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Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

ABSTRACT: Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

SUBMITTER: Obejero-Paz CA

PROVIDER: S-EPMC2234021 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

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Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

Obejero-Paz Carlos A CA Gray I Patrick IP Jones Stephen W SW

The Journal of general physiology 20041201 6

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by st ...[more]

PMID: 15572343

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Project description: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 11 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 12 regions encoding alternative splice isoforms of Cav3.1/alpha1G T-type calcium channels. Of the 12 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 Cav3.1/alpha1G 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.

Dataset Information

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

Publications

Y3+ block demonstrates an intracellular activation gate for the alpha1G T-type Ca2+ channel.

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