Parkinson’s disease (PD) is one of the most common neurodegenerative disorders. Its motor symptoms are characterized by progressive degeneration of dopamine (DA)-releasing neurons in the Substantia nigra (SN), while neighboring DA neurons in the ventral tegmental area (VTA) remain largely unaffected (Damier et al., 1999; Giguère et al., 2018; Surmeier et al., 2017). Current PD therapy is only symptomatic and primarily based on the substitution of striatal DA by administration of L-DOPA or dopamine D2 receptor agonists. Unfortunately, none of the existing therapeutic approaches for PD patients is disease-modifying and can prevent disease progression (for review see Liss and Striessnig, 2019; Surmeier et al., 2011; Surmeier et al., 2017).

The development of novel neuroprotective strategies for the treatment of early PD requires the understanding of the cellular mechanisms responsible for the high vulnerability of SN DA neurons. Among these mechanisms, elevated metabolic stress appears to play a central role (for review see Liss and Striessnig, 2019), eventually triggering lysosomal, proteasomal and mitochondrial dysfunction (Burbulla et al., 2017; Surmeier et al., 2017). Intrinsic physiological properties of SN DA neurons, in particular increased cytosolic DA levels and high energy demand due to large axonal arborization favor metabolic stress (Bolam and Pissadaki, 2012; Liss and Striessnig, 2019). In addition, these neurons must handle a constant intracellular Ca²⁺-load resulting from dendritic and somatic Ca²⁺-oscillations triggered during their continuous electrical activity (Ortner et al., 2017; Surmeier et al., 2011). Dendritic Ca²⁺-transients largely depend on the activity of voltage-gated Ca²⁺ channels, in particular Cav1.3 L-type (LTCCs) and T-type channels (Guzman et al., 2018). Cav1.3 channels can activate at subthreshold membrane potentials (Koschak et al., 2001; Lieb et al., 2014; Xu and Lipscombe, 2001) and do not completely inactivate during continuous pacemaking activity (Guzman et al., 2018; Guzman et al., 2009; Ortner et al., 2017). Some, but not all, in vivo studies (Liss and Striessnig, 2019) showed neuroprotection by the systemic administration of dihydropyridine (DHP) LTCC blockers in 6-OHDA and MPTP animal models of PD thus further supporting a role of LTCCs as potential neuroprotective drug target. Based on these preclinical data and supporting observational clinical evidence (Liss and Striessnig, 2019), the neuroprotective potential of the DHP isradipine (ISR) was tested in a double-blind, placebo-controlled, parallel-group phase 3 clinical trial (“STEADY-PD III”, NCT02168842; Biglan et al., 2017). This trial reported no evidence for neuroprotection by ISR. Several explanations have been offered for this negative outcome (Investigators, 2020). One likely explanation is that voltage-gated Ca²⁺-channels (Cavs) other than LTCCs also contribute to Ca²⁺-transients in SN DA neurons. This is supported by the observation that only about 50% of the Ca²⁺-transients are blocked by ISR in the dendrites of SN DA neurons (Guzman et al., 2018) and that action potential-associated Ca²⁺-transients in the soma appear to be even resistant to ISR (Ortner et al., 2017). Therefore, in addition to L-type, other types of Cavs expressed in SN DA neurons (Branch et al., 2014; Evans et al., 2017; Philippart et al., 2016) may also contribute to Ca²⁺-induced metabolic stress in SN DA neurons. Cav2.3 (R-type) Ca²⁺-channels are very promising candidates. We have recently shown that SN DA neurons in mice lacking Cav2.3 channels were fully protected from neurodegeneration in the chronic MPTP-model of PD. Moreover, we found that Cav2.3 is the most abundant Cav expressed in SN DA neurons, and substantially contributes to activity-related somatic Ca²⁺-oscillations (Benkert et al., 2019). These findings make Cav2.3 R-type Ca²⁺ channels a promising target for neuroprotection in PD.

SN DA neurons are spontaneously active, pacemaking neurons, either firing in a low-frequency single-spike mode or transiently in a high-frequency burst mode (Grace and Bunney, 1984; Paladini and Roeper, 2014). During regular pacemaking their membrane potential is, on average, rather depolarized ranging from about –70 mV after an action potential (AP) to about –40 mV at firing threshold (Gantz et al., 2018; Guzman et al., 2018; Ortner et al., 2017). The contribution of a particular Cav channel to Ca²⁺-entry is largely determined by its steady-state inactivation properties, which determines its availability at these depolarized voltages. Therefore, in SN DA neurons steady-state inactivation of Cav2.3 channels must occur within a voltage-range preventing inactivation of a substantial fraction of channels during the positive operating range of these continuously firing neurons. However, Cav2.3 α1-subunits have originally been cloned as a low-voltage-gated channel with a negative steady-state inactivation voltage-range (V_0.5,inact –78 mV; Soong et al., 1993) with almost complete inactivation at voltages positive to –50 mV. Interestingly, SNX-482-sensitive R-type currents mediated by Cav2.3 α1 subunits (Newcomb et al., 1998) recorded from different neuronal cell types reveal a wide voltage-range of steady-state inactivation despite similar recording conditions (5–10 mM Ba²⁺ as charge carrier): half-maximal voltages for steady-state inactivation (V_0.5,inact) range from –90 to –70 mV in cerebellar, cortical and myenteric neurons (Bian et al., 2004; Sochivko et al., 2002; Tottene et al., 2000) to –58 mV in neurohypophyseal terminals (Wang et al., 1999) and to even as positive as –40 mV in GnRH-releasing neurons or GT1-7 cells (Kato et al., 2003; Watanabe et al., 2004). This raises the important question about potential molecular mechanisms accounting for this heterogeneity and its role for stabilization of R-type currents in SN DA neurons.

While alternative splicing of Cav2.3 α1 subunits is unlikely to account for such large shifts in steady-state inactivation (Pereverzev et al., 2002), accessory β-subunits appear as obvious candidates for several reasons. First, like for other high-voltage-activated Ca²⁺ channels (including Cav2.1 and Cav2.2, Liu et al., 1996; Scott et al., 1996) they form part of the Cav2.3 channel complex and thus affect its membrane targeting and gating properties (Zamponi et al., 2015). Second, previous work has shown that β-subunits tightly control V_0.5,inact of Cav2 channels, including Cav2.3. While all cytosolic β-subunit isoforms (β1 – β4 and cytosolic splice variants of β2) induce steady-state inactivation at negative potentials, being almost complete at voltages positive to –50 mV, the membrane-anchored β2 subunit splice variants β2a and β2e induce strong positive shifts of V_0.5,inact of Cav2.3 channels and slow the time course of voltage-dependent inactivation (Jones et al., 1998; Pereverzev et al., 2002; Soong et al., 1993; Williams et al., 1994; Yasuda et al., 2004). However, a specific physiological role for this modulatory effect has so far not been reported and the relative abundance of β2a and β2e subunits in neurons, including SN DA neurons, is unknown.

Here, we directly addressed the question if association of Cav2.3 channels with β2a or β2e but not with cytosolic β-subunits can prevent channel inactivation during SN DA activity patterns and could therefore serve as a mechanism allowing these channels to contribute to SN DA function. When co-expressed together with auxiliary α2δ1-subunits in tsA-201 cells we indeed found that during simulated SN DA neuron-like continuous tonic pacemaking or brief burst activity (Ortner et al., 2017) Cav2.3 channels remained active only when associated with β2a or β2e, but not the cytosolic β3 and β4 subunits. In contrast, steady-state inactivation of Cav1.3 channels was largely unaffected by β2a, suggesting that Cav1.3 availability is much less dependent on the presence of β2a subunits. Using RNAscope we confirmed the presence of both β2a and β2e transcripts in identified mouse SN and VTA DA neurons and quantitative PCR analysis showed that β2-subunits represent about 25% of all β-subunit transcripts in the mouse SN and VTA with about 50% comprising β2a and β2e variants. In patch-clamp recordings of SN DA neurons in mouse brain slices, we detected SNX-482-sensitive R-type Ca²⁺-currents with voltage-dependent inactivation properties suggesting a contribution of β2a- or β2e-stabilized Cav2.3-currents. Recordings in cultured DA midbrain neurons confirmed R-type current activity during the pacemaking cycle. Taken together, our data further support a role of Cav2.3 in SN DA neuron Ca²⁺-signaling and imply a potential (patho)physiological role of β-subunit alternative splicing.

To explore how Cav2.3 channels can contribute to DA neuron Ca²⁺-entry during sustained neuronal activity we expressed Cav2.3 α1-subunits together with its accessory α2δ1 and different β-subunits in tsA-201 cells under near-physiological conditions (Figure 1). For this purpose, we employed physiological extracellular Ca²⁺ (2 mM), weak intracellular Ca²⁺-buffering (0.5 mM EGTA, see methods), and, in addition to square pulse protocols, used typical AP waveforms previously recorded from SN DA neurons in mouse midbrain slices (2.5 Hz) or simulated bursts as command voltages as described (Ortner et al., 2017; see methods). Moreover, we specifically employed the Cav2.3e α1-subunit splice variant for our recordings, which among the six major Cav2.3 α1-subunit splice variants, was the only one detected in UV laser-microdissected mouse SN DA neurons in experiments using a qualitative single-cell RT-qPCR approach (Figure 1—figure supplement 1A, B).

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β-subunit transcripts in mouse SN and VTA

This prompted us to investigate if β2a and β2e splice variants are indeed expressed in SN tissue. First, we investigated β1 - β4 subunit expression patterns in the SN (and VTA for comparison, Figure 2A) dissected from brain slices of 12- to 14-week-old male C57Bl/6 N mice (Figure 2C) using a standard-curve-based absolute RT-qPCR assay (Schlick et al., 2010; Figure 2—figure supplement 1, Supplementary file 1, Supplementary file 2, Supplementary file 3). In both SN and VTA tissue, β4 (SN:~64%; VTA:~57%) and β2 (SN:~27%; VTA:~29%) represented the most abundant β-subunit transcripts, followed by β1 and β3 (~4–7%) (Figure 2A). Our findings are in excellent agreement (β2: 31–35%, β4: 41–45%) with cell-type-specific RNA sequencing data from identified mouse midbrain DA neurons (Brichta et al., 2015; Shin, 2015).

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Since the relative abundance of β2-subunit splice variants in the brain is unknown, we used our standard curve-based RT-qPCR assay to specifically quantify their expression in the SN and VTA (Figure 2B; for alignment of N-terminal β2 splice variants see Figure 2—figure supplement 1A). Assays were designed to specifically discriminate between β2a, β2b, and β2e. β2c and β2d N-termini were detected together (the β2d N-terminus is also present in β2c but with different alternative splicing in the HOOK region; Buraei and Yang, 2010; see methods). In SN and VTA, β2a (~30%) and β2e (~26%) transcripts together comprised about half of all tested β2-subunit splice variants, cytosolic β2c and β2d-species about 42% and β2b only about 3% (Figure 2B). Therefore, β2a and β2e together should be able to form a substantial fraction of Cav2.3 channel complexes in these neurons.

We further confirmed the presence of the various N-terminal β2 splice variants in individual UV-laser microdissected mouse SN DA neurons at the mRNA level using a qualitative PCR approach (Figure 2—figure supplement 2, Supplementary file 4, Supplementary file 5). Moreover, quantitative RNAscope analysis confirmed the expression of β2e and β2a in identified mouse SN and VTA DA neurons with β2e more abundantly expressed compared to β2a (Figure 2—figure supplement 2).

β2 splice variant-dependent regulation of Cav2.3 activity during SN DA neuron-like regular pacemaking activity

To test a possible role of β-subunits for Cav2.3 channel availability in SN DA neurons, we mimicked their electrical activity in tsA-201 cells because individual Ca²⁺-current components arising from Cav2.3 channel complexes associated with different β-subunits or even different splice variants cannot be isolated during continuous pacemaking in patch-clamp recordings of identified SN DA neurons. With co-expressed β3 and β4 isoforms simulated SN DA neuron regular pacemaker activity (initiated from a holding potential of –89 mV) induced large inward currents in response to single AP waveforms (Figure 3A and B). Cav2.3 channels conducted I_Ca during the repolarization phase of the AP (I_AP) without evidence for inward current during the interspike interval (ISI, Figure 3A and B bottom inset). However, I_AP decreased rapidly during continuous activity and almost completely disappeared after 1 (co-transfected β3)–2 min (co-transfected β4; Figure 3A, B, E and F). The time course of I_AP decrease was best fitted by a bi-exponential function (see legend to Figure 3). Our data, therefore, suggest that Cav2.3e α1-subunits, in complex with α2δ1 and β3 or β4, cannot support substantial inward currents during continuous SN DA neuron pacemaking activity. This is in contrast to our previously published finding of stable Cav1.3 Ca²⁺-channel activity persisting under near identical experimental conditions (Cav1.3 α1/α2δ1/β3 previously published data; Ortner et al., 2017; illustrated for comparison in Figure 3E and F, in gray).

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In contrast, the depolarizing shifts in steady-state inactivation and the slowing of inactivation kinetics by β2a and β2e subunits are sufficient to stabilize Cav2.3e currents during simulated regular pacemaking activity. When analyzed in parallel under identical experimental conditions I_AP decreased with a much slower time course with about 40% of the maximal initial I_AP still remaining even after 5 min of continuous activity (Figure 3C–F). The time course of I_AP decrease could be fitted by a bi-exponential function predicting a steady-state reached at 32.3% ± 0.77% (n=12) of the initial I_AP for β2a and 25.0% ± 0.12% (n=9) for β2e (see legend to Figure 3). Similar to co-transfected β3 or β4 subunits, β2a or β2e supported Cav2.3 I_Ca predominantly during the repolarization phase after the AP spike. However, in accordance with enhanced window currents at negative voltages (Figure 1D) these subunits also supported an inward current during the interspike interval (ISI) as evident from the first few sweeps (with the largest current amplitudes) (Figure 3C and D, bottom insets). I_AP persisting after 2 min (β3, β4) or 5 min (β2a, β2e) of pacemaking was completely blocked by 100 µM Cd²⁺ with almost complete recovery upon washout (Figure 3A–E).

Irreversible loss of I_Ca, a phenomenon also known as current "run-down" widely described for both native and recombinant Cavs (Kepplinger et al., 2000; Ortner et al., 2017; Schneider et al., 2018) during patch-clamp recordings, may also contribute to the current decay observed during simulated pacemaking. We, therefore, quantified the contribution of current run-down for Cav2.3 co-expressed with α2δ1 and β2a or β3 subunits first by applying short (20 ms) square pulses to V_max (holding potential –89 mV) with a frequency of 0.1 Hz (Figure 4A). With this protocol (short pulse, long inter-sweep interval, hyperpolarized holding potential) activity- and voltage-dependent inactivation of Cav2.3 channels should be minimized. While β3 co-transfected Cav2.3 channels showed a time-dependent current run-down to about 60% of the peak I_Ca after 5 min, β2a prevented the current decline during this period (Figure 4A). To further quantify the current run-down during simulated pacemaking, we interrupted the pacemaking protocol after different time periods by 20 s long pauses at –89 mV to allow channel recovery from inactivation (Figure 4B). Thus, the percent run-down can be estimated from the non-recovering current component (Figure 4B, horizontal dashed lines). After 30 s of pacemaking the current amplitude during the first AP after the pause was similar to I_AP during the initial AP with both β-subunits (β3: 96.9% ± 3.63%, 95% CI: 86.8–107.0, n=5, N=2; β2a: 107.3% ± 3.66%, 95% CI: 99.3–115.4, n=12, N=2, of the initial I_AP). After the pause that followed another 1 min of pacemaking, 83.6% ± 7.33% (95% CI: 63.2–103.9) of the initial current was still recovered with co-transfected β3 but after 2 more minutes of pacemaking recovery decreased to 54.7% ± 6.27% (95% CI: 34.7–74.7, Figure 4B;~45% run-down after 4.5 min in total, n=5). This time course is in good agreement with values obtained using the square-pulse protocol (Figure 4A). Again, run-down was largely prevented by co-expressed β2a-subunits (Figure 4B;~16% run-down after 4.5 min in total, n=12).

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These data clearly demonstrate that the I_AP decrease in response to simulated SN DA neuron pacemaking (2.5 Hz) is almost completely due to the reversible accumulation of Cav2.3 channels in inactivated states, an effect partially prevented by β2a and β2e.

Cav2.3 Ca²⁺ currents during simulated SN DA burst firing activity

In addition to regular pacemaking activity (in vitro) or irregular single spike mode (in vivo), burst firing with transient increases in intracellular Ca²⁺-load has been associated with neurodegeneration and selective neuronal vulnerability in Parkinson’s disease (Dragicevic et al., 2015; Schiemann et al., 2012). Thus, we investigated to which extent Cav2.3 Ca²⁺-channels can contribute to Ca²⁺-entry during bursts and after post-burst hyperpolarizing pauses of SN DA neurons. After reaching steady-state I_AP during simulated SN DA neuron pacemaking (β4: 1–2 min, β2a/β2e: 5–6 min, see also Figure 3E and F) we applied a computer modeled typical three-spike burst, followed by a 1.5 s long afterhyperpolarization-induced pause at more negative potentials as a command voltage as previously described (Ortner et al., 2017; Figure 5A). First, we quantified to which extent total burst Ca²⁺-charge, that is I_Ca integrated over the duration of the burst, changes as compared to total Ca²⁺-charge during the same duration of a single AP in steady-state (calculated as the mean of 3 APs preceding the burst). Integrated I_Ca during the burst was four- to sixfold higher than the mean integrated I_Ca during a steady-state AP with all co-expressed β-subunits (β2a, β2e, β4) (Figure 5B, left panel). However, it has to be considered that with co-expressed β4 only ~6% of the initial I_AP remained in steady-state during regular pacemaking (Figure 3E and F). Therefore, this relative increase will cause a much smaller rise in absolute Ca²⁺ charge compared to β2a/β2e-associated Cav2.3 where ~ 40% of I_AP persisted in steady-state (Figure 5B, Figure 3E and F).

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We also investigated if post-burst afterhyperpolarizations would allow Cav2.3 channels to recover from inactivation and thus mediate increased Ca²⁺-entry during the first APs after the burst. We first determined the recovery from inactivation of Cav2.3 channels co-expressed with α2δ1 and β4 or β2a using a square-pulse protocol (Figure 5C, Table 2; 1 s conditioning prepulse to V_max to inactivate Cav2.3 channels followed by a 10 ms step to V_max after different time periods at –89 mV). About 30% of currents recovered under these experimental conditions after 1.5 s at –89 mV with both co-expressed β4 and β2a (~sixfold increase of the remaining I_Ca after 1 s at V_max). In contrast, recovery during the hyperpolarizing pause after the burst of the AP protocol was much less pronounced (β4) or absent (β2a, β2e; Figure 5B, right panel). This may be due to the different pulse protocols inducing channel inactivation over different time periods and may stabilize inactivated states with different recovery times.

Table 2

Cav2.3 - recovery from inactivation 2 mM Ca2+
β-subunit	r100 [%]	r1500 [%]	r4000 [%]	r10000 [%]	n
β2a	15.7±2.4	39.3±4.6	53.4 ±4.5	74.2±3.6	17
β4	12.7±1.7	35.6±2.4	51.3 ±2.8	72.0±1.9	9

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Taken together, these data predict that β2a- and β2e-associated Cav2.3 channels can contribute to enhanced Ca²⁺-entry during brief burst activity but not during post-burst APs.

Steady-state activation and inactivation of R-type currents in mouse DA neurons

We have recently shown in whole-cell patch-clamp recordings that 100 nM SNX-482 inhibit ~30% of total Cav currents in mouse SN DA neurons (Benkert et al., 2019) when stimulated from a negative holding potential of –70 mV. If V_0.5,inact of Cav2.3 channels in SN DA neurons is indeed stabilized at more positive potentials, then the voltage-dependent inactivation of the R-type currents should allow a fraction of channels to be available even at voltages positive to –40 mV.

Using whole-cell patch-clamp recordings (Figure 6), we therefore measured the steady-state inactivation of R-type I_Ca in identified SN DA neurons in mouse midbrain slices. First, R-type current (i.e. current reminaing after blocking other Cav channels) was isolated as reported in previous publications by preincubation of slices with selective inhibitors of Cav1 (1 µM ISR), Cav2 (Cav2.1, Cav2.2; 1 µM ω-conotoxin MVIIC) and Cav3 (10 µM Z941) (Figure 6A, blue traces/symbols, see also methods section). R-type current evoked from –100 mV activated at ~8 mV more positive voltages than total I_Ca (Figure 6A, Table 3 for statistics). This observation is in accordance with the positive activation voltage-range of Cav2.3e channels measured in tsA-201 cells. The R-type current V_0.5,inact was ~5 mV more positive compared to total I_Ca (–47.5 mV vs. –52.7 mV; Figure 6A, Table 3) and therefore ~7 mV more negative than recombinant Cav2.3 currents expressed with β2a or β2e subunits (~ –40 mV, Table 1) and ~23–29 mV more positive than recombinant Cav2.3 currents expressed with β4 or β3. Since R-type currents may include current components not mediated by Cav2.3 (Newcomb et al., 1998; Sochivko et al., 2002; Tottene et al., 2000; Wilson et al., 2000), we also isolated SNX-482-sensitive currents, which must be mediated by Cav2.3 (Zamponi et al., 2015) by subtracting current remaining in the presence of SNX-482 from control current (Figure 6B). Although the slope of the voltage-dependence of activation was different for the SNX-482-sensitive current as compared to R-type current, steady-state inactivation was again observed at significantly more positive voltages as compared to control current (–44.6 mV vs. –52.9 mV, Figure 6B, Table 3), and ~5 mV more negative than recombinant Cav2.3 currents associated with β4 or β3 (Table 3). Although these experiments do not directly prove the contribution of β2a and/or β2e to R-type current in SN DA neurons, the strong requirement of β-subunits for Cav2.3 channel activity and the inability of other cytosolic β-subunit variants to account for the observed voltage-dependence of R-type current (Figure 1A and B) strongly suggests a contribution of these membrane-associated β-subunits for preventing complete inactivation of Cav2.3-mediated currents during typical activity patterns in these cells.

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Table 3

	Activation			Inactivation
	V0.5,act (95% CI) [mV]	kact (95% CI) [mV]	n/N	V0.5, inact (95% CI) [mV]	kinact (95% CI) [mV]	n/N
Control A (without Cav-blocker cocktail)	–37.8 (−39.3,–36.3)	9.17 (7.90, 10.6)	16/8	–52.7 (−53.9,–51.5)	10.5 (−11.6,–9.48)	16/8
+Cav blocker cocktail ("R-type")	–29.9 (−30.8,–29.1)***	5.49 (4.73, 6.30)***	19/5	–47.5 (−48.7,–46.4)***	9.15 (−10.2,–8.16)	19/5
Control B (before SNX-482)	–34.4 (−35.7,–33.2)	9.68 (8.59, 10.8)	9/6	–52.9 (−53.7,–52.1)	9.30 (−10.9,–8.61)	9/6
SNX-482 - sensitive current	–34.9 (−37.7,–32.0)	10.6 (8.26, 13.3)	9/6	–44.6 (−46.9,–42.2)+++	10.1 (−12.1,–8.24)	9/6

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We independently also tested the availability of SNX-482-sensitive R-type currents at more depolarized voltages using perforated patch recordings (Figure 6—figure supplement 1), which allow very stable recordings in identified SN DA neurons (Figure 6—figure supplement 1I, J). We held cells at a more positive holding potential of –60 mV, a voltage within the spontaneous depolarization phase of the ISI, where a substantial fraction of Cav2.3 channels must already be inactivated (Figure 6). Indeed, under these conditions still about 13% of the total I_Ca was inhibited by acute bath application of SNX-482 and inhibition was partially reversible upon washout (Figure 6—figure supplement 1A-D, Supplementary file 7). Under these conditions, about 10% of current were blocked reversibly by the L-type channel blocker nifedipine (10 µM, Figure 6—figure supplement 1E-H).

To further confirm the presence of inactivation-resistant R-type currents, we also recorded SNX-482-sensitive Ca²⁺ currents elicited from a holding potential of –70 mV in primary cultures of mouse DA midbrain neurons after 8–9 days in vitro. When added after the complete block of LTCC currents by 3 µM isradipine (comprising 24.7% ± 4.2% of total Ca²⁺-current, n=15), 100 nM SNX-482 significantly reduced non-LTCC currents by 41% ± 4% (95% CI: 33–49, n=20, N=4, paired Student’s t-test; P<0.001) (Figure 7A–D). All residual I_Ca components were blocked by adding 2 μM Cd²⁺ to the bath solution (Figure 7A and B).

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We also determined the voltage-dependence of steady-state activation (Figure 7E) and inactivation (Figure 7F) before (non-L, control) and during application of 100 nM SNX-482 and calculated parameters for the SNX-482-sensitive R-type current by subtracting the current resistant to 100 nM SNX-482 from the control current (legend to Figure 7 for details). SNX-482-sensitive currents (blue) activated with a half maximal activation voltage (V_0.5,act –9.2±0.6 mV) slightly more positive than non-LTCC current (V_0.5,inact = -12.5 ± 0.6 mV). To determine the steady-state inactivation, test pulses of 50 ms to 0 mV were preceded by 1 min prepulses to voltages from –80 to –10 mV (Figure 7F). SNX-482-sensitive current inactivation was best fit with a V_0.5,inact of –58.1±0.6 mV, which was 12–18 mV more positive than recombinant Cav2.3 currents expressed with β4 or β3 (Table 1). Despite an ~13 mV more negative inactivation range compared to SN DA neurons (Figure 6, Table 3), this still allowed substantial channel availability at voltages positive to –50 mV (Figure 7F, in contrast to currents with co-expressed cytosolic β-subunits Figure 1D).

Finally, we tested if SNX-482 also affects the spontaneous AP firing of these cultured midbrain neurons. In current-clamp recordings 100 nM SNX-482 significantly reduced the spontaneous firing frequency from 4.1±0.8 Hz (control, n=10, N=3; 95% CI: 2.1–6.1) to 1.1±0.2 Hz (SNX-482, n=10, 95% CI: 0.2–2.1, p=0.0036, paired Student’s t-test; Figure 7—figure supplement 1A, B), decreased the regularity of pacemaking and also caused changes in AP shape (Figure 7—figure supplement 1A-C). Cav2.3-mediated currents may therefore also contribute to the pacemaking cycle in cultured DA neurons.

Although the modulation of Cavs by β-subunits and the characteristic gating changes induced by N-terminally membrane-anchored β2-subunit splice variants have been well described in the literature, the physiological significance of these findings remained underexplored. Here, we provide strong evidence for an important role of β-subunit alternative splicing for Cav2.3 Ca²⁺-channel signaling during continuous SN DA neuron-like regular pacemaking activity. We show that only membrane-bound β2a and β2e stabilize Cav2.3 channel complexes with voltage-dependent inactivation properties preventing complete inactivation during the on-average depolarized membrane potentials encountered during the pacemaking cycle. This cannot only explain our previous finding of a substantial contribution of SNX-482-sensitive R-type currents to activity-dependent somatic Ca²⁺-oscillations in SN DA neurons in brain slices (Benkert et al., 2019) but also to pacemaking in cultured DA neurons. We also provide evidence for the expression of β2a and β2e subunit splice variants in highly vulnerable mouse SN DA and in more resistant VTA DA neurons. Together with our finding that R-type currents in SN DA neurons are available within a voltage-range more positive than expected for association with β4-, β3- or other cytosolic β2-subunits, our data strongly suggest that β2a and β2e contribute to non-inactivating Cav2.3 R-type currents required for normal DA neuron function. This may therefore also be of pathophysiological relevance given the previously reported pathogenic potential of Cav2.3 Ca²⁺-channels in PD pathophysiology (Benkert et al., 2019).

The large shift of V_0.5,inact of Cav2.3 R-type current by membrane-anchored β2a and β2e subunits would predict that Cav2.3 channel availability can be adjusted along a wide range of potentials (about 30 mV; V_0.5,inact –40 to –70 mV, Table 1) just by varying the abundance and local availability of these subunits relative to cytosolic β-subunits within a cell or nanodomain. This range is also reflected by an ~11–14 mV difference in V_0.5,inact of R-type currents in our recordings from mouse midbrain DA neurons in culture and ex vivo slice recordings from SN DA neurons. However, the voltage-range of steady-state inactivation in both preparations is clearly more positive than predicted from heterologous expression with β4- or β3-subunits. This could be due to a different contribution of β2a and/or β2e to Cav2.3 channel complexes in these preparations. Our data predict that this contribution is higher in SN DA neurons than in cultured cells.

Our quantitative PCR analysis of β-subunit expression demonstrate that, although β4 (60%) is the predominant one in the SN and VTA, β2-subunit transcripts are also abundant and comprise about 27% of all β-subunits. These data are in excellent agreement with RNAseq data from mouse midbrain DA neurons with expression levels of about 33% for β2% and 43% for β4 (Brichta et al., 2015; Shin, 2015) and transcriptomic analysis of laser-captured SN DA neurons from 18 human postmortem brains (30% β2, 45% β4; 15% β3, 8% β1; Aguila et al., 2021). While these transcriptomic data do not provide quantitative information about the expression of β2-splice variants, we clearly demonstrated the presence of both membrane-bound isoforms in identified SN DA neurons using PCR and RNAscope analysis.

Our transcript data (and confirmatory RNAseq data) show that β2a and β2e are present in SN DA neurons but still represent only a small fraction of all β-subunits present. However, our experimental data find a positive inactivation voltage-range suggesting that a majority of SNX-482-sensitive channels is associated with these β-subunits. At present biochemical data regarding preferential targeting of β-subunits to specific subcellular neuronal compartments and/or Cav-subtypes is absent but cannot be excluded. For example, β4-subunits may extensively target to channels at axonal presynaptic locations (as reported in cultured hippocampal neurons) or even to the nucleus (where they regulate gene transcription) and thus contribute less to Cav2.3 channel complexes at somatodendritic sites (Etemad et al., 2014). Moreover, differences in the affinity of β-subunits for their α1-subunit interaction domain in the I-II-linker may also support preferential association with β2 (De Waard et al., 1995).

Based on the finding that β-subunit association is required for expression of robust Cav2.3 channel currents (Figure 1A, Table 1), they must also play a key role in determining the gating properties of individual channel complexes. However, this does not exclude a modulatory role of other posttranslational modifications of Cav2.3 channels or protein interaction partners expressed at somatodendritic locations of SN DA neurons that could enhance Cav2.3 availability at more positive membrane potentials (such as Rab3-interacting proteins at axonal sites, Kiyonaka et al., 2007; Robinson et al., 2019).

A limitation of our work is that we cannot provide direct proof for a role of β2-subunit splice variants for R-type current modulation in DA neurons. Direct proof for the existence of such complexes would require biochemical studies requiring immunoprecipitation of Cav2.3 complexes from laser-dissected SN DA neurons. However, such studies, as well as other approaches, such as proximity ligation assays are hampered by the fact, that suitable β2-splice variant-specific antibodies are not available. We also found no suitable combination of antibodies raised in different species for such studies. Two mouse anti-β2 (not even splice variant-specific) antibodies that could be combined with our previously used rabbit anti-Cav2.3 α1 subunit antibody turned out to bind to other nonspecific bands or cross-reacted with β3 (not shown, data available upon request). Alternatively, direct proof for a role of β2-subunit splice variants could be obtained by a splice variant-specific gene knockout or siRNA-mediated knock-down of both β2a and β2e subunits in SN DA neurons, followed by isolation of Cav2.3 current components or Ca²⁺ transients of which only a fraction would be mediated by β2a/β2e-associated channels. This would be methodologically extremely challenging in these neurons.

We have previously shown that Cav2.3-knockout mice are protected from the selective loss of SN DA neurons in the chronic MPTP/probenecid PD model (Benkert et al., 2019). At least in this rodent model of PD, the observed protection provides strong evidence for a role of these channels in PD pathology. Pharmacological inhibition of Cav2.3 alone or together with other Cavs may therefore confer beneficial disease-modifying effects and a novel approach for neuroprotection in PD. The recent failure of the LTCC blocker ISR in the STEADY-PD III trial (Investigators, 2020) to prevent disease progression in early PD patients indicates that inhibition of LTCCs alone may not be sufficient for neuroprotection. Our previous preclinical findings identified also Cav2.3 channels as novel drug targets for neuroprotective PD-therapy. Therefore, the inhibition of Cav2.3 in addition to Cav1.3 may be required for clinically meaningful neuroprotection. However, Cav2.3-mediated R-type currents are notoriously drug-resistant (Schneider et al., 2013), and so far no selective potent small-molecule Cav2.3/R-type blocker has been reported.

Our findings reported here provide the rationale for exploring novel neuroprotective strategies based on Cav2.3 channel inhibition. These could take advantage of the predicted strong dependence of continuous Cav2.3 channel activity on gating properties such as those stabilized by β2a and/or β2e. Rather than inhibiting Ca²⁺-entry through the pore-forming α1-subunit, such a strategy could aim at reducing its contribution to the R-type current component persisting during continuous activity in SN DA neurons by interfering with the association of β2a and β2e subunits. Even if other β-subunits would replace them in the channel complex and ensure its stable expression, our data suggest that they would not be able to shift the steady-state inactivation voltage into the operating voltage-range of SN DA neurons like β2a and β2e. Such an approach appears realistic due to the availability of novel genetically encoded Ca²⁺-channel inhibitors for a cell-type-specific gene therapeutic intervention. One such approach (CaVablator) has elegantly been used to specifically target Ca²⁺-channel β-subunits for degradation by fusing β-specific nanobodies with the catalytic HECT domain of Nedd4L, an E3 ubiquitin ligase (Morgenstern et al., 2019). This strongly reduces Cav1- and Cav2-mediated Ca²⁺-currents in different cell types. At present, it is unclear to which extent other high-voltage-activated Cav2 channels (Cav2.1, Cav2.2) also contribute to the high vulnerability of SN DA neurons. However, our recent quantitative RNAscope analyses in mature SN DA neurons (Benkert et al., 2019) clearly demonstrate that Cav2.3 α1-subunit (Cacna1e) transcripts are the most abundant α1-subunit expressed in these cells, in excellent agreement with cell-type-specific RNAseq data of identified midbrain DA neurons (Brichta et al., 2015; Shin, 2015).

We also show that in cultured mouse DA neurons, 100 nM SNX-482 slows pacemaking. While SNX-482 is highly suitable to isolate Cav2.3-mediated Ca²⁺ current components, effects on pacemaking also must take into account that this toxin is also a potent blocker of Kv4.3 channels (Kimm and Bean, 2014) underlying A-type K⁺-currents (I_A). 60 nM nearly fully block reconstituted Kv4.3 currents in HEK cells (IC₅₀ ~3 nM). Therefore, one can argue that in current-clamp recordings, 50–300 nM SNX-482 could alter pacemaking or the AP shape by effectively blocking Kv4.3 channels. However, the observed SNX-482-induced shortening of APs and the reduction of spontaneous firing are difficult to reconcile with a block of I_A channels, which typically induces a broadening of APs in several neuronal preparations (Kim et al., 2005) and an increased frequency in DA neurons (Liss et al., 2001).

Therefore, the observed SNX-482 effects in cultured neurons provided no final answer to the question about a potential role of Cav2.3 on pacemaking. In brain slices of Cav2.3 knockout mice, small changes in AP spike waveforms were observed, without major changes in pacemaking activity (Benkert et al., 2019). Similarly, inconsistent findings for the contribution of LTCC currents for pacemaking in slice recordings (Dragicevic et al., 2014; Guzman et al., 2009; Mercuri et al., 1994; Ortner et al., 2017) and cultured neurons (Puopolo et al., 2007) have been reported but the reasons for this remain unclear.

Despite providing no direct biochemical evidence, we here show the first example for a potential physiological and perhaps even pathophysiological role of β2-subunit alternative splicing emphasizing a need for further investigation in other types of neurons.

All data generated or analyzed during this study are included in the manuscript and supporting files. Raw data have been provided for mean population data shown in figures and tables.

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Author details

1. Anita Siller

   Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

   Contribution

   Conceptualization, Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   The authors declare that they have no financial and non-financial competing interests

2. Nadja T Hofer

   Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

3. Giulia Tomagra

   Department of Drug Science, NIS Centre, University of Torino, Torino, Italy

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

4. Nicole Burkert

   Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

   Contribution

   Data curation, Writing – review and editing, Formal analysis

   Competing interests

   No competing interests declared

5. Simon Hess

   Institute for Zoology, Biocenter, University of Cologne, Cologne, Germany

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

6. Julia Benkert

   Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

7. Aisylu Gaifullina

   Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   No competing interests declared

8. Desiree Spaich

   Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   The authors declare that they have no financial and non-financial competing interests

9. Johanna Duda

   Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

   Contribution

   Data curation, Formal analysis, Writing – review and editing

   Competing interests

   The authors declare that they have no financial and non-financial competing interests

10. Christina Poetschke

    Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany

    Contribution

    Data curation, Formal analysis, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

11. Kristina Vilusic

    Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

    Contribution

    Data curation, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

12. Eva Maria Fritz

    Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

    Contribution

    Methodology, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

13. Toni Schneider

    Institute of Neurophysiology, University of Cologne, Cologne, Germany

    Contribution

    Resources, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

14. Peter Kloppenburg

    Institute for Zoology, Biocenter, University of Cologne, Cologne, Germany

    Contribution

    Data curation, Formal analysis, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

    "This ORCID iD identifies the author of this article:" 0000-0002-4554-404X

15. Birgit Liss

    1. Institute of Applied Physiology, University of Ulm, Ulm, Germany, Ulm, Germany
    2. Linacre College & New College, University of Oxford, Oxford, United Kingdom

    Contribution

    Supervision, Writing – review and editing, Formal analysis, Funding acquisition

    Competing interests

    No competing interests declared

16. Valentina Carabelli

    Department of Drug Science, NIS Centre, University of Torino, Torino, Italy

    Contribution

    Formal analysis, Supervision, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

17. Emilio Carbone

    Department of Drug Science, NIS Centre, University of Torino, Torino, Italy

    Contribution

    Formal analysis, Supervision, Funding acquisition, Writing – review and editing

    Competing interests

    The authors declare that they have no financial and non-financial competing interests

    "This ORCID iD identifies the author of this article:" 0000-0003-2239-6280

18. Nadine Jasmin Ortner

    Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Methodology, Writing – original draft, Writing – review and editing

    For correspondence

    nadine.ortner@uibk.ac.at

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3882-3283

19. Jörg Striessnig

    Department of Pharmacology and Toxicology, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    joerg.striessnig@uibk.ac.at

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9406-7120

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