav channels, Ba2+ is often substituted for Ca2+ in the extracellular recording solution in part to minimize Ca2+-dependent pathways that could complicate analysis of intrinsic channel properties. However, ICa can differ from IBa in physiologically relevant ways. A prominent example is Ca2+-dependent inactivation (CDI), which is a characteristic of all Cav1 and Cav2 channels and manifests as faster decay of ICa compared to IBa [36, 43]. Cav2 channels also undergo CDF [33–35], which for Cav2.2 requires CaMKII and is reduced following nerve injury [38]. The VDF of ICa for Cav2.2 in our study differed from CaMKII-dependent CDF since it was not blocked by high BAPTA or the CaMKII inhibitor (Fig 6). The BAPTA-insensitivity suggests that Ca2+ elevations within a nanodomain of the Cav2.2 channel are needed to support VDF when G-proteins are inhibited. Considering that Ca2+ can increase the enzymatic activity of PLC [44, 45], Ca2+ influx through Cav2.2 could amplify the effects of Gαq coupling to PLC, leading to greater reductions in PIP2 levels and reduced channel function than when using Ba2+ as the permeant ion. The persistence of VDF in the presence of GDPβS could then be viewed as a disinhibition of Cav2.2 by stabilizing PIP2 levels that support channel function (Fig 8B). Some PLC isoforms are membrane-associated and can form macromolecular complexes with ion channels and GPCRs to allow for fast and localized signaling [46, 47]. Cav channels interact with a variety of proteins [1] including those that may scaffold PLC and position it for regulation by incoming Ca2+ ions. In addition, micromolar concentrations of Ca2+ can cluster PIP2 in nanodomains [48, 49] which might make PIP2 a more appealing substrate for hydrolysis by PLC than in the presence of Ba2+ ions.

PIP2 has complex actions on Cav2 channels, causing both a stimulation and inhibition of function [40]. According to one model, PIP2 binds to an “R” domain which, like G-proteins, causes channels to enter a “reluctant” (i.e., inhibited) mode of gating at intermediate voltages. In contrast, PIP2 binding to a stimulatory “S” domain is required for channel activation [40]. Structural and functional studies show that the S domain likely corresponds to a PIP2 binding site in domain II S4 [50–52]. Additionally, a second site in the cytoplasmic I-II linker is important for stimulatory effects of PIP2 on Cav2.2 channels containing the cytosolic β2c but not the membrane-tethered β2a subunit [52]. Apparently, the palmitoylation of β2a allows it to compete with PIP2 binding to the “S” domain, perhaps biasing its interaction with the “R” domain [52–54]. Compared to Cav2.2 channels with other β subunits, those containing β2a are less sensitive to the stimulatory effects, and more sensitive to the inhibitory effects, of PIP2; an increase in current density is seen upon Gαq-linked receptor activation of β2a-containing channels [53, 54]. The conversion of “reluctant” channels to “willing” channels upon Dr-VSP activation could contribute to the reduced VDF of ICa if “willing” channels represent a “pre-facilitated” state. Based on this logic, the effects of Gαq-mediated PIP2 depletion on VDF are expected to differ for Cav2.2 channels containing β2a vs. cytosolic β subunits (i.e., β2c or β3), which may further diversify the modulatory properties of these channels in neurons. PIP2 has been shown to support VDF of Cav2.2 channels in hypothalamic neurons [55]. Moreover, Ca2+ and PIP2 have been found to strengthen Gβγ-mediated inhibition of Cav2 channels [56–58]. Future studies are needed to dissect the mechanisms whereby alterations in PIP2 enable gating transitions that underlie VDF, and the interplay of Ca2+, G-proteins, and Cavβ subunits in this process.

Supporting information

S1 File. Datasets for Figs 1–7.

Datasets for analysis presented in Figs 1–7 of the article.

https://doi.org/10.1371/journal.pone.0314839.s001

(XLSX)

Acknowledgments

The authors thank these individuals for gifts of cDNAs: Paul Kammermeier for MAS-GRK2-ct, Byung Chang Suh for the Dr-VSP, Diane Lipscombe for Cav2.2.

References

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