Modulation of Voltage- and Ca2+-dependent Gating of CaV1.3 L-type Calcium Channels by Alternative Splicing of a C-terminal Regulatory Domain

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.

Gating defects of disease-causing de novo mutations in Cav1.3 Ca2+ channels

“…Like for G407R L the inactivation time course during 5-s depolarizations was also dramatically slowed for G407R S (Figure 3, Table 2). G407R S , in contrast to G407R L [10] retained an initial fast inactivating component, most likely representing residual CDI (Figure 3(c,d)), which is more pronounced in short Ca v 1.3 splice variants [25,31,32]. Despite reduced maximal current amplitudes, the slow inactivation resulted in larger absolute current amplitudes during prolonged depolarization than in WT S channels (Figure 3(c,d)).…”

“…Ca v 1.3 also undergoes extensive alternative splicing, giving rise to a major C-terminal long splice variant (Ca v 1.3 L ) and a number of C-terminal short splice variants, with Ca v 1.3 43s (Ca v 1.3 S ) being the most abundant one in the brain [25,28–30]. C-terminal splicing removes a modulatory domain allowing channels to undergo major changes in voltage-and Ca 2+ -dependent gating resulting in activation at lower voltages, enhanced Ca 2+ -dependent inactivation and increased channel open probability [25,27,28,31]. We have previously reported gating effects of the germline mutations A749G and G407R (situated at the intracellular end of IS6 and IIS6, respectively) only in the human C-terminally long (Ca v 1.3 L ) isoform [10].…”

Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

“…Compared with Ca V 1.3 channels studied in heterologous expression systems, Ca V 1.3 channels in IHCs show little inactivation, which has been attributed to inhibition of calmodulin-mediated Ca 2+ -dependent inactivation (CDI) (14-17) by Ca 2+ -binding proteins (CaBPs) (18,19) and/or the interaction of the distal and proximal regulatory domains of the Ca V 1.3α1 C terminus (20)(21)(22). This "noninactivating" phenotype of IHC Ca V 1.3 enables reliable excitation-secretion coupling during ongoing stimulation (23-25).…”

“…Ca 2+ channels at the IHC presynaptic active zone are key signaling elements because they couple the sound-evoked IHC receptor potential to the release of glutamate. IHC Ca 2+ -channel complexes are known to contain Ca V 1.3 α1 subunit (Cav1.3α1) (3-5), betasubunit 2 (Ca V β2) (6), and alpha2-delta subunit 2 (α2δ2) (7) to activate at around −60 mV (8-10), and are partially activated already at the IHC resting potential in vivo [thought to be between −55 and −45 mV (11, 12)], thereby mediating "spontaneous" glutamate release during silence (13).Compared with Ca V 1.3 channels studied in heterologous expression systems, Ca V 1.3 channels in IHCs show little inactivation, which has been attributed to inhibition of calmodulin-mediated Ca 2+ -dependent inactivation (CDI) (14-17) by Ca 2+ -binding proteins (CaBPs) (18,19) and/or the interaction of the distal and proximal regulatory domains of the Ca V 1.3α1 C terminus (20)(21)(22). This "noninactivating" phenotype of IHC Ca V 1.3 enables reliable excitation-secretion coupling during ongoing stimulation (23-25).…”

Voltage-Gated Calcium Channels

“…Deletion of the gene encoding Ca V 1.3 channels causes deafness in mice (Platzer et al 2000). The distal carboxy-terminal domain plays an autoregulatory role in both Ca V 1.3 and Ca V 1.4 channels (Singh et al 2006(Singh et al , 2008, but it is not known whether it is subject to proteolytic processing in vivo. Ca V 1.3 channels are regulated by multiple interacting proteins (Cui et al 2007;Jenkins et al 2010), which may be important in tuning their activity to fit the specific requirements of hair cells transmitting auditory information at different frequencies.…”