β2-subunit alternative splicing stabilizes Cav2.3 Ca2+ channel activity during continuous midbrain dopamine neuron-like activity

Figures

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  Figure 1.. 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…

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  Figure 1—figure supplement 1.. Determination of the expressed Cav2.3 splice variants in SN DA neurons. (A) Cartoon of the exon structure of all Cav2.3 splice variants. The murine Cacna1e gene, coding for the Cav2.3 α1-subunits contains 48 exons with six major alternative splice variants (right panel). The splicing events include the variable use of exon 19, 21 nucleotides of exon 20 and exon 45. The outer and inner primer pairs chosen for identification of the different splice variants are located in the II-III loop and the C-terminus, covering the three described splicing sites. Expressed exons are shown as white boxes and splicing sites are indicated in red. (B). Agarose gel electrophoresis image showing two PCR products (363 bp II-III loop nested PCR fragment and 498 bp C-terminus nested PCR fragment) coding for the Cav2.3e splice variant of Cav2.3 (Cacna1e) found in mouse laser-dissected SN DA neuron derived cDNA (n=40, left). In contrast, all five PCR products (363 bp, 399 bp, and 420 bp in the II-III loop and 369 bp and 498 bp in the C-terminus nested PCR fragments) coding for all six major splice variants were found in whole brain tissue-derived cDNA (as positive control, right panel).

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  Figure 1—figure supplement 2.. Effect of β2a palmitoylation on the biophysical properties of Cav2.3 and Cav1.3 Ca2+ channels in tsA-201 cells. Since palmitoylation is reversible and regulated in an activity-dependent localized manner (Bijlmakers and Marsh, 2003; Matt et al., 2019), we also investigated the contribution of palmitoylation of β2a for Cav2.3e modulation under our experimental conditions. To mimic the de-palmitoylated form, we replaced the two N-terminal cysteines to serines (C3S/C4Sβ2a) which prevents plasma membrane anchoring of β2a (Gebhart et al., 2010; Qin et al., 1998). Data are shown for Cav1.3 (C-terminally long splice variant, Bock et al., 2011) or Cav2.3 α1-subunits co-expressed with α2δ1 and β2a (orange), C3S/C4Sβ2a (red) or β3 (green). Respective command voltages are given in each panel. To disrupt palmitoylation-mediated membrane anchoring, the two N-terminal cysteines of β2a (see Figure 2—figure supplement 1) were replaced by serines in C3S/C4Sβ2a. (A, B) Inactivation kinetics during a 5 s long depolarizing step to Vmax for Cav1.3L (A), 15 mM Ca2+, holding potential –89 mV or Cav2.3 (B), 2 mM Ca2+, holding potential –119 mV. Curves represent means ± SEM for the indicated number of…

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  Figure 2.. 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.

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  Figure 2—figure supplement 1.. Binding specificity of β1-β4 and β2a-β2e TaqMan assays and expression stability of endogenous control genes. (A) Alignment of the N-terminal amino acid sequence of the investigated β-subunits (β2a, β2d, β2e, β3, or β4). Residues responsible for membrane anchoring of β2a (palmitoylated cysteine residues are highlighted in orange) and β2e (positively charged amino acids forming a lipid binding motif are highlighted in blue) are indicated. Homologous exons are shaded in gray. Sequence accession numbers (human CACNB2 (β2) splice variants): β2a (NP_000715.2), β2d (NP_963887.2), β2e (NP_963864.1), β3 (NP_000716.2), β4e (NP_001307651.1). (B) The indicated β-subunit isoforms (β1-β4; n=3; gene names in parenthesis) and β2 splice variants (n=3) were recognized with high specificity (low CT value) by the corresponding RT-qPCR assay even in the presence of a 10-fold higher concentration of the mismatching DNA fragments corresponding to other isoforms/splice variants (β1–4: n=1; β2 splice variants: n=1). High binding specificity was confirmed by inefficient detection (high CT value) of all non-matching DNA fragments. We additionally performed experiments with a mixture of the specific with…

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  Figure 2—figure supplement 2.. Expression of β2-subunit splice variants in UV-LMD SN DA neurons. (A) Left: Overview of a cresyl violet (CV) stained coronal midbrain section of a juvenile wildtype mouse after UV-LMD of 10 SN DA neurons (scale bar: 250 μm, upper). Image of one CV stained SN DA neuron of a juvenile wildtype mouse before and after UV-LMD (scale bar: 10 μm, lower). Middle: β2-subunit transcript relative mRNA quantification by reverse transcription quantitative PCR-based in UV-LMD mouse SN DA neurons (n=10). Right: Capillary gel electrophoresis image illustrating PCR products of β2-subunit (Cacnb2) splice variants amplified from a UV-LMD cDNA template corresponding to 2.9 cells. (B) Left: Representative images showing β2a and β2e (red) or tyrosine hydroxylase (TH (Th), green) RNAscope fluorescence signals (combined with nuclear DAPI staining, blue) of individual SN DA neurons from an adult wildtype mouse. Scale bar: 5 μm. Right: Absolute mRNA transcript numbers per cell for β2 (Cacnb2) - variants β2a or β2e in adult SN DA and VTA DA neurons of adult wildtype mice (N=6, each), as indicated. Tukey’s boxplots are shown. Significances are indicated by asterisks: *** p<0.001, **** p<0.0001. For…

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  Figure 3.. 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).…

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  Figure 4.. 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 normalized to the inward current of the first AP. The current run-down component can be estimated from the non-recovering current component (horizontal dashed lines). Traces represent means ± SEM from the indicated number of experiments (N=2). BS, bath solution.

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  Figure 5.. Effects of different β-subunits on Cav2.3 currents during a simulated SN DA neuron three-spike burst and post-burst APs in tsA-201 cells. The burst command voltage was elicited after ~5–6 min (β2a, β2e) or ~1–2 min (β4) of regular pacemaking to reach steady-state IAP (β2a and β2e:~30% of the initial IAP, β4:~6% of the initial IAP, see Figure 3). (A) Normalized current responses of Cav2.3 channels co-expressed with β2a, β2e or β4 subunits (and α2δ1) induced by a command voltage (top panel) simulating a typical three-spike burst followed by a hyperpolarization phase at hyperpolarized potentials (lowest voltage: –82 mV) for 1.5 seconds. Remaining Cd2+-insensitive current components (100 µM Cd2+) were subtracted off-line to extract pure Cav2.3 mediated ICa. One of at least four experiments with similar results is shown. (B) The integrated ICa during a single AP before the burst (obtained as the mean of the three preceding APs) was set to 100% and compared with ICa during the three-spike burst integrated over the time period equivalent to one AP (left) or the first APs after the pause (right). All investigated β-subunits resulted in increased integrated ICa during the burst. Data…

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  Figure 6.. Voltage-dependence of gating of R-type currents in mouse SN DA neurons recorded in brain slices. (A) ICa recorded in SN DA neurons without (Control, black, n=16) or after preincubation ("R-type", blue, n=19) of slices with a Cav channel blocker cocktail to inhibit Cav3 (10 µM Z941), Cav1 (1 µM I), Cav2.1 and Cav2.2 (1 µM ω-conotoxin-MVIIC) (see Methods). Left panel: representative current traces of recordings at –20 mV test potentials (holding potential –100 mV); notice that similar amplitudes of control and R-type current were chosen to facilitate comparison of current kinetics. Right panel: voltage-dependence of steady-state activation and inactivation, curve fits to a Boltzman equation of all individual data points are shown. Data are means ± SEM. For parameters and statistics see Table 3. (B). SNX-482-sensitive currents (light blue) were obtained by subtracting current measured in the presence of SNX-482 (green) from the respective control ICa before addition of SNX-482 (black) (n=9). Left panel: Representative recording of a SNX-482 sensitive current component compared to the respective control (holding potential –100 mV, –10 mV test potential). Steady-state activation and…

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  Figure 6—figure supplement 1.. Inhibition of ICa in SN DA neurons of adult (12 weeks) mice by 100 nM SNX-482. To further prove the presence of SNX-482-sensitive R-type currents in SN DA neurons, we performed perforated-patch recordings. These allow stable recordings over a long period of time and to test for reversibility of channel block by 100 nM SNX-482. Recordings were performed with the pulse protocol shown in A (holding potential –60 mV) after blocking IA K+-currents known to be present in these cells to exclude block of IA by SNX-482 (Kimm and Bean, 2014) as described previously (Benkert et al., 2019). 100 nM SNX-482 significantly reduced current to 87% ± 4% of control ICa (n=8, p=0.012, one-sample t-test, for statistics see Supplementary file 7) and this inhibition was slowly reversible. (A) ICa before (black), during (red) and after (blue) SNX-482 application. (B) Example of peak ICa plotted over time during SNX-482 application. The asterisks mark the time points of the recordings shown on the left. Single depolarizing voltage steps to 0 mV were applied from a holding potential of –60 mV every 10 s. (C) Mean effect of SNX-482 on peak ICa. *, p<0.05 (n=8; two-tailed paired t-test). (D) Remaining peak…

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  Figure 7.. SNX-482–sensitive R-type currents in cultured mouse midbrain DA neurons. (A) Representative traces illustrating the inhibition of non-L-type ICa by 100 nM SNX-482 (red). Cells were initially perfused with a bath solution containing 3 μM isradipine (black). Full block was obtained using 2 μM Cd2+ (blue). Square pulses (50 ms) were applied to 0 mV from a holding potential of –70 mV (top) (B) Current amplitude values plotted as a function of time. After stabilization of ICa with ISR (black circles), 100 nM SNX-482 was applied. The remaining currents was blocked by 2 μM Cd2+. Current run-down in the absence of drugs during 150 s was less than 1% (0.48 ± 0.18%; n=4 cells). (C) SNX-482 inhibition expressed as % of control ICa after LTCC block using 3 μM ISR. (D) Mean current amplitude at the end of ISR application and at the end of SNX-482 application. Absolute current amplitude decreased from 529±57 pA (95% CI: 409–649) to 313±33 pA (95% CI: 245–381, n=20, p<0.01, paired Students t-test). Data represent the means ± SEM for the indicated number of experiments (N=4). Statistical significance was determined using paired Student’s t-test: *** p<0.001; **p<0.01; *p<0.05. (E) Left:…