BACKGROUND
alternative splicing of pre-mrnas is a critical regulatory mechanism augmenting mrna transcript diversity and expanding protein function  <cit> .
within the nervous system, alternative splicing affects neuronal development and function by regulating various proteins involved in synaptic connectivity and cellular excitation, including ion channels  <cit> .
voltage-gated ion channels control the flow of charged ions across the cell membrane and the resulting membrane potential fluctuations sculpt the excitability and overall firing patterns of the cell.
several mechanisms, including alternative splicing of channel transcripts, maintain a highly diversified population of ion channels needed to coordinate complex cellular activities and functions  <cit> .
calcium ion channel pore-forming α-subunits are encoded by ten genes, and of these, three express low-voltage-activated cav <dig> /α1g, cav <dig> /α1h, and cav <dig> /α1i t-type channels  <cit> .
t-type calcium channels open at low membrane potentials, regulate calcium influx, and trigger rebound action potential bursting following prolonged hyperpolarizations.
these channels are widely expressed in the nervous system, the heart, and other tissues  <cit> , and contribute to various physiological activities, suggesting the utility of a heterogeneous population of t-type channels.
extensive alternative splice isoform profiling of the human cacna1g, cacna1h, and cacna1i genes demonstrates highly diversified t-type channel mrna transcript populations with distinct electrophysiology properties  <cit> .
the mouse cacna1g gene encodes α1g channels ranging from ~240– <dig> kda  <cit> , and alternative splicing profiling of the orthologous human cacna1g gene shows four cassette exons , two alternative splice donor sites , four alternative splice acceptor sites , and one protein-coding intron   <cit> .
to evaluate the expression patterns of the mouse cacna1g gene regulated by alternative splicing, we profiled the cacna1g mrna using overlapping rt-pcr amplicons to confirm the orthologous human α1g splicing events and to identify potential mouse-specific splicing events.
compared to human α1g alternative splicing, six of the  <dig> splicing events are conserved in the mouse, and six additional splicing events appear exclusively in the mouse transcriptome.
we have also identified two brain region-specific alternative splice patterns in the cerebellum.
our results further define the scope of alternative splicing of the mouse cacna1g gene and provide potential evidence of tissue-specific mrna transcripts in generating functionally distinct α1g t-type channel proteins.
RESULTS
increased cacna1g mrna transcript diversity
to determine the alternative splicing sites and events occurring within the mouse brain cacna1g mrna population, we utilized a transcript scanning approach using overlapping rt-pcr amplicons to cover the full coding sequence .
using isolated brain mrna samples, rt-pcr products representing alternative splicing variants were isolated, cloned, and sequenced .
six of the  <dig> potential orthologous human α1g alternative splicing events were verified in the mouse transcriptome.
the common splicing events include four cassette exons , one alternative splice donor site , and one alternative splice acceptor site   <cit>  .
we were unable to detect the other five alternative splice variants  in the mouse and these appear to be human-specific.
analysis of mouse cacna1g mrna transcripts by rt-pcr identifies  <dig> total alternative splicing events.
the primer combinations  are listed along with the scanned exons, the assayed splice isoforms, and the generated amplicon sizes.
newly-characterized splicing events and amplicon sizes are designated in bold, italic type.
orthologous mouse cacna1g alternative splice isoforms are comparable to human cacna1g splice sites, as previously identified by amittman et al.
and bemerick et al.
.
we confirmed six novel mouse-specific cacna1g alternative splice variants, including one intron retention site , three alternative acceptor sites , one alternative donor site , and one case of exon exclusion  .
use of the Δ5' e <dig> splice site causes skipping of the first  <dig> base pairs  resulting in a predicted in-frame truncation of  <dig> amino acids  in exon  <dig> .
the alternatively spliced form localizes to the di-s <dig> region of the channel.
the second newly-identified splice variant, +i6- <dig>  results in the retention of the intronic sequence between exons  <dig> and  <dig> .
inclusion of this intron adds  <dig> bp to mrna transcripts and a predicted  <dig> aa into the di-p-loop .
exon  <dig> undergoes two independent alternative splicing events that utilize separate splice acceptor  and donor  sequences.
the Δ5' e <dig> acceptor site signals splicing from the end of exon  <dig> into exon  <dig> after  <dig> bp, while the Δ5' e <dig> donor site initiates splicing into exon  <dig>  <dig> bp before the end of exon  <dig> .
both alternative splice sites generate in-frame truncations causing loss of the first  <dig> aa within di-s <dig>  or the final  <dig> aa in the di-ii linker  .
the exclusion of exon  <dig>  in cacna1g transcripts results in the loss of the entire  <dig> bp segment that normally encodes the end of the dii-s <dig> region and the beginning of the dii p-loop .
however, alternative splicing produces an exon 11– <dig> junction that forms a novel mrna sequence that prematurely adds a stop codon and truncates the channel protein.
the final additional instance of alternative splicing occurs in exon  <dig>  a previously characterized alternatively spliced exon that excludes the full sequence  <cit> .
exon  <dig> also contains an alternate splice acceptor site  that omits the first  <dig> bp of the exon  and removes the  <dig> aa from the c-terminal tail .
brain region-specific alternative splicing
to assess the patterns of cacna1g mrna alternative splicing within distinct structures of the brain, we performed rt-pcr reactions on thalamus, cortex, cerebellum, and hippocampus rna samples using the primer sets detailed in table  <dig>  comparisons of the thalamic, cortical, and hippocampal samples revealed similar patterns of expression for each of the  <dig> alternative splice sites .
analysis of splicing within the cerebellum of c57bl/ <dig>  stargazer, lethargic, and tottering mice identified two atypical patterns.
the first occurred at exon  <dig>  which encodes part of the dii-iii linker, and results in elevated alternative splicing.
as shown in whole brain , the alternative splice product is normally the more weakly expressed variant, yet within the cerebellum, the Δe <dig> splice isoform selectively predominated transcript expression.
a second alteration of cacna1g mrna splicing is observed at exon  <dig>  which encodes portions of the diii-iv linker.
in this instance, all transcripts lacked exon  <dig> due to exclusive alternative splicing in the cerebellum.
note the absence of the normally spliced product that includes exon  <dig> .
in some experiments, rt-pcr amplification of regions subject to alternative splicing produced hybrid, heteroduplex dnas.
these rt-pcr products were identified after cloning strategies failed, due to their altered structure.
alternative splicing at Δ3' e <dig> and Δe <dig> generated intermediary heteroduplex dimers and digestion of purified rt-pcr products using the t <dig> endonuclease i enzyme eliminated these dna complexes .
unaltered alternative splicing patterns in absence epilepsy mouse models
identification of the full profile of alternative splicing events in cacna1g transcripts creates a valuable tool to assess the contribution of this regulatory mechanism to pathogenic t-type channel function in the mouse brain.
we compared the splicing profiles within whole brain, cortex, thalamus, hippocampus, and cerebellum samples between wildtype and four absence epilepsy mutant mouse models previously shown in our laboratory to express elevated thalamic t-type currents, lethargic, tottering, stargazer, and coloboma  <cit> .
as presented in figures  <dig> and  <dig>  lethargic and tottering mutant mice show no apparent dysregulation of whole brain cav <dig> /α1g channel alternative splicing in comparison to the wildtype pattern of expression.
similar results were observed within whole brain samples collected from stargazer and coloboma mice .
additionally, comparisons of dissected brain region samples showed identical splicing patterns between each mutant mouse and wildtype controls .
discussion
the lethargic, tottering, stargazer, coloboma, and cacna1a-/- mutant mouse models of absence epilepsy each exhibit significant increases in thalamic t-type calcium currents despite the absence of evidence for enhanced t-type channel subunit expression  <cit> .
the cellular mechanism evoking this biophysical change remains unknown, but since t-type channels are subject to alternative splicing  <cit>  and cav <dig> /α1g channels distinctly localize to the thalamic relay nuclei where the enhanced t-type current densities are present  <cit> , we reasoned that alternative splicing of t-type channels might underlie this pathogenic hyperexcitability phenotype.
in this study, complete alternative splice isoform scanning of the full open reading frame of the mouse cacna1g gene revealed extensive molecular diversity of cav <dig> /α1g t-type calcium channels.
previously,  <dig> alternative splice variants were identified within eight exons of the orthologous human cacna1g gene  <cit> .
overlapping rt-pcr amplification of mouse cacna1g transcripts identified six splicing events comparable to those known in human cacna1g: Δe <dig>  Δ3' e <dig>  Δe <dig>  Δ5' e <dig>  Δe <dig>  and Δe <dig>  in addition, six novel mouse-specific splice variants were identified, resulting in 5' end truncations of exons  <dig>   <dig>  and  <dig>  through use of alternative splice acceptors, inclusion of the intron between exons  <dig> and  <dig> , 3' end truncation of exon  <dig>  via utilization of an alternative splice donor, and exclusion of exon  <dig> .
the  <dig> total splicing events in the mouse gene, occurring at  <dig> locations within cacna1g mrna transcripts, theoretically could generate  <dig>  distinct full-length transcripts.
two splice sites, Δe <dig> and Δ5' e <dig>  however, create modified exon-exon junctions that form premature nonsense stop codon sequences, thus reducing the pool of potential functional cacna1g transcripts from  <dig> to  <dig> .
analysis of full-length human cacna1g transcripts from fetal and adult brain samples identified only  <dig> total transcripts,  <dig> of which were distinct in fetal samples and  <dig> that are unique in adult samples  <cit> .
in addition to the highly regulated developmental expression of transcripts, alternative splicing controls the systematic combinations of expressed exons  <cit> .
these factors suggest that the vast number of potential combinations of alternatively spliced exons when forming complete transcripts is regulated to generate specific cav <dig> /α1g t-type channel function.
the developmental and activity-dependent control of these regulatory mechanisms in the mouse nervous system remain unknown and warrant further investigation.
nevertheless, the extensive potential diversity of mouse cacna1g transcripts through alternative splicing may produce channel variants capable of redefining the functional identity of a cell.
two of the newly-identified splice events localize to regions of t-type channels previously shown to alter channel kinetics.
alternative splicing at the mouse Δ3' e <dig> splice site truncates a large portion  of the i-ii linker domain.
splicing of this domain produces functional differences observed through the alternative splicing of human exon  <dig> in cav <dig> /α1i t-type channels.
the inclusion of this exon produced different effects dependent upon other splice variants.
in the presence of full-length exon  <dig>  channels exhibit a shifted voltage dependence of ssi and an increased recovery rate from inactivation near the resting potential, while alternative splicing of exon  <dig> increases the activation and inactivation rates at depolarized potentials  <cit> .
a second related splicing event in the mouse occurs at exon  <dig>  that removes  <dig> aa encoding a portion of the c-terminal tail of the channel.
splicing at the c-terminus of cav <dig> /α1i t-type channels also alters electrophysiological properties, as the spliced exon  <dig> produces slower activation and faster inactivation rates  <cit> .
the observed kinetic changes induced by alternative splicing of cav <dig> /α1i demonstrate that splicing at analogous sites of the cav <dig> /α1g channels, Δ3' e <dig> and Δ5' e <dig>  can promote altered channel kinetics.
alternative splicing occurring at the Δ5' e <dig> and Δ5' e <dig> sites truncates the s <dig> and s <dig> transmembrane segments of domain i, respectively.
splicing within these transmembrane segments may perturb proper protein structure; however, the functional consequences of splicing within these regions are unknown.
furthermore, retention of the intronic sequence between exons  <dig> and  <dig> inserts  <dig> aa into the p-loop of domain i. while splicing does not affect the composition of the ion selectivity filter of the channel, expression of intron 6– <dig> may alter the physical structure of the pore by obstructing the alignment of the filter residues and therefore modify channel function.
the identification of an additional six alternative splicing events, excluding the Δe <dig> variant that generates a premature stop codon, now necessitates the cloning and expression of these transcripts, along with the electrophysiological analysis of their biophysical properties to fully comprehend their individual and combinatorial effects on cav <dig> /α1g channel activity.
expression profiling of the alternative splice patterns within the brain identified distinct regional splice variant expression within the cerebellum when compared to the thalamus, hippocampus, and neocortex.
cerebellar-specific splicing patterns observed due to the Δe <dig> and Δe <dig> splice variants result in preferential alternative splicing of exon  <dig> and exclusive splicing out of exon  <dig>  rebound burst firing activity in several cerebellar neurons arises in part from the coordinate expression of t-type calcium channel isoforms  <cit> , and altered cav <dig> /α1g channel function mediated by alternative splicing may initiate abnormal burst firing leading to movement disorders, such as the ataxia and paroxysmal dyskinesia phenotypes associated with absence epilepsy in several of these mutant mouse models.
although several studies of absence epilepsy mutants have identified striking increases in thalamic t-type calcium currents, these elevations were not the result of clear increases in t-type channel expression  <cit> .
dysregulation of t-type cacna1g channels induced from steady-state expression levels of t-type channels can account for the low-threshold burst firing associated with the absence epilepsy phenotype  <cit> .
specific cav <dig> /α1g splice variants localize to various regions of the thalamocortical network  <cit> ; therefore, comparative analysis of cav <dig> /α1g splicing between mutant mice and wildtype controls was performed to determine whether this mechanism enhances t-type currents.
the results of profiling alternative splicing of cacna1g in the tottering, lethargic, stargazer, and coloboma revealed unaltered splicing regulation of cacna1g transcripts .
this result indicates that even though extensive cacna1g alternative splicing generates transcript variants that can enhance the cav <dig> /α1g channel kinetics in vitro  <cit> , in vivo splice isoform variation does not explain the elevated t-type currents in these models.
CONCLUSIONS
in summary,  <dig> alternative splicing events found within the coding region of the mouse cacna1g gene enhance the known diversity of t-type channel molecular structure.
although no alteration in splicing patterns of cav <dig> /α1g channel mrna was detected that could account for the elevated t-type currents linked to the generation of absence epilepsy in several known mouse mutant models, this mechanism may contribute to t-type calcium current variation in other developmental or pathological conditions.

 alternative splicing of pre-mrnas is a critical regulatory mechanism augmenting mrna transcript diversity and expanding protein function. within the nervous system, alternative splicing affects neuronal development and function by regulating various proteins involved in synaptic connectivity and cellular excitation, including ion channels. voltage-gated ion channels control the flow of charged ions across the cell membrane and the resulting membrane potential fluctuations sculpt the excitability and overall firing patterns of the cell. several mechanisms, including alternative splicing of channel transcripts, maintain a highly diversified population of ion channels needed to coordinate complex cellular activities and functions. calcium ion channel pore-forming α-subunits are encoded by ten genes, and of these, three express low-voltage-activated cav /α1g, cav /α1h, and cav /α1i t-type channels. t-type calcium channels open at low membrane potentials, regulate calcium influx, and trigger rebound action potential bursting following prolonged hyperpolarizations. these channels are widely expressed in the nervous system, the heart, and other tissues, and contribute to various physiological activities, suggesting the utility of a heterogeneous population of t-type channels. extensive alternative splice isoform profiling of the human cacna1g, cacna1h, and cacna1i genes demonstrates highly diversified t-type channel mrna transcript populations with distinct electrophysiology properties. the mouse cacna1g gene encodes α1g channels ranging from ~240– kda, and alternative splicing profiling of the orthologous human cacna1g gene shows four cassette exons, two alternative splice donor sites, four alternative splice acceptor sites, and one protein-coding intron. to evaluate the expression patterns of the mouse cacna1g gene regulated by alternative splicing, we profiled the cacna1g mrna using overlapping rt-pcr amplicons to confirm the orthologous human α1g splicing events and to identify potential mouse-specific splicing events. compared to human α1g alternative splicing, six of the splicing events are conserved in the mouse, and six additional splicing events appear exclusively in the mouse transcriptome. we have also identified two brain region-specific alternative splice patterns in the cerebellum. our results further define the scope of alternative splicing of the mouse cacna1g gene and provide potential evidence of tissue-specific mrna transcripts in generating functionally distinct α1g t-type channel proteins.  increased cacna1g mrna transcript diversity to determine the alternative splicing sites and events occurring within the mouse brain cacna1g mrna population, we utilized a transcript scanning approach using overlapping rt-pcr amplicons to cover the full coding sequence. using isolated brain mrna samples, rt-pcr products representing alternative splicing variants were isolated, cloned, and sequenced. six of the potential orthologous human α1g alternative splicing events were verified in the mouse transcriptome. the common splicing events include four cassette exons, one alternative splice donor site, and one alternative splice acceptor site. we were unable to detect the other five alternative splice variants in the mouse and these appear to be human-specific. analysis of mouse cacna1g mrna transcripts by rt-pcr identifies total alternative splicing events. the primer combinations are listed along with the scanned exons, the assayed splice isoforms, and the generated amplicon sizes. newly-characterized splicing events and amplicon sizes are designated in bold, italic type. orthologous mouse cacna1g alternative splice isoforms are comparable to human cacna1g splice sites, as previously identified by amittman et al. and bemerick et al.. we confirmed six novel mouse-specific cacna1g alternative splice variants, including one intron retention site, three alternative acceptor sites, one alternative donor site, and one case of exon exclusion. use of the Δ5' e splice site causes skipping of the first base pairs resulting in a predicted in-frame truncation of amino acids in exon. the alternatively spliced form localizes to the di-s region of the channel. the second newly-identified splice variant, +i6- results in the retention of the intronic sequence between exons and. inclusion of this intron adds bp to mrna transcripts and a predicted aa into the di-p-loop. exon undergoes two independent alternative splicing events that utilize separate splice acceptor and donor sequences. the Δ5' e acceptor site signals splicing from the end of exon into exon after bp, while the Δ5' e donor site initiates splicing into exon bp before the end of exon. both alternative splice sites generate in-frame truncations causing loss of the first aa within di-s or the final aa in the di-ii linker. the exclusion of exon in cacna1g transcripts results in the loss of the entire bp segment that normally encodes the end of the dii-s region and the beginning of the dii p-loop. however, alternative splicing produces an exon 11– junction that forms a novel mrna sequence that prematurely adds a stop codon and truncates the channel protein. the final additional instance of alternative splicing occurs in exon a previously characterized alternatively spliced exon that excludes the full sequence. exon also contains an alternate splice acceptor site that omits the first bp of the exon and removes the aa from the c-terminal tail. brain region-specific alternative splicing to assess the patterns of cacna1g mrna alternative splicing within distinct structures of the brain, we performed rt-pcr reactions on thalamus, cortex, cerebellum, and hippocampus rna samples using the primer sets detailed in table comparisons of the thalamic, cortical, and hippocampal samples revealed similar patterns of expression for each of the alternative splice sites. analysis of splicing within the cerebellum of c57bl/ stargazer, lethargic, and tottering mice identified two atypical patterns. the first occurred at exon which encodes part of the dii-iii linker, and results in elevated alternative splicing. as shown in whole brain, the alternative splice product is normally the more weakly expressed variant, yet within the cerebellum, the Δe splice isoform selectively predominated transcript expression. a second alteration of cacna1g mrna splicing is observed at exon which encodes portions of the diii-iv linker. in this instance, all transcripts lacked exon due to exclusive alternative splicing in the cerebellum. note the absence of the normally spliced product that includes exon. in some experiments, rt-pcr amplification of regions subject to alternative splicing produced hybrid, heteroduplex dnas. these rt-pcr products were identified after cloning strategies failed, due to their altered structure. alternative splicing at Δ3' e and Δe generated intermediary heteroduplex dimers and digestion of purified rt-pcr products using the t endonuclease i enzyme eliminated these dna complexes. unaltered alternative splicing patterns in absence epilepsy mouse models identification of the full profile of alternative splicing events in cacna1g transcripts creates a valuable tool to assess the contribution of this regulatory mechanism to pathogenic t-type channel function in the mouse brain. we compared the splicing profiles within whole brain, cortex, thalamus, hippocampus, and cerebellum samples between wildtype and four absence epilepsy mutant mouse models previously shown in our laboratory to express elevated thalamic t-type currents, lethargic, tottering, stargazer, and coloboma. as presented in figures and lethargic and tottering mutant mice show no apparent dysregulation of whole brain cav /α1g channel alternative splicing in comparison to the wildtype pattern of expression. similar results were observed within whole brain samples collected from stargazer and coloboma mice. additionally, comparisons of dissected brain region samples showed identical splicing patterns between each mutant mouse and wildtype controls. discussion the lethargic, tottering, stargazer, coloboma, and cacna1a-/- mutant mouse models of absence epilepsy each exhibit significant increases in thalamic t-type calcium currents despite the absence of evidence for enhanced t-type channel subunit expression. the cellular mechanism evoking this biophysical change remains unknown, but since t-type channels are subject to alternative splicing and cav /α1g channels distinctly localize to the thalamic relay nuclei where the enhanced t-type current densities are present, we reasoned that alternative splicing of t-type channels might underlie this pathogenic hyperexcitability phenotype. in this study, complete alternative splice isoform scanning of the full open reading frame of the mouse cacna1g gene revealed extensive molecular diversity of cav /α1g t-type calcium channels. previously, alternative splice variants were identified within eight exons of the orthologous human cacna1g gene. overlapping rt-pcr amplification of mouse cacna1g transcripts identified six splicing events comparable to those known in human cacna1g: Δe Δ3' e Δe Δ5' e Δe and Δe in addition, six novel mouse-specific splice variants were identified, resulting in 5' end truncations of exons and through use of alternative splice acceptors, inclusion of the intron between exons and, 3' end truncation of exon via utilization of an alternative splice donor, and exclusion of exon. the total splicing events in the mouse gene, occurring at locations within cacna1g mrna transcripts, theoretically could generate distinct full-length transcripts. two splice sites, Δe and Δ5' e however, create modified exon-exon junctions that form premature nonsense stop codon sequences, thus reducing the pool of potential functional cacna1g transcripts from to. analysis of full-length human cacna1g transcripts from fetal and adult brain samples identified only total transcripts, of which were distinct in fetal samples and that are unique in adult samples. in addition to the highly regulated developmental expression of transcripts, alternative splicing controls the systematic combinations of expressed exons. these factors suggest that the vast number of potential combinations of alternatively spliced exons when forming complete transcripts is regulated to generate specific cav /α1g t-type channel function. the developmental and activity-dependent control of these regulatory mechanisms in the mouse nervous system remain unknown and warrant further investigation. nevertheless, the extensive potential diversity of mouse cacna1g transcripts through alternative splicing may produce channel variants capable of redefining the functional identity of a cell. two of the newly-identified splice events localize to regions of t-type channels previously shown to alter channel kinetics. alternative splicing at the mouse Δ3' e splice site truncates a large portion of the i-ii linker domain. splicing of this domain produces functional differences observed through the alternative splicing of human exon in cav /α1i t-type channels. the inclusion of this exon produced different effects dependent upon other splice variants. in the presence of full-length exon channels exhibit a shifted voltage dependence of ssi and an increased recovery rate from inactivation near the resting potential, while alternative splicing of exon increases the activation and inactivation rates at depolarized potentials. a second related splicing event in the mouse occurs at exon that removes aa encoding a portion of the c-terminal tail of the channel. splicing at the c-terminus of cav /α1i t-type channels also alters electrophysiological properties, as the spliced exon produces slower activation and faster inactivation rates. the observed kinetic changes induced by alternative splicing of cav /α1i demonstrate that splicing at analogous sites of the cav /α1g channels, Δ3' e and Δ5' e can promote altered channel kinetics. alternative splicing occurring at the Δ5' e and Δ5' e sites truncates the s and s transmembrane segments of domain i, respectively. splicing within these transmembrane segments may perturb proper protein structure; however, the functional consequences of splicing within these regions are unknown. furthermore, retention of the intronic sequence between exons and inserts aa into the p-loop of domain i. while splicing does not affect the composition of the ion selectivity filter of the channel, expression of intron 6– may alter the physical structure of the pore by obstructing the alignment of the filter residues and therefore modify channel function. the identification of an additional six alternative splicing events, excluding the Δe variant that generates a premature stop codon, now necessitates the cloning and expression of these transcripts, along with the electrophysiological analysis of their biophysical properties to fully comprehend their individual and combinatorial effects on cav /α1g channel activity. expression profiling of the alternative splice patterns within the brain identified distinct regional splice variant expression within the cerebellum when compared to the thalamus, hippocampus, and neocortex. cerebellar-specific splicing patterns observed due to the Δe and Δe splice variants result in preferential alternative splicing of exon and exclusive splicing out of exon rebound burst firing activity in several cerebellar neurons arises in part from the coordinate expression of t-type calcium channel isoforms, and altered cav /α1g channel function mediated by alternative splicing may initiate abnormal burst firing leading to movement disorders, such as the ataxia and paroxysmal dyskinesia phenotypes associated with absence epilepsy in several of these mutant mouse models. although several studies of absence epilepsy mutants have identified striking increases in thalamic t-type calcium currents, these elevations were not the result of clear increases in t-type channel expression. dysregulation of t-type cacna1g channels induced from steady-state expression levels of t-type channels can account for the low-threshold burst firing associated with the absence epilepsy phenotype. specific cav /α1g splice variants localize to various regions of the thalamocortical network; therefore, comparative analysis of cav /α1g splicing between mutant mice and wildtype controls was performed to determine whether this mechanism enhances t-type currents. the results of profiling alternative splicing of cacna1g in the tottering, lethargic, stargazer, and coloboma revealed unaltered splicing regulation of cacna1g transcripts. this result indicates that even though extensive cacna1g alternative splicing generates transcript variants that can enhance the cav /α1g channel kinetics in vitro, in vivo splice isoform variation does not explain the elevated t-type currents in these models.  in summary, alternative splicing events found within the coding region of the mouse cacna1g gene enhance the known diversity of t-type channel molecular structure. although no alteration in splicing patterns of cav /α1g channel mrna was detected that could account for the elevated t-type currents linked to the generation of absence epilepsy in several known mouse mutant models, this mechanism may contribute to t-type calcium current variation in other developmental or pathological conditions. 
to test whether enhanced t-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models. the determination of six novel alternative splice sites within the coding region of the mouse cacna1g gene greatly expands the potential biophysical diversity of voltage-gated t-type channels in the mouse central nervous system. although alternative splicing of cav /α1g channels does not explain the enhancement of t-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of t-type channels through this mechanism may influence other developmental neurological phenotypes. in mouse models of absence epilepsy, elevated t-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic t-type currents and wildtype controls failed to reveal differences in cacna1g splicing patterns. of the splice sites, six displayed homology to the human cacna1g splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. in addition, two brain region-specific alternative splice patterns were observed in the cerebellum.
 alternative splicing of low-voltage-activated t-type calcium channels contributes to the molecular and functional diversity mediating complex network oscillations in the normal brain. transcript scanning of the human cacna1g gene has revealed the presence of regions within the coding sequence subjected to alternative splicing, some of which enhance t-type current. in mouse models of absence epilepsy, elevated t-type calcium currents without clear increases in channel expression are found in thalamic neurons that promote abnormal neuronal synchronization. to test whether enhanced t-type currents in these models reflect pathogenic alterations in channel splice isoforms, we determined the extent of alternative splicing of mouse cacna1g transcripts and whether evidence of altered transcript splicing could be detected in mouse absence epilepsy models.  transcript scanning of the murine cacna1g gene detected regions encoding alternative splice isoforms of cav /α1g t-type calcium channels. of the splice sites, six displayed homology to the human cacna1g splice sites, while six novel mouse-specific splicing events were identified, including one intron retention, three alternative acceptor sites, one alternative donor site, and one exon exclusion. in addition, two brain region-specific alternative splice patterns were observed in the cerebellum. comparative analyses of brain regions from four monogenic absence epilepsy mouse models with altered thalamic t-type currents and wildtype controls failed to reveal differences in cacna1g splicing patterns.  the determination of six novel alternative splice sites within the coding region of the mouse cacna1g gene greatly expands the potential biophysical diversity of voltage-gated t-type channels in the mouse central nervous system. although alternative splicing of cav /α1g channels does not explain the enhancement of t-type current identified in four mouse models of absence epilepsy, post-transcriptional modification of t-type channels through this mechanism may influence other developmental neurological phenotypes. 