Abstract
Low voltage activation of CaV1.3 L-type Ca2+ channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. CaV1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in CaV1.4 L-type Ca2+ channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the CaV1.3 α1 subunit C terminus gives rise to a long (CaV1.342) and a short form (CaV1.342A), we investigated if a C-terminal modulatory mechanism also controls CaV1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with β3 and α2δ1 subunits in HEK-293 cells. Activation of calcium current through CaV1.342A channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several CaV1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting CaV1.3ΔC116 channels showed gating properties similar to CaV1.342A that were reverted by co-expression of the corresponding C-terminal peptide C116. Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of CaV1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control CaV1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates CaV1.3 channel activation at lower voltages expected to favor CaV1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.
Highlights
Ca2ϩ influx through voltage-gated L-type Ca2ϩ channels (LTCCs)2 regulates numerous physiological functions, including muscle contraction, hormone release, neuronal firing and plasticity, sensory function, and cardiac pacemaking (1)
3 subunits were selected because we have previously shown that they form a large fraction of dihydropyridine-sensitive LTCCs in the brain
In the absence of the C-terminal gating modulator (CTM), V0.5,act is shifted about 10 mV to more negative voltages by decreasing the slope factor of the activation curve but without measurable effects on the activation threshold
Summary
To generate splice variant CaV1.342A, the alternatively spliced exon 42A was introduced into CaV1.3 cDNA (CaV1.342 ϭ human CaV1.38A as in Ref. 4, GenBankTM accession number EU363339) by co-ligating an EcoRI-BamHI-cut PCR product containing exon 42A from a rat brain ␣1 subunit (GenBankTM accession number M57682) with HindIII-EcoRI-cut CaV1.3 cDNA into HindIII-BamHI (4622–7100)-cut CaV1.3-pBS vector. For analyzing the effects of exon 44, amino acid residues RTRYYETYI were introduced into an untagged construct containing the long CaV1.3 C terminus after position 1787 using standard PCR techniques. N-terminally EYFP-labeled constructs 5Ј-HindIII* and 3Ј-SalI* restriction sites flanking nucleotide sequences corresponding to the following CaV1.3 amino acid positions were introduced and cloned into HindIII-SalI-cut vector pEYFP-C1 as follows: peptide EF-preIQ-IQ-postIQ, amino acids 1453– 2137; EF-preIQ-IQ, amino acids 1453–1623; and EF-preIQ-IQPCRD, amino acids 1453–1664. For C158-CFP and C116-CFP, 5Ј-EcoRI* and 3Ј-BamHI* restriction sites were introduced to flanking nucleotide sequences corresponding to CaV1.3 amino acid positions 1980 –2137 and 2022–2137 and cloned into EcoRI-BamHI-cut vector pECFP-N1. To amplify specific cDNA templates from the analyzed tissues for the standard curves of each assay, primer pairs were as follows: exon 42 fwd primer, 5Ј-CAACCCTGTTTGCTTTGGTC-3Ј, rev primer, 5Ј-TGATTGACATGGTTTCCAAGC-3Ј; exon 42A fwd primer, 5Ј-CAACCCTGTTTGCTTTGGTC-3Ј, rev primer, 5Ј-CTTCCTTCCGGAGGAGTGC-3Ј.
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