CACNA1I is a candidate schizophrenia risk gene. It encodes the pore-forming human Ca V 3.3 α1 sub... more CACNA1I is a candidate schizophrenia risk gene. It encodes the pore-forming human Ca V 3.3 α1 subunit, a subtype of voltage-gated calcium channel that contributes to T-type currents. Recently, two de novo missense variations, T797M and R1346H, of hCa V 3.3 were identified in individuals with schizophrenia. Here we show that R1346H, but not T797M, is associated with lower hCa V 3.3 protein levels, reduced glycosylation, and lower membrane surface levels of hCa V 3.3 when expressed in human cell lines compared to wild-type. Consistent with our biochemical analyses, whole-cell hCa V 3.3 currents in cells expressing the R1346H variant were ~50% of those in cells expressing WT hCa V 3.3, and neither R1346H nor T797M altered channel biophysical properties. Employing the NEURON simulation environment, we found that reducing hCa V 3.3 current densities by 22% or more eliminates rebound bursting in model thalamic reticular nucleus (TRN) neurons. Our analyses suggest that a single copy of Chr22: 39665939G > A CACNA1I has the capacity to disrupt Ca V 3.3 channel-dependent functions, including rebound bursting in TRN neurons, with potential implications for schizophrenia pathophysiology.
cGMP is a second messenger widely used in the nervous system and other tissues. One of the major ... more cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG sig-naling cascade. Our results indicate that the Ca V 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K +. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphoryla-tion sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion.
Using exome sequencing and linkage analysis in a three-generation family with a unique dominant m... more Using exome sequencing and linkage analysis in a three-generation family with a unique dominant myoclonus-dystonia-like syndrome with cardiac arrhythmias, we identified a mutation in the CACNA1B gene, coding for neuronal voltage-gated calcium channels CaV2.2. This mutation (c.4166G>A;p.Arg1389His) is a disruptive missense mutation in the outer region of the ion pore. The functional consequences of the identified mutation were studied using whole-cell and single-channel patch recordings. High-resolution analyses at the single-channel level showed that, when open, R1389H CaV2.2 channels carried less current compared with WT channels. Other biophysical channel properties were unaltered in R1389H channels including ion selectivity, voltage-dependent activation or voltage-dependent inactivation. CaV2.2 channels regulate transmitter release at inhibitory and excitatory synapses. Functional changes could be consistent with a gain-of-function causing the observed hyperexcitability charac...
Voltage-gated calcium ion channels are essential for numerous biological functions of excitable c... more Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cell-specific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains ca...
Voltage-gated calcium ion channels are essential for numerous biological functions of ex-citable ... more Voltage-gated calcium ion channels are essential for numerous biological functions of ex-citable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique Ca V 1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cell-specific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among Ca V isoforms expressed in different tissues, and such differences may be greater than those that exist across Ca V channel homologs of closely related species. Here we highlight examples of Ca V isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of Ca V isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of Ca V channel inhibitors and agonists with improved tissue-specificity.
CACNA1I is a candidate schizophrenia risk gene. It encodes the pore-forming human Ca V 3.3 α1 sub... more CACNA1I is a candidate schizophrenia risk gene. It encodes the pore-forming human Ca V 3.3 α1 subunit, a subtype of voltage-gated calcium channel that contributes to T-type currents. Recently, two de novo missense variations, T797M and R1346H, of hCa V 3.3 were identified in individuals with schizophrenia. Here we show that R1346H, but not T797M, is associated with lower hCa V 3.3 protein levels, reduced glycosylation, and lower membrane surface levels of hCa V 3.3 when expressed in human cell lines compared to wild-type. Consistent with our biochemical analyses, whole-cell hCa V 3.3 currents in cells expressing the R1346H variant were ~50% of those in cells expressing WT hCa V 3.3, and neither R1346H nor T797M altered channel biophysical properties. Employing the NEURON simulation environment, we found that reducing hCa V 3.3 current densities by 22% or more eliminates rebound bursting in model thalamic reticular nucleus (TRN) neurons. Our analyses suggest that a single copy of Chr22: 39665939G > A CACNA1I has the capacity to disrupt Ca V 3.3 channel-dependent functions, including rebound bursting in TRN neurons, with potential implications for schizophrenia pathophysiology.
cGMP is a second messenger widely used in the nervous system and other tissues. One of the major ... more cGMP is a second messenger widely used in the nervous system and other tissues. One of the major effectors for cGMP is the serine/threonine protein kinase, cGMP-dependent protein kinase (PKG), which catalyzes the phosphorylation of a variety of proteins including ion channels. Previously, it has been shown that the cGMP-PKG signaling pathway inhibits Ca 2+ currents in rat vestibular hair cells and chromaffin cells. This current allegedly flow through voltage-gated Ca V 1.3L-type Ca 2+ channels, and is important for controlling vestibular hair cell sensory function and catecholamine secretion, respectively. Here, we show that native L-type channels in the insulin-secreting RIN-m5F cell line, and recombinant Ca V 1.3 channels heterologously expressed in HEK-293 cells, are regulatory targets of the cGMP-PKG sig-naling cascade. Our results indicate that the Ca V 1 ion-conducting subunit of the Ca V 1.3 channels is highly expressed in RIN-m5F cells and that the application of 8-Br-cGMP, a membrane-permeable analogue of cGMP, significantly inhibits Ca 2+ macroscopic currents and impair insulin release stimulated with high K +. In addition, KT-5823, a specific inhibitor of PKG, prevents the current inhibition generated by 8-Br-cGMP in the heterologous expression system. Interestingly, mutating the putative phosphoryla-tion sites to residues resistant to phosphorylation showed that the relevant PKG sites for Ca V 1.3 L-type channel regulation centers on two amino acid residues, Ser793 and Ser860, located in the intracellular loop connecting the II and III repeats of the Ca V 1 pore-forming subunit of the channel. These findings unveil a novel mechanism for how the cGMP-PKG signaling pathway may regulate Ca V 1.3 channels and contribute to regulate insulin secretion.
Using exome sequencing and linkage analysis in a three-generation family with a unique dominant m... more Using exome sequencing and linkage analysis in a three-generation family with a unique dominant myoclonus-dystonia-like syndrome with cardiac arrhythmias, we identified a mutation in the CACNA1B gene, coding for neuronal voltage-gated calcium channels CaV2.2. This mutation (c.4166G>A;p.Arg1389His) is a disruptive missense mutation in the outer region of the ion pore. The functional consequences of the identified mutation were studied using whole-cell and single-channel patch recordings. High-resolution analyses at the single-channel level showed that, when open, R1389H CaV2.2 channels carried less current compared with WT channels. Other biophysical channel properties were unaltered in R1389H channels including ion selectivity, voltage-dependent activation or voltage-dependent inactivation. CaV2.2 channels regulate transmitter release at inhibitory and excitatory synapses. Functional changes could be consistent with a gain-of-function causing the observed hyperexcitability charac...
Voltage-gated calcium ion channels are essential for numerous biological functions of excitable c... more Voltage-gated calcium ion channels are essential for numerous biological functions of excitable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique CaVα1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cell-specific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains ca...
Voltage-gated calcium ion channels are essential for numerous biological functions of ex-citable ... more Voltage-gated calcium ion channels are essential for numerous biological functions of ex-citable cells and there is wide spread appreciation of their importance as drug targets in the treatment of many disorders including those of cardiovascular and nervous systems. Each Cacna1 gene has the potential to generate a number of structurally, functionally, and in some cases pharmacologically unique Ca V 1 subunits through alternative pre-mRNA splicing and the use of alternate promoters. Analyses of rapidly emerging deep sequencing data for a range of human tissue transcriptomes contain information to quantify tissue-specific and alternative exon usage patterns for Cacna1 genes. Cell-specific actions of nuclear DNA and RNA binding proteins control the use of alternate promoters and the selection of alternate exons during pre-mRNA splicing, and they determine the spectrum of protein isoforms expressed within different types of cells. Amino acid compositions within discrete protein domains can differ substantially among Ca V isoforms expressed in different tissues, and such differences may be greater than those that exist across Ca V channel homologs of closely related species. Here we highlight examples of Ca V isoforms that have unique expression patterns and that exhibit different pharmacological sensitivities. Knowledge of expression patterns of Ca V isoforms in different human tissues, cell populations, ages, and disease states should inform strategies aimed at developing the next generation of Ca V channel inhibitors and agonists with improved tissue-specificity.
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