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• W4286703413 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract In dopaminergic (DA) Substantia nigra (SN) neurons Cav2.3 R-type Ca2+-currents contribute to somatodendritic Ca2+-oscillations. This activity may contribute to the selective degeneration of these neurons in Parkinson’s disease (PD) since Cav2.3-knockout is neuroprotective in a PD mouse model. Here, we show that in tsA-201-cells the membrane-anchored β2-splice variants β2a and β2e are required to stabilize Cav2.3 gating properties allowing sustained Cav2.3 availability during simulated pacemaking and enhanced Ca2+-currents during bursts. We confirmed the expression of β2a- and β2e-subunit transcripts in the mouse SN and in identified SN DA neurons. Patch-clamp recordings of mouse DA midbrain neurons in culture and SN DA neurons in brain slices revealed SNX-482-sensitive R-type Ca2+-currents with voltage-dependent gating properties that suggest modulation by β2a- and/or β2e-subunits. Thus, β-subunit alternative splicing may prevent a fraction of Cav2.3 channels from inactivation in continuously active, highly vulnerable SN DA neurons, thereby also supporting Ca2+ signals contributing to the (patho)physiological role of Cav2.3 channels in PD. Editor's evaluation This study finds that voltage-gated calcium channel auxiliary β2a and β2e splice variants confer gating properties on Cav2.3 channels that enable them to contribute sustained Ca2+ influx during pacemaking in substantia nigra dopaminergic neurons. This sustained Ca2+ influx may contribute to the selective vulnerability of substantia nigra dopaminergic neurons to neurodegeneration in Parkinson's disease. The work will be of great interest to ion channel biophysicists and neuroscientists interested in mechanisms of neurodegeneration. https://doi.org/10.7554/eLife.67464.sa0 Decision letter Reviews on Sciety eLife's review process Introduction 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 (V0.5,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 (V0.5,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 V0.5,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 V0.5,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. Results 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). Figure 1 with 2 supplements see all Download asset Open asset Biophysical properties of Cav2.3 channels co-transfected with different β-subunits (and α2δ1) in tsA-201 cells. (A) Current densities (pA/pF) with or without (gray) co-transfection of indicated β-subunits. Color code and n-numbers are given in the graphs. (B) Voltage-dependence of steady-state activation (normalized conductance G, right axis, solid lines) and inactivation (normalized ICa of test pulses, left axis, dashed lines, left n-numbers in parentheses). (C) Inactivation time course during 5 s depolarizing pulses to Vmax starting from a holding potential of –119 mV. Inset shows the first 200 ms of the 5 s pulse. Respective stimulation protocols are shown above each graph. The curves represent the means ± SEM for the indicated number of experiments (N = β2a: 5; β2d, β2e: 2; β3, β4, no β: 3). For statistics see Table 1. Vmax, voltage of maximal inward current. (D) Window currents measured in the presence of the indicated β-subunits were calculated by multiplying mean current densities (pA/pF) of I-V-relationships by the fractional current inactivation from steady-state inactivation curves at the indicated voltages. Data represent the means ± SEM for the indicated number of experiments (N = β2a: 5; β2d, β2e: 2; β3, β4: 3). Statistical significance was determined using one-way ANOVA with Bonferroni post-hoc test and is indicated: *** p<0.001; ** p<0.01; * p<0.05. Source data provided in Figure 1—source data 1. Figure 1—source data 1 Source data for data shown in Figure 1. https://cdn.elifesciences.org/articles/67464/elife-67464-fig1-data1-v4.xlsx Download elife-67464-fig1-data1-v4.xlsx β-subunit isoform-dependent regulation of Cav2.3 channel gating We first used standard square pulse protocols to quantify the effect of different β-subunits on the voltage-dependence of channel gating under our experimental conditions. In addition to β3, β4, and β2a analyzed in previous studies (Jones et al., 1998; Yasuda et al., 2004), we also included membrane-anchored β2e and β2d, a cytosolic β2 splice variant, in our head-to-head comparison (Figure 1). In agreement with earlier studies (Jones et al., 1998; Yasuda et al., 2004), we found that recombinant Cav2.3 channels when associated with cytosolic β3 or β4 subunits inactivate at negative voltages (V0.5,inact < –70 mV) and with a rapid inactivation time course (≥50% within 50 ms, Figure 1B–C, Table 1). The cytosolic β2d splice variant also showed a gating behavior very similar to β4. In contrast, β2a, which is anchored to the plasma membrane through N-terminal palmitoylation, slowed the inactivation time course and shifted Cav2.3 voltage-dependent inactivation by ~30 mV to more positive potentials compared to β4 (and β3). Likewise, with co-expressed β2e, which is membrane anchored through N-terminal phospholipid interaction (Buraei and Yang, 2010; Miranda-Laferte et al., 2014), gating properties were similar to β2a-containing Cav2.3 channel complexes (Figure 1A–C, Table 1). In the case of β2a, the shift in the voltage-dependence of inactivation, but not the slowing of the inactivation time course, was largely dependent on N-terminal palmitoylation as shown with the palmitoylation-deficient mutant C3S/C4Sβ2a (Figure 1—figure supplement 2). The voltage-dependence of activation was similar for all tested β-subunits (Figure 1A, Table 1). Due to the voltage-dependent inactivation at more depolarized potentials and the resulting overlap of the steady-state activation and inactivation curves (Figure 1B), β2a and β2e subunits induce window currents, i.e. steady-state Ca2+ influx, at negative potentials (Figure 1D). Our findings confirm and extend previous observations that membrane-anchored β2 subunits give rise to slowly inactivating Cav2.3 currents available at more positive voltages that could participate in the formation of such SNX-482-sensitive current components in neurons. Table 1 Voltage-dependence of activation and inactivation, and time course of inactivation of Cav2.3 co-transfected with α2δ1 and different β subunits in tsA-201 cells. β-subunitCav2.3 - Activation 2 mM Ca2+Cav2.3 - Inactivation 2 mM Ca2+V0.5[mV]k[mV]Vrev[mV]act thresh[mV]current density[pA/pF]nV0.5, inact[mV]kinact[mV]plateau[%]nno β–10.7±1.123β2a–14.8±1.24.8±0.237.1±0.9–32.0±0.9–130.0+++±15.226–40.6±1.67.3±0.50.4±1.113C3S/C4Sβ2a–13.9±1.74.7±0.338.2±1.1–30.9±0.7–105.7+++±21.412–62.6***/###/§§§±1.68.3##±0.43.8±1.69β2d–15.9§±1.14.6±0.237.2±1.1–32.1§±0.7–107.7+++±13.417–69.9***/###±2.08.2##±0.22.0±0.710β2e–14.1±0.94.8±0.240.3*±0.6–31.5±0.7–96.8+++±14.332–39.9±1.96.5±0.33.8±1.224β3–10.5±0.75.2±0.239.8±0.9–29.2±0.7–64.6++±12.817–76.2***/###±1.07.0±0.14.1±0.714β4–13.1±0.75.0±0.238.5±0.5–31.0±0.5–74.8+++±11.917–70.3***/###±1.27.5±0.12.4±0.411Cav2.3–5 s Inactivation time course 2 mM Ca2+β-subunitr50 [%]r100 [%]r250 [%]r500 [%]r1000 [%]r5000 [%]nβ2a71.3±2.152.4±2.827.8±2.914.3±2.36.5±1.21.3±0.317C3S/C4Sβ2a67.4§§§/%±5.247.3#/§§§/%%±6.423.4§±5.311.6±3.36.2±1.73.4±1.111β2d57.4*/###/§§§±2.832.6***/###/§§±2.411.3*/###±1.74.4**/###±0.92.4**/###±0.61.7±0.521β2e77.5±2.964.2±3.341.1±3.222.3±2.810.1±2.03.3±0.827β332.9***/###±2.914.2***/###±1.56.1***/###±1.04.4**/###±0.72.9#±0.51.6±0.316β450.3***/###/§§±2.727.1***/###±2.58.6**/###±1.43.7**/###±0.82.0**/###±0.51.0±0.215 Table 1—source data 1 Source data for data shown in Table 1. https://cdn.elifesciences.org/articles/67464/elife-67464-table1-data1-v4.xlsx Download elife-67464-table1-data1-v4.xlsx Since Cav2.3 α1-subunits have also been reported to mediate ICa even when expressed in the absence of β-subunits (Jones et al., 1998; Yasuda et al., 2004), we also tested to which extent β-subunit modulated channels are expected to contribute to Cav2.3 currents. In our experiments, all five tested β-subunits (β2a, β2d, β2e, β3, β4) caused a robust and highly significant (6–12-fold; Figure 1A, Table 1) increase in current densities. This implies that β-associated Cav2.3 channels contribute more to overall Cav2.3-mediated currents than channel complexes devoid of β-subunits and thus can be subject to differential modulation by β-subunits. Taken together, these data suggest that through their effect on inactivation gating β2a- and β2e-subunits could contribute to sustained Cav2.3 activity and AP-associated Ca2+ transients previously recorded in SN DA neurons (Benkert et al., 2019). 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). Figure 2 with 2 supplements see all Download asset Open asset Transcript expression of various β-subunits and β2-subunit splice variants in mouse SN and VTA tissue. (A) Expression of β1-β4 subunit transcripts in SN (n=3, N=3) (left) and VTA (n=3, N=3) (right) determined by RT-qPCR as described in Methods. (B) Expression of β2a-β2e subunit transcripts in SN (n=3, N=3) (left) and VTA (n=3, N=3) (right). Data are shown as the mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Bonferroni post-hoc test: *** p<0.001; *p P<0.01; * p<0.05. Data was normalized to Gapdh and Tfrc determined by geNorm. (C) Example for four SN (left) and two VTA (right) tissue punches obtained for cDNA preparation with diameters of 0.5 mm each (left) or 0.8 mm each (right) from 7 to 8 successive 100-μm-sections between Bregma –3.00 mm and –3.80 mm, stained with Cresyl violet. Source data provided in Figure 2—source data 1. Figure 2—source data 1 Source data for data shown in Figure 2. https://cdn.elifesciences.org/articles/67464/elife-67464-fig2-data1-v4.xlsx Download elife-67464-fig2-data1-v4.xlsx 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 ICa during the repolarization phase of the AP (IAP) without evidence for inward current during the interspike interval (ISI, Figure 3A and B bottom inset). However, IAP 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 IAP 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). Figure 3 Download asset Open asset Activity-dependent inactivation of Cav2.3 channels co-transfected with different β subunits (and α2δ1) during simulated SN DA neuron regular pacemaking activity in tsA-201 cells. (A–D) Top panel: The SN DA neuron-derived command voltage was applied with a frequency of 2.5 Hz (only a time interval around the AP-spike is shown). Middle panel: Corresponding representative Ca2+ current traces (2 mM charge carrier) for Cav2.3 channels co-expressed with α2δ1 and β3 (A, green), β4 (B, purple), β2a (C, orange), or β2e (D, blue) are shown for the indicated sweep number (1st, 10th, 100th, 295th, 720th). Cav2.3 currents were completely blocked by 100 μM Cadmium (Cd2+), and remaining Cd2+-insensitive current components were subtracted off-line (bottom panel). ISI, interspike interval. (E) Current decay during simulated 2.5 Hz SN DA neuron pacemaking. Normalized peak inward current during APs (IAP) is plotted against time as mean ± SEM for the indicated number of experiments. IAP amplitudes were normalized to the IAP amplitude of the first AP after holding the cell at –89 mV. Cav1.3L co-expressed with α2δ1 and β3 (gray, mean only) is shown for comparison (data taken from Ortner et al., 2017). The IAP decay was fitted to a bi-exponential function (Cav2.3 β3: Aslow = 39.4% ± 0.65 %, τslow = 22.2 ± 0.15 min, Afast = 54.2 ± 0.76 %, τfast = 2.86 ± 0.07 min, non-inactivating=4.47% ± 0.13%; β4: Aslow = 48.5 ± 0.26 %, τslow = 90.3 ± 1.07 min, Afast = 41.8 ± 0.40 %, τfast = 8.39 ± 0.16 min, non-inactivating=5.12% ± 0.12%; β2a: Aslow = 52.6 ± 0.47 %, τslow = 299.3 ± 10.2 min, Afast = 13.4 ± 0.53 %, τfast = 18.2 ± 1.47 min, non-inactivating=32.3% ± 0.77%; β2e: Aslow = 67.7 ± 0.11 %, τslow = 294.1 ± 1.77 min, Afast = 7.10.0±0.27 %, τfast = 16.6 ± 1.24 min, non-inactivating=25.0% ± 0.12%). Curves represent the means ± SEM for the indicated number of experiments (independent transfections, N=Cav2.3/β3: 4; Cav2.3/β4: 2; Cav2.3/β2a: 4; Cav2.3/β2e: 2; Cav1.3L/β3: 6). (F) Normalized IAP decay after predefined time points for Cav2.3 with β3, β4, β2a or β2e, and Cav1.3L (with β3). Color-code and n-numbers as in panel E. Statistical significance was determined using one-way ANOVA followed by Bonferroni post-hoc test: *** p<0.001; ** p<0.01; * p<0.05. Source data provided in Figure 3—source data 1. Figure 3—source data 1 Source data for data shown in Figure 3. https://cdn.elifesciences.org/articles/67464/elife-67464-fig3-data1-v4.xlsx Download elife-67464-fig3-data1-v4.xlsx 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 IAP decreased with a much slower time course with about 40% of the maximal initial IAP still remaining even after 5 min of continuous activity (Figure 3C–F). The time course of IAP decrease could be fitted by a bi-exponential function predicting a steady-state reached at 32.3% ± 0.77% (n=12) of the initial IAP 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 ICa 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). IAP 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 ICa, 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 Vmax (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 ICa 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 IAP 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 IAP). 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). Figure 4 Download asset Open asset β-subunit-dependent run-down of Cav2.3 channel Ca2+ current in tsA-201 cells. Data for Cav2.3 co-expressed with α2δ1 and β2a (orange) or β3 (green) are shown. (A). Run-down during a 0.1 Hz square pulse protocol (20 ms to Vmax, holding potential –89 mV). Currents were normalized to the ICa of the sweep with the maximal peak inward current observed during the recording. After a full block with 100 µM Cd2+ currents recovered to the amplitude preceding the Cd2+ application. (B) Cells were held at –89 mV and then stimulated using the regular SN DA neuron pacemaking protocol for 30 s, 1 min, and 2 min each followed by 20 s long pauses (vertical dashed lines) at hyperpolarized potentials (–89 mV) to allow channel recovery from inactivation. IAP of individual APs was norma" @default.
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• W4286703413 date "2022-06-24" @default.
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• W4286703413 title "Author response: β2-subunit alternative splicing stabilizes Cav2.3 Ca2+ channel activity during continuous midbrain dopamine neuron-like activity" @default.
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