Functional Characterization of Alternative Splicing in the C Terminus of L-type CaV1.3 Channels

Tan, Bao Zhen; Feng, Jinteng; Tan, Ming; Yu, Dejie; Huang, Hongtao; Shen, Yiru; Soong, Tuck Wah

doi:10.1074/jbc.m111.265207

Cited by 84 publications

(109 citation statements)

References 35 publications

(49 reference statements)

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“…Together with three other mutations (V401L, G407D, I750M) that cause an even more severe neurodevelopmental syndrome [9,15], this strongly suggests that these mutations confer a high risk for these diseases [18]. 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].…”

Section: Resultsmentioning

confidence: 99%

“…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].…”

Section: Resultsmentioning

confidence: 99%

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Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

et al. 2018

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Recently, we and others identified somatic and germline de novo gain-of-function mutations in CACNA1D, the gene encoding the α1-subunit of voltage-gated Cav1.3 Ca2+-channels. While somatic mutations identified in aldosterone producing adenomas (APAs) underlie treatment-resistant hypertension, germline CACNA1D mutations are associated with a neurodevelopmental disorder characterized by a wide symptomatic spectrum, including autism spectrum disorder. The number of newly identified CACNA1D missense mutations is constantly growing, but their pathogenic potential is difficult to predict in silico, making functional studies indispensable to assess their contribution to disease risk.Here we report the functional characterization of previously identified CACNA1D APA mutations F747L and M1354I using whole-cell patch-clamp electrophysiology upon recombinant expression in tsA-201 cells. We also investigated if alternative splicing of Cav1.3 affects the aberrant gating of the previously characterized APA mutation R990H and two mutations associated with autism spectrum disorder (A479G and G407R). Splice-variant dependent gating changes are of particular interest for germline mutations, since the relative expression of Cav1.3 splice variants differs across different tissues and within brain regions and might therefore result in tissue-specific phenotypes. Our data revealed a complex gain-of-function phenotype for APA mutation F747L confirming its pathogenic role. Furthermore, we found splice-variant dependent gating changes in R990H, A749G and G407R. M1354I did not change channel function of Cav1.3 splice variants and should therefore be considered a rare non-pathogenic variant until further proof for its pathogenicity is obtained. Our new findings together with previously published data allow classification of pathogenic CACNA1D mutations into four categories based on prototypical functional changes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

et al. 2018

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“…IHC Ca V 1.3 Ca 2+ channels stand out by reason of their hyperpolarized activation range and modest inactivation (10,18,19,22,45). These properties have been attributed to the molecular composition of the native Ca 2+ -channel complexes that, in addition to the specific pore-forming Ca V 1.3α1 splice variant (46), auxiliary Ca V β2 (6,47), and Ca V α 2 δ2 (7) subunit, also contain interactions partners, such as Rab3-interacting molecule (RIM) (48,49); harmonin (50, 51); calmodulin; and, notably, CaBPs (this study and refs.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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

Picher

Gehrt

Meese

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Ca 2+ -binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca 2+ channels of type 1.3 (Ca V 1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2 LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2 LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2 LacZ/LacZ IHCs revealed enhanced Ca 2+ -channel inactivation. The voltage dependence of activation and the number of Ca 2+ channels appeared normal in Cabp2 LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits Ca V 1.3 Ca 2+ -channel inactivation, and thus sustains the availability of Ca V 1.3 Ca 2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.H earing relies on faithful transmission of information at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs; recently reviewed in refs. 1, 2). 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). In fact, postsynaptic spike rate adaptation during ongoing sound stimulation is thought to reflect primarily presynaptic vesicle pool depletion, with minor contributions of Ca V 1.3 inactivation or AMPA-receptor desensitization (23-26). CaBPs are calmodulin-like proteins that use three functional out of four helix-loop-helix domains (EF-hand) for Ca 2+ binding (27). They are thought to function primarily as signaling proteins (28) and differentially modulate calmodulin effectors (29,30). In addition, CaBPs m...

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Spectroscopic, Hirshfeld Surface Analysis, and Molecular Docking Studies on Potent Anti‐Inflammatory and Analgesic Activity Methyl 3‐methyl‐2‐((pyridin‐2‐ylcarbonyl)amino)Benzoate

Eşme

2020

ChemistrySelect

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The spectral analysis of methyl 3‐methyl‐2‐((pyridin‐2‐ylcarbonyl)amino)benzoate (L1) is investigated using the FT‐IR, 1H and 13C NMR and UV‐Vis analysis using the DFT methods (B3LYP and PBEPBE) with the 6‐311+G(d,p) basis set. The Hirshfeld and fingerprint analyses were used to investigate the intermolecular interactions in the crystal structure. The vibrational wavenumbers were fulfilled in the form of potential energy distribution. The absorption wavelengths, excitation energies, and oscillator strengths for the UV‐Vis were performed by the TD‐DFT calculations in the gas phase and DMSO, chloroform solvents with the major contributions to the electronic transitions. The chemical shifts values of the NMR in CDCl3 solvent were computed using the gauge independent atomic orbital (GIAO) method and compared with the experimental data. The NLO properties such as mean polarizability, the anisotropy of the polarizability and the mean first‐order hyperpolarizability were computed by using the finite field method. The high β values (2.617×10−30 and 6.481×10−30 esu using the B3LYP and PBEPBE) indicate that a title compound is an attractive object for NLO properties. Reduced density gradient was also given to study the weak interaction, strong attraction, and strong repulsion interactions of the molecule. Molecular docking studies with different receptors (3IX3, 3UW8, and 1P93) have been performed to demonstrate that L1 has good anti‐inflammatory and analgesic activity.

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Functional Characterization of Alternative Splicing in the C Terminus of L-type CaV1.3 Channels

Cited by 84 publications

References 35 publications

Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

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

Spectroscopic, Hirshfeld Surface Analysis, and Molecular Docking Studies on Potent Anti‐Inflammatory and Analgesic Activity Methyl 3‐methyl‐2‐((pyridin‐2‐ylcarbonyl)amino)Benzoate

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Functional Characterization of Alternative Splicing in the C Terminus of L-type CaV1.3 Channels

Cited by 84 publications

References 35 publications

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

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

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

Spectroscopic, Hirshfeld Surface Analysis, and Molecular Docking Studies on Potent Anti‐Inflammatory and Analgesic Activity Methyl 3‐methyl‐2‐((pyridin‐2‐ylcarbonyl)amino)Benzoate

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Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

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