Autism spectrum disorders (ASD) are neurodevelopmental conditions characterized by impaired social interaction, communication skills, and restricted and repetitive behavior. The genetic causes for autism are largely unknown. Previous studies implicate CACNA1C (L-type Ca V 1.2) calcium channel mutations in a disorder associated with autism (Timothy syndrome). Here, we identify missense mutations in the calcium channel gene CACNA1H (T-type Ca V 3.2) in 6 of 461 individuals with ASD. These mutations are located in conserved and functionally relevant domains and are absent in 480 ethnically matched controls (p ‫؍‬ 0.014, Fisher's exact test). Non-segregation within the pedigrees between the mutations and the ASD phenotype clearly suggest that the mutations alone are not responsible for the condition. However, functional analysis shows that all these mutations significantly reduce Ca V 3.2 channel activity and thus could affect neuronal function and potentially brain development. We conclude that the identified mutations could contribute to the development of the ASD phenotype.Autism spectrum disorders affect ϳ0.5% of children in the general population and cause great morbidity (1, 2). Epidemiologic studies estimate that up to 400,000 children are affected in the United States alone (3). The primary features of ASD are severe difficulties in social interaction, communication deficits, and unusual behaviors, including repetitive and/or ritualistic actions. There is considerable variation in the severity of phenotypes in autism spectrum disorders, which include autism, Asperger syndrome, childhood disintegrative disorder, Rett syndrome, and pervasive developmental disorder not otherwise specified. Despite the high prevalence and importance of autism spectrum disorders, very little is known about underlying molecular and cellular mechanisms (4).Timothy syndrome (TS) 3 is a complex physiological and developmental disorder, which includes autism spectrum disorders. We discovered that TS resulted from a recurrent, de novo CACNA1C calcium (Ca 2ϩ ) channel mutation, G406R. Our findings that individuals with TS met the criteria for autism, or had severe deficits of language and social development, suggest that abnormal Ca 2ϩ signaling may cause these disorders (5). Based on these results we hypothesized that mutations in other Ca 2ϩ channel genes might be responsible for non-syndromic forms of ASD.Neuroanatomical studies of autistic patients have found histological abnormalities in the major regions of the limbic system including the hippocampus and amygdala and in the cerebellum and cerebral cortex (6). Ca V 3.2, a T-type calcium channel encoded by the CACNA1H gene, is abundantly expressed in these and other regions. T-type Ca 2ϩ channels activate with relatively small depolarization of the neuron membrane triggering low threshold spikes that contribute to rebound burst firing and oscillatory behavior in central neurons (7). In the thalamus, this behavior maintains normal transitions in sensory gating, sleep, and arousal (8,...

Background Apabetalone (RVX-208) is a bromodomain and extraterminal protein inhibitor (BETi) that in phase II trials reduced the relative risk (RR) of major adverse cardiac events (MACE) in patients with cardiovascular disease (CVD) by 44% and in diabetic CVD patients by 57% on top of statins. A phase III trial, BETonMACE, is currently assessing apabetalone’s ability to reduce MACE in statin-treated post-acute coronary syndrome type 2 diabetic CVD patients with low high-density lipoprotein C. The leading cause of MACE is atherosclerosis, driven by dysfunctional lipid metabolism and chronic vascular inflammation (VI). In vitro studies have implicated the BET protein BRD4 as an epigenetic driver of inflammation and atherogenesis, suggesting that BETi may be clinically effective in combating VI. Here, we assessed apabetalone’s ability to regulate inflammation-driven gene expression and cell adhesion in vitro and investigated the mechanism by which apabetalone suppresses expression. The clinical impact of apabetalone on mediators of VI was assessed with proteomic analysis of phase II CVD patient plasma. Results In vitro, apabetalone prevented inflammatory (TNFα, LPS, or IL-1β) induction of key factors that drive endothelial activation, monocyte recruitment, adhesion, and plaque destabilization. BRD4 abundance on inflammatory and adhesion gene promoters and enhancers was reduced by apabetalone. BRD2-4 degradation by MZ-1 also prevented TNFα-induced transcription of monocyte and endothelial cell adhesion molecules and inflammatory mediators, confirming BET-dependent regulation. Transcriptional regulation by apabetalone translated into a reduction in monocyte adhesion to an endothelial monolayer. In a phase II trial, apabetalone treatment reduced the abundance of multiple VI mediators in the plasma of CVD patients (SOMAscan® 1.3 k). These proteins correlate with CVD risk and include adhesion molecules, cytokines, and metalloproteinases. Ingenuity® Pathway Analysis (IPA®) predicted that apabetalone inhibits pro-atherogenic regulators and pathways and prevents disease states arising from leukocyte recruitment. Conclusions Apabetalone suppressed gene expression of VI mediators in monocytes and endothelial cells by inhibiting BET-dependent transcription induced by multiple inflammatory stimuli. In CVD patients, apabetalone treatment reduced circulating levels of VI mediators, an outcome conducive with atherosclerotic plaque stabilization and MACE reduction. Inhibition of inflammatory and adhesion molecule gene expression by apabetalone is predicted to contribute to MACE reduction in the phase III BETonMACE trial. Electronic supplementary material The online version of this article (10.1186/s13148-019-0696-z) contains supplementary material, which is available to authorized users.

Specialized proteins in the plasma membrane, endoplasmic reticulum (ER), and mitochondria tightly regulate intracellular calcium. A unique mechanism called store-operated calcium entry is activated when ER calcium is depleted, serving to restore intra-ER calcium levels. An ER calcium sensor, stromal interaction molecule 1 (STIM1), translocates within the ER membrane upon store depletion to the juxtaplasma membrane domain, where it interacts with intracellular domains of a highly calcium-selective plasma membrane ion channel, Orai1. STIM1 gates Orai1, allowing calcium to enter the cytoplasm, where it repletes the ER store via calcium-ATPases pumps. Here, we performed affinity purification of Orai1 from Jurkat cells to identify partner of STIM1 (POST), a 10-transmembrane–spanning segment protein of unknown function. The protein is located in the plasma membrane and ER. POST-Orai1 binding is store depletion-independent. On store depletion, the protein binds STIM1 and moves within the ER to localize near the cell membrane. This protein, TMEM20 (POST), does not affect store-operated calcium entry but does reduce plasma membrane Ca 2+ pump activity. Store depletion promotes STIM1–POST complex binding to smooth ER and plasma membrane Ca 2+ ATPases (SERCAs and PMCAs, respectively), Na/K-ATPase, as well as to the nuclear transporters, importins-β and exportins.

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

L-type, voltage-gated Ca2؉ channels (Ca L ) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle Ca L channel isoforms are acutely potentiated following ␣5␤1 integrin activation. Only the ␣ 1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by ␣5␤1 integrin requires phosphorylation of ␣ 1C C-terminal residues Ser 1901 and Tyr 2122 . These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following ␣5␤1 integrin activation, native Ca L channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of Ca L channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.Voltage-gated calcium channels play critical roles in the regulation of calcium entry across the plasma membranes of excitable cells. L-type calcium channels (Ca L ), 5 which are highly expressed in brain and muscle, are heteromeric transmembrane proteins composed of a poreforming ␣ 1C (Cav1.2) subunit along with accessory ␤, ␣ 2 , ␦, and sometimes ␥ subunits (1, 2). The ␣ 1C subunit contains four highly conserved repeat regions with 24 membrane-spanning domains, in addition to a variable length N terminus and relatively long, intracellular C terminus. The three ␣ 1C isoforms (neuronal, Cav1.2c; smooth muscle, Cav1.2b; cardiac, Cav1.2a) exhibit significant sequence differences in their N and C termini but all are regulated by intracellular kinases in ways that uniquely determine calcium entry and cell excitability.The regulation of Ca L channels by serine-threonine kinases has been extensively investigated. PKG phosphorylates a conserved serine reside in the cytoplasmic I-II linker (3) of all three ␣ 1C isoforms, leading to inhibition of current. PKC phosphorylates N-terminal threonine residues in cardiac and smooth muscle isoforms (4 -6) leading in most cases to potentiation of current. PKA phosphorylates all three ␣ 1C isoforms at a conserved C-terminal serine (Ser 1901 in Cav1.2c; Ser 1928 in Cav1.2a), thereby mediating ␤-adrenergic potentiation of the calcium current in cardiac myocytes and neurons (7-9). PKA also regulates ␣ 1C in smooth muscle, but the functional consequences on calcium current are complicated by crossover activation of PKG, which is expressed at high levels in that tissue (10).We recently demonstrated that Ca L currents in vascular smooth muscle (VSM) are acutely regulated by the integrin class of cel...

Voltage-dependent inactivation of calcium channels is a key mechanism for regulating intracellular calcium levels and neuronal excitability. In sodium and potassium channels, the molecular determinants that govern fast inactivation involve pore block by a cytoplasmic gating particle. As we discuss here, there is an increasing body of evidence that is consistent with a qualitatively similar inactivation mechanism in high-voltage-activated calcium channels. Work from a number of laboratories has implicated both cytoplasmic regions and the pore-lining S6 transmembrane helices in the inactivation process. Together with our recent findings, this leads us to propose a model in which the intracellular domain I-II linker region acts as a 'hinged lid' that physically occludes the pore by docking to the cytoplasmic ends of the S6 segments. We further propose that the ancillary calcium channel β subunits differentially modulate inactivation kinetics by binding to and thereby regulating the mobility of the putative inactivation gate. Indeed, additional evidence suggests that the carboxy terminus, amino terminus and domain III-IV linker regions of the channel modulate inactivation rates through interactions with the I-II linker per se, or indirectly via the ancillary β subunits. Taken together, the fast voltagedependent inactivation of calcium channels appears reminiscent of that of sodium channels, but appears to show a more complex regulation through intramolecular interactions between the putative inactivation gate and other cytoplasmic regions.

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428 -22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type ␣ 1C and ␣ 1E calcium channels. Substitution of the II S6 and/or III S6 segments of ␣ 1E into the ␣ 1C backbone resulted in rapid inactivation rates that closely approximated those of wild type ␣ 1E channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type ␣ 1E channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of ␣ 1E S6 segments of domains II, III, or II؉III with those of ␣ 1C was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of ␣ 1E were concomitantly replaced with ␣ 1C sequence could inactivation be abolished. Conversely, introduction of the ␣ 1E domain I-II linker sequence into ␣ 1C conferred ␣ 1E -like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.Calcium entry through voltage-dependent calcium channels is important for a range of cellular processes, including neurotransmitter release and activation of Ca 2ϩ -dependent enzymes. Molecular cloning has identified the primary structures of at least 9 different neuronal Ca 2ϩ channel ␣ 1 subunits (termed ␣ 1A through ␣ 1I (1-15)) that encode the previously identified native L-, P-, N, -Q-, T-, and R-types (for review, see Refs. 16 and 17). Calcium channels, like many other voltage-dependent ion channels, undergo a series of conformational changes in response to voltage, resulting in their opening, closing, and inactivation. Voltage-dependent inactivation of calcium channels is an important intrinsic process that prevents the breakdown of the calcium gradient as well as excessive calcium entry that is toxic to most cells (18 -20). In addition, many pharmacological agents interact predominantly with inactivated channels (21, 22). Unlike sodium (23, 24) and potassium (25-27) channels, the mechanisms that govern calcium channel voltagedependent inactivation are not fully understood. Although a number of structural moieties of the calcium channel ␣ 1 subunit have been implicated in being important in fast calcium channel inactivation (7, 22, 28 -30, 32, 33), the detailed mechanism underlying the inactivation process remain unknown, and there have been few systematic attempts to resolve this issue. By creating a series o...

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