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Modulation of CaV1.3b L-type calcium channels by M1 muscarinic receptors varies with CaVβ subunit expression

Objectives We examined whether two G protein-coupled receptors (GPCRs), muscarinic M1 receptors (M1Rs) and dopaminergic D2 receptors (D2Rs), utilize endogenously released fatty acid to inhibit L-type Ca2+ channels, CaV1.3. HEK-293 cells, stably transfected with M1Rs, were used to transiently transfect D2Rs and CaV1.3b with different CaVβ-subunits, allowing for whole-cell current measurement from a pure channel population. Results M1R activation with Oxotremorine-M inhibited currents from CaV1.3b coexpressed with α2δ-1 and a β1b, β2a, β3, or β4-subunit. Surprisingly, the magnitude of inhibition was less with β2a than with other CaVβ-subunits. Normalizing currents revealed kinetic changes after modulation with β1b, β3, or β4, but not β2a-containing channels. We then examined if D2Rs modulate CaV1.3b when expressed with different CaVβ-subunits. Stimulation with quinpirole produced little inhibition or kinetic changes for CaV1.3b coexpressed with β2a or β3. However, quinpirole inhibited N-type Ca2+ currents in a concentration-dependent manner, indicating functional expression of D2Rs. N-current inhibition by quinpirole was voltage-dependent and independent of phospholipase A2 (PLA2), whereas a PLA2 antagonist abolished M1R-mediated N-current inhibition. These findings highlight the specific regulation of Ca2+ channels by different GPCRs. Moreover, tissue-specific and/or cellular localization of CaV1.3b with different CaVβ-subunits could fine tune the response of Ca2+ influx following GPCR activation. Electronic supplementary material The online version of this article (10.1186/s13104-018-3783-x) contains supplementary material, which is available to authorized users.

Although present in MSNs, M 1 R signaling has been characterized most thoroughly in superior cervical ganglion (SCG) neurons. M 1 Rs couple to Gα q and phospholipase C (PLC) to inhibit native L-and N-VGCC currents [7][8][9]. This signal transduction cascade, referred to as the slow or diffusible second messenger pathway, is characterized as pertussis toxin (PTX)-insensitive, voltageindependent, and requiring intracellular Ca 2+ to function [10]. Our laboratory has identified arachidonic acid (AA) as a critical effector in the slow pathway [9]. Exogenously applied AA inhibits L-current [11][12][13], which in SCG neurons most likely arises from Ca V 1.3 [14]. Moreover, Ca 2+ -dependent cytosolic phospholipase A 2 (cPLA 2 ) appears critical for release of AA from phospholipids following M 1 R activation; loss of cPLA 2 activity by pharmacological antagonists or gene knockout ablates L-current inhibition [15,16].
Additionally, D 2 Rs inhibit L-current via a diffusible second messenger pathway involving phospholipase C (PLC), InsP 3 , and calcineurin in MSNs [3]. While both GPCRs signal through PLC, they share another commonality: their activation releases AA from striatal neurons [17,18] and transfected cell lines [19,20]. Therefore, D 2 Rs may also inhibit L-(Ca V 1.3) and N-(Ca V 2.2) currents via a pathway utilizing cPLA 2 to release AA. In the present study, we tested whether the M 1 R and D 2 R pathways converge to modulate recombinant L-VGCC activity.

Electrophysiology
Whole-cell currents were recorded following the methods of Liu et al. [11]. High resistance seals were established in Mg 2+ Tyrode's (in mM): 5 MgCl 2 , 145 NaCl, 5.4 KCl, and 10 HEPES, brought to pH 7.50 with NaOH. Once a seal was established and the membrane ruptured, the Tyrode's solution was exchanged for external bath solution (in mM): 125 NMG-aspartate, 20 Ba-acetate, 10 HEPES, brought to pH 7.50 with CsOH. Only cells with ≥ 0.2 nA of current were used. Data were acquired using Signal 2.14 software (CED) and stored for later analysis on a personal computer. Linear leak and capacitive currents were subtracted from all traces.

Drugs
All chemicals were purchased from Sigma unless otherwise noted. FPL 64176 (FPL), nimodipine (NIM), and oleoyloxyethyl phosphorylcholine (OPC, Calbiochem) were prepared as stock solutions in 100% ethanol. Quinpirole (quin) and Oxotremorine-M (Oxo-M, Tocris) were dissolved in DDW and stored as 10 mM stock solutions at − 70 °C. Stocks were diluted daily to the final concentration by at least 1000-fold with external solution. For ethanol-prepared stocks, the final ethanol concentration was less than 0.1%.

Statistical analysis
Data are presented as the mean ± s.e.m. Data were analyzed for significance using a Student's paired t-test for two means, or a one-way ANOVA followed by a Tukey multiple-comparison post hoc test. Statistical significance was set at p < 0.05 or < 0.001. Analysis programs included Signal (CED), Excel (Microsoft), and Origin (OriginLab).

Characterization of recombinant Ca V 1.3 current as L-type in HEK-M1 cells
Whole-cell L-currents, from β 3 -containing L-channels, elicited from a holding potential of − 60 mV to a test potential of − 10 mV, averaged − 4699 ± 279 pA (n = 3) compared to − 9 ± 1 pA for HEK-M1 cells transfected with only accessory subunits (n = 10, P < 0.001). Lack of current from cells transfected without Ca V 1.3b, confirmed that HEK-M1 cells exhibit little endogenous Ca 2+ current and transfection of accessory subunits does not upregulate endogenous Ca 2+ channels. Recombinant current was confirmed as L-type by showing sensitivity to the L-VGCC antagonist NIM. NIM inhibited β 3 -containing currents (Additional file 1A) in a concentration-dependent manner (Additional file 1B). Currents were also sensitive to FPL, which enhanced current from β 2a -and β 3 -containing channels and produced long-lasting tail currents upon repolarization (Additional file 1C, D). Additionally, FPL produced a slight hyperpolarizing voltage shift in the peak inward current and enhanced current amplitude at all voltages (Additional file 1E). Additional file 1F demonstrates that FPL enhanced the long-lasting tail current in a concentration-dependent manner. These pharmacological and biophysical properties show that transfection of HEK-M1 cells with Ca V 1.3b and accessory subunits produce currents with L-type characteristics.

The Ca V β-subunit varies the magnitude of Ca V 1.3 current inhibition by M 1 Rs
In MSNs, M 1 R stimulation inhibits L-current in Ca V 1.2 knockout animals [4]. Only Ca V 1.2 and Ca V 1.3 constitute the L-type Ca V α 1 subunits expressed in brain [23], implying that M 1 Rs specifically inhibit Ca V 1.3 current. Using a cell line transfected with only Ca V 1.3 channels provides molecular proof for the identity of the inhibited channel. Therefore, to determine if activation of M 1 Rs inhibits Ca V 1.3 activity, peak current amplitudes were measured prior to and following application of the M 1 R agonist Oxo-M. Figure 1a compares representative current traces for Ca V 1.3b coexpressed with β 1b , β 2a , β 3 , or β 4 -subunits in the absence or presence of Oxo-M.  Ca V 1.3b current inhibition and kinetic changes produced by M 1 R stimulation are Ca V β-subunit dependent. a Representative current traces from Ca V 1.3b coexpressed with β 1b , β 2a , β 3 or β 4 before (black) or 1 min after applying 10 μM Oxo-M (red). b Current traces from a were normalized to the end of the test pulse. c Summary of Oxo-M inhibition of Ca V 1.3b with different Ca V β-subunits. Maximal inward current amplitudes were measured after the onset of the test pulse using a trough seeking function (peak current). Percent of current inhibition was calculated as: %I inhib = 100*(I CTL -I DRUG )∕I CTL , where I CTL and I DRUG are the average maximum current amplitude of 5 traces prior to and after 1 min of application of test material (unless otherwise noted). d Schematic of quantification of kinetic changes. e, f Summary of kinetic changes (n = 4-6, ***P < 0.001, **P < 0.05) open bars, control; hatched bars, Oxo-M. e Time to peak (TTP) was measured using a minimum seeking function in Signal within the test pulse duration. f Current remaining (r40) was measured from an average of five normalized current traces per condition using the equation: r40 = 100*I end ∕I peak , where r40 is the percent of the maximum inward current remaining at the end of a 40 ms test pulse; I end is the current amplitude at the end of the test pulse; I peak is the maximum inward current measured during the test pulse Roberts-Crowley and Rittenhouse BMC Res Notes (2018) 11:681 After 1 min, Oxo-M significantly inhibited L-current by 58 ± 8% with β 1b ; 36 ± 12% with β 2a ; 66 ± 6% with β 3 ; and 72 ± 10% with β 4 (Fig. 1c). Oxo-M elicited kinetic changes that were visualized by normalizing individual traces to the end of the 40 ms test pulse (Fig. 1b), which were quantified by measuring TTP and r40 (Fig. 1d). TTP (Fig. 1e) and r40 (Fig. 1f ) decreased following Oxo-M with β 1b , β 3 , or β 4 ; however, no changes were detected with β 2a (P ≥ 0.11 for TTP; P ≥ 0.40 for r40). These differences in the magnitude of current inhibition and kinetics suggest that the Ca V β-subunit affects M 1 R modulation of Ca V 1.3b.

Dopamine D 2 receptors inhibit Ca V 2.2 but not Ca V 1.3 currents
Both M 1 Rs and D 2 Rs activate pathways involving G proteins, PLC, and AA release (Fig. 2a). However, whether L-current inhibition by D 2 Rs shows varied inhibition depending on Ca V β-subunit expression has not been examined. Therefore, we coexpressed D 2 Rs with Ca V 1.3b, α 2 δ-1 and different Ca V β-subunits. While Oxo-M inhibited Ca V 1.3b-β 2a currents over time (Fig. 2b), quin, a D 2 R agonist, had no effect on current amplitude (Fig. 2c) or kinetics (Fig. 2c inset, g). Since Ca V 1.3b-β 2a current shows less inhibition and no kinetic changes with Oxo-M, we tested whether Ca V 1.3b-β 3 current was sensitive to modulation by quin. Figure 2d shows a time course of Ca V 1.3b-β 3 current inhibition by Oxo-M whereas the time course with quin ( Fig. 2e) shows no inhibition or kinetic change (Fig. 2e inset, g). Several concentrations of quin were tested but did not inhibit L-current to the same extent as Oxo-M (Fig. 2f ). D 2 Rs appeared to desensitize with 10 μM quin. Application of quin for 1 min to cells co-transfected with the D 2 R-like family member, D 4.4 R, inhibited L-current by 8.5 ± 2.5% and did not produce changes in TTP or r40 (Additional file 2).
To confirm that lack of L-current inhibition was not due to poor expression of D 2 Rs, we repeated the experiment but substituted Ca V 2.2 for Ca V 1.3b to serve as a positive control since activated D 2 Rs also inhibit Ca V 2.2 [24][25][26]. Quin inhibited Ca V 2.2 by 45 ± 7% after 30 s and 48 ± 4% after 1 min (Fig. 3a). Inhibition occurred specifically by activating transfected D 2 Rs because cells transfected without D 2 Rs showed no response to quin (Fig. 3a,  n = 3). Moreover, N-current inhibition by quin occurred in a concentration-dependent manner (Fig. 3b, n = 3-5). Compared to lower concentrations, 10 μM quin resulted in less inhibition; inhibited current did not recover upon wash, suggesting this concentration causes receptor desensitization (data not shown). Thus, our findings indicate that transfected D 2 Rs functionally express in HEK-M1 cells to modulate Ca V 2.2, but not Ca V 1.3b VGCC activity.

M 1 R and D 2 R pathways use different signaling mechanisms to inhibit N-current
To compare D 2 Rs and M 1 Rs signaling pathways on Ca V 2.2 current, we first confirmed that activation of the stably transfected M 1 Rs could suppress N-current. Indeed, Oxo-M inhibited currents from β 3 -containing channels by 70 ± 5% after 30 s (Fig. 3c). When incubated with the PLA 2 antagonist OPC, cells showed less N-current inhibition by Oxo-M, 14 ± 8% inhibition after 30 s (Fig. 3c). In contrast, low concentrations of quin still suppressed N-current in the presence of OPC (Fig. 3d). Inhibition was relieved by pre-pulse facilitation (Fig. 3e,  g, h) and occurred in the presence of BSA, which acts as a scavenger of free AA (Fig. 3f-h), suggesting that quin mediates membrane-delimited inhibition of N-current. These findings suggest that M 1 Rs and D 2 Rs do not share a common pathway leading to N-current inhibition.

Discussion
Previously, the Ca V 1.3b splice variant of L-VGCCs, found in MSNs, had not been specifically tested for modulation by GPCRs. Here, using HEK-M1 cells, we present the novel finding that M 1 R stimulation inhibits Ca V 1.3b L-current with the accessory Ca V β-subunit determining the magnitude of inhibition. In contrast, stimulation of transfected D 2 Rs with quin does not recapitulate L-current inhibition observed in MSNs [3]. Pharmacological sensitivity to both FPL and NIM confirmed that Ca V 1.3b expressed in HEK-M1 cells behaves similarly to other recombinant Ca V 1.3 VGCCs [22,27].
We also report that N-current modulation by the D 2 R short splice variant appears similar to membrane-delimited inhibition by the D 2 R long form [24]. In this form of modulation, when G proteins are activated, Gβγ directly binds to and inhibits Ca V 2.2 which can be reversed by strong prepulses [10,28]. Indeed, D 2 R-mediated inhibition of Ca V 2.2 was independent of PLA 2 , whereas blockers of PLA 2 abolished inhibition by M 1 Rs. Thus, the membrane-delimited pathway may be at least partially responsible for the inhibition of Ca V 2.2 by D 2 Rs in MSNs [25].
In our experiments, the short splice variant of Ca V 1.3 (Ca V 1.3b) was unaffected by activation of D 2 Rs, expressed in HEK-293 cells, similar to a previous report on Ca V 1.3a, which has a longer C-terminus [24]. Since neither D 2 R-long inhibited Ca V 1.3a [24], nor D 2 R-short inhibited Ca V 1.3b (Fig. 2f ), one possibility is that another channel/ receptor combination occurs in vivo; however, D 2 R-long and short equally couple to G i proteins [29]. On the other hand, Ca V 1.3a binds a scaffolding protein found in the postsynaptic density of synapses known as Shank [30]. In MSNs, Ca V 1.3a requires an association with Shank for current inhibition by D 2 Rs [4]. Although lack of the longer

Conclusions
These findings highlight the specific regulation of Ca 2+ channels in a Ca V β-subunit dependent manner by different neurotransmitters. While M 1 R and D 2 R pathways contain similar signaling molecules and share a common functional output of inhibiting Ca 2+ channels, differences between the two cascades exist. Expression and localization of Ca V 1.3b associated with different Ca V β-subunits in a tissue or cell may dictate how Ca 2+ influx is modulated by nearby GPCRs, ultimately affecting Ca 2+ -dependent processes.

Limitations
Further experiments are needed to determine the differences in signaling between successful Ca V 1.3b inhibition by M 1 Rs versus none with D 2 Rs.

Additional files
Additional file 1. Pharmacological characterization of Ca V 1.3b L-current. HEK-M1 cells were washed with DMEM and the DNA mixture of Ca V 1.3b, α 2 δ-1, a β 3 -subunit and GFP was added and incubated for 1 h at 37 °C in a 5% CO 2 incubator. Supplemented media, without antibiotics, was then returned to the cells to bring the volume up to 1 ml (normal medium volume). After 2 h, cells were washed with supplemented media and washed a final time 2 h later. 10 mM MgSO 4 was added to the medium to block basal activity of Ca V 1.3b, which helped minimize excitotoxicity of transfected cells. Cells were transferred 24-72 h post-transfection using 2 mM EDTA in 1X PBS, to poly-l-lysine-coated coverslips. Recording began 1 h after transfer to coverslips. A Individual traces of Ca V 1.3b-β 3 current before (CTL) and after exposure to 0.3 µM NIM. B Concentration-response curve of L-current inhibition to NIM (n = 4-8). C Ca V 1.3b-β 2a currents before and after exposure to FPL (1 µM). Cells were stepped to a test potential of − 10 mV from a holding potential of − 90 mV followed by repolarization to − 90 or − 50 mV. Control (CTL) currents from β 2acontaining L-VGCCs show little to no inactivation as observed previously [31]. D Ca V 1.3b-β 3 currents before and after FPL. Cells were stepped to a test potential of − 10 mV from a holding potential of − 60 mV followed by repolarization to − 60 mV. Following FPL, both β 2a -and β 3 -containing channels exhibited slower activation and deactivation kinetics, hallmarks of agonist action on L-current [32]. E FPL enhancement of the Ca V 1.3b-β 2a current-voltage plot from a holding potential of − 90 mV (CTL, filled circles; FPL, open circles, n = 3, *P < 0.05). F Concentration-response curve of Ca V 1.3b-β 3 tail current enhancement to FPL (n = 4-8). Currents inhibited by NIM and enhanced by FPL fully recovered by washing with bath solution (data not shown).