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.

Tonic neurotransmitter release at sensory cell ribbon synapses is mediated by calcium (Ca2+) influx through L-type voltage-gated Ca2+ channels. This tonic release requires the channels to inactivate slower than in other tissues. Ca(v)1.4 L-type voltage-gated Ca2+ channels (LTCCs) are found at high densities in photoreceptor terminals, and alpha1 subunit mutations cause human congenital stationary night blindness type-2 (CSNB2). Ca(v)1.4 voltage-dependent inactivation is slow and Ca2+-dependent inactivation (CDI) is absent. We show that removal of the last 55 or 122 (C122) C-terminal amino acid residues of the human alpha1 subunit restores calmodulin-dependent CDI and shifts voltage of half-maximal activation to more negative potentials. The C terminus must therefore form part of a mechanism that prevents calmodulin-dependent CDI of Ca(v)1.4 and controls voltage-dependent activation. Fluorescence resonance energy transfer experiments in living cells revealed binding of C122 to C-terminal motifs mediating CDI in other Ca2+ channels. The absence of this modulatory mechanism in the CSNB2 truncation mutant K1591X underlines its importance for normal retinal function in humans.

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Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage‐activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3‐deficient (Cav1.3−/−) mice degenerate shortly after the onset of hearing. We therefore studied whole‐cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mm Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at −50 mV with Vmax=−1.9 ± 6.9 mV, V0.5=−15.0 ± 7.1 mV and k= 8.2± 1.1 mV (n= 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mm Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 ± 10.3 % (n= 14) with 10 μm nifedipine and increased to 178.5 ± 57.8 % (n= 14) by 5 μm Bay K 8644. A dose‐response curve for nifedipine revealed an IC50 of 2.3 μm, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 ± 10.8 pA pF−1 (n= 105) which is only 38 % of the value in inner hair cells. Single cell RT‐PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3−/− mice, Ba2+ current density was reduced to 0.6 ± 0.5 pA pF−1 (n= 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.