Domain II (S5-P) region in Lymnaea T-type calcium channels and its role in determining biophysical properties, ion selectivity and drug sensitivity
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Invertebrate T-type calcium channels cloned from the great pond snail, Lymnaea Stagnalis (LCav3) possess highly sodium permeant ion channel currents by means of alternative splicing of exon 12. Exon 12 is located on the extracellular turret and the descending helix between segments 5 and segments 6, upstream of the ion selectivity filter in Domain II. Highly-sodium permeant T-type channels are generated without altering the selectivity filter locus, the primary regulatory domain known to govern ion selectivity for calcium and sodium channels. Comparisons of exon 12 sequences between invertebrates and vertebrate T-type channels reveals a conserved pattern of cysteine residues. Calcium-selective mammalian T-type channels possess a single cysteine in exon 12 in comparison to invertebrate T-type channels with either a tri- or penta- cysteine framework. Cysteine residues in exon 12 were substituted with a neutral amino acid, alanine in LCav3 channels harbouring exon 12a and 12b to mimic the turret structure of vertebrate T-type channels. The results generated T-type channels that were even more sodium-permeable than the native T-type channels in snails. Furthermore, permeant divalent ions similar in structure to calcium (eg. barium) were unable to sufficiently block the monovalent ion current of channels lacking cysteines in Domain II, suggesting that the pore is highly sodium permeant, and has weak affinity and block by permeant divalent ions other than calcium. Besides ion selectivity, the cysteine mutated T-type channels were 10 to 100 fold more sensitive to inhibition by nickel and zinc, respectively. The cysteine mutation data highly suggests that the cysteines form an extracellular structure that regulates ion selectivity and shields T-type channels from block by nickel and zinc. In addition, we replaced exon 12 from the sodium permeant snail T-type channel with exon 12 from human Cav3.2 channels. The snail T-type channel with exon 12 from human T-type channels produced a T-type channel that was modestly sodium permeable, but did not confer the high calcium permeability of Cav3.2 channels. These findings suggest that the cysteine containing extracellular domains in exon 12 are not sufficient to generate calcium selective channels similar to human Cav3.2 and likely work in concert with other extracellular domains to regulate the calcium or sodium selectivity of T-type channels.