CaV1.1 is a slowly activating voltage-gated Ca2+ channel that exists in two splice variants with different voltage sensitivities. By making chimeras of these variants, Tuluc et al. show that activation kinetics and voltage dependence are controlled by distinct molecular mechanisms in the voltage-sensing domains of repeats I and IV, respectively.
The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (Ca V ) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with Ca V 1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in Ca V 1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal Ca V 1 channels remained unknown. Using Ca V 1.2/2.1 chimeras expressed in dysgenic (Ca V 1.1) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of Ca V 1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of Ca V 1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with Ca V 1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the Ca V 1.2 IQ domain with that of Ca V 2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the Ca V 1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of Ca V 1.2 currents.ecently STAC3 (SH3 and cysteine-rich containing protein 3) has been identified as an essential regulator of calcium channel trafficking and function in skeletal muscle excitationcontraction (EC) coupling and a mutation in STAC3 has been linked to the severe muscle disease Native American myopathy (NAM) (1, 2). Expression of STAC3 is restricted to skeletal muscle, where STAC3 associates with the voltage-gated calcium channel Ca V 1.1 and is involved in mediating voltage-induced calcium release from the sarcoplasmic reticulum (3, 4). In nonmuscle cells, STAC3 was shown to facilitate functional membrane expression of Ca V 1.1 and alter the current properties of Ca V 1.2 (5), suggesting a role of STAC proteins as an L-type calcium channel (Ca V 1) regulator. STAC3 belongs to a family of adaptor proteins that comprises two additional isoforms, STAC1 and STAC2, which are highly expressed in the brain (1).All three STAC proteins contain one C1 domain and two SH3 protein interaction domains (6). We previously demonstrated that the stable association of STAC3 to the Ca V 1 channel complex in the triads of skeletal muscle relies on the C1 domain (7). In contrast, the NAM mutation, which impairs EC coupling and is located in the SH3-1 domain of STAC3 (2-4), did not abolish the interaction with ...
Highlights d PA produced by PLD1 is involved in distinct steps of neuroendocrine exocytosis d Secretagogue-evoked stimulation leads to the production of several PA species d Mono-unsaturated PA regulates the number of exocytotic events d Poly-unsaturated PA regulates fusion pore stability and expansion
P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming Ca V 2.1 α 1 subunit and the auxiliary α2δ-2 and β 4 subunits. β 4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126-Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β 4b cDNA. Heterologously expressed wild-type β 4b associated with GFP-Ca V 1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β 4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with Ca V 2.1 in tsA201 cells, β 4b and β 4b-L125P augmented the calcium current amplitudes, however, β 4b-L125P failed to stably complex with α 1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β 4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β 4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β 4b in cultured myotubes and hippocampal neurons. While binding of β 4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β 4b-L125P and the neuronal TRAF2 and NCK
Voltage-dependent calcium channels (CaV) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of CaV1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of CaV1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of CaV1.3 and in VSDs II and IV of CaV1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of CaV1.3 and in two splice variants of CaV1.2. In both channels the D4N (VSD IV) mutation resulted in a ̴5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in CaV1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined CaV1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle CaV1.1 channel.
-Although originally restricted to their structural role as major constituents of membranes, lipids are now well-defined actors to integrate intracellular or extracellular signals. Accordingly, it has been known for decades that lipids, especially those coming from diet, are important to maintain normal physiological functions and good health. This is especially the case to maintain proper cognitive functions and avoid neuronal degeneration. But besides this empiric knowledge, the exact molecular nature of lipids in cellular signaling, as well as their precise mode of action are only starting to emerge. The recent development of novel pharmacological, molecular, cellular and genetic tools to study lipids in vitro and in vivo has contributed to this improvement in our knowledge. Among these important lipids, phosphatidic acid (PA) plays a unique and central role in a great variety of cellular functions. This article will review the different findings illustrating the involvement of PA generated by phospholipase D (PLD) and diacylglycerol kinases (DGK) in the different steps of neuronal development and neurosecretion. We will also present lipidomic evidences indicating that different species of PA are synthesized during these two key neuronal phenomena.Keywords: exocytosis / neuroendocrine / neuron / phospholipase D / phosphatidic acid Résumé -Différentes formes d'acide phosphatidique sont produites au cours de la croissance neuronale et la neurosécrétion. Bien qu'originalement restreints à leur rôle majeur de constituants principaux des membranes, il est maintenant communément admis que certains lipides possèdent également une fonction importante d'intégration des signaux intra-ou extra-cellulaires. En accord avec cette notion, cela fait des décennies qu'il est reconnu que les lipides, principalement via l'alimentation, jouent un rôle crucial dans le maintien de l'homéostasie de nombreuses fonctions physiologiques et dans la santé humaine au sens large. Ceci est particulièrement le cas pour la conservation d'un niveau optimal des fonctions cognitives et pour lutter contre la dégénérescence neuronale. Toutefois, malgré ces connaissances empiriques, la nature exacte des lipides impliqués, ainsi que les mécanismes d'action mis en jeu peinent à émerger. Le développement récent de nouveaux outils génétiques, moléculaires, et pharmacologiques pour étudier les lipides in vitro et in vivo permettent à présent d'améliorer nos connaissances. Parmi ces lipides, l'acide phosphatidique joue un rôle particulier et central dans diverses fonctions cellulaires essentielles. Cet article résume les observations récentes qui illustrent que l'acide phosphatidique, produit par deux voies enzymatiques distinctes impliquant les phospholipases D et les diacylglycérol-kinases, est impliqué dans le développement neuronal et la neurosécrétion. Pour finir, nous présentons des résultats d'analyses lipidomiques qui indiquent que différentes formes de l'acide phosphatidique sont produites au cours de ces deux processus neuronaux majeur...
and different types of learning and memory. Recently, large-scale genetic analysis revealed de-novo missense mutations in their pore-forming a 1 -subunit (CACNA1D gene) in 6 patients associated with a neurodevelopmental syndrome including varying degrees of sporadic autism spectrum disorder (ASD, G407R), intellectual disability (A749G), neurological manifestations (including seizures, V401L) and endocrine symptoms (G403D, I750M). A typical hallmark of these mutations are severe gating changes compatible with a gain-of-channel-function. Here we investigated if similar gating changes are observed in a de-novo CACNA1D mutation (IIS4-S5 linker, Ca v 1.3 a 1mut ) which could explain symptoms in a patient diagnosed with a severe developmental disorder of unknown cause. Methods: Mutant (Ca v 1.3 a 1mut ) and wild-type Ca v 1.3 a 1 were co-expressed together with b 3 and a 2 d-1 subunits in tsA-201 cells and calcium currents (15mM) were measured using the whole cell patch-clamp technique. Results: Very similar to the previously characterized mutation V401L (IS6), A749G and I750M (IIS6), Ca v 1.3 a 1mut dramatically shifted the voltagedependence of Ca v 1.3 steady-state activation and inactivation to more negative voltages ($20 mV) without slowing of inactivation. A complete biophysical analysis revealed that these changes are compatible with a mutational gainof-function phenotype. Conclusion: By demonstrating the typical gating changes previously shown by us for CACNA1D de-novo missense mutations we propose that Ca v 1.3 a 1mut also explains the symptoms in this patient with a severe developmental disorder. Patients carrying such mutations may benefit from treatment with already available L-type Ca 2þ -channel blockers, such as nimodipine. Such CACNA1D missense mutations are likely underreported in large-scale genetic analyses. Support: Austrian Science Fund (FWF F4402, W1101).
Highlights d PA produced by PLD1 is involved in distinct steps of neuroendocrine exocytosis d Secretagogue-evoked stimulation leads to the production of several PA species d Mono-unsaturated PA regulates the number of exocytotic events d Poly-unsaturated PA regulates fusion pore stability and expansion
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