Stephens, Robert2016-07-202016-07-202016-07-202016-07-15http://hdl.handle.net/10012/10590The T-type calcium channel from the pond snail Lymnaea stagnalis, LCav3, undergoes alternative splicing in exon 12. Exon 12 codes for the L5 structure (S5-P) in Domain II (DII), which is dubbed the "turret". The turret is a cysteine-rich extracellular loop near the selectivity filter. The selectivity filter is critical for ion selectivity. The DII turret is capable of influencing ion selectivity in the LCav3 channel, possibly through interactions with the selectivity filter. LCav3 with exon 12a is highly sodium-selective, whereas LCav3-12b is more calcium-selective. The turret of exon 12a is different from the turret of exon 12b in its size (number of amino acids) and cysteine configuration. Genome and transcriptome analyses have revealed that some cnidarians possess two versions of the genes encoding T-type calcium channels, and that these gene products differ from one another based on the size and cysteine configurations of their Domain IV turrets. We hypothesize that the Domain IV turret is playing a role in determining ion selectivity in T-type calcium channels. We created T-type calcium channel chimeras and investigated their ion selectivity in whole-cell patch clamp recordings of transfected HEK293T cells. Chimeras were created, in which the Domain IV turret from LCav3 was replaced by the Domain IV turret from Cav3.2. Cav3.2 is a highly calcium-selective human T-type channel. Each of the chimeras possessed increased calcium selectivity. Calcium selectivity in the LCav3-12b DIVα1hL chimera approached the levels of calcium-selectivity observed in human channels. However, we found that the Domain II turret has a dominant influence on selectivity. The research presented here provides support for a role in ion selectivity for extracellular turrets. A structural model, based on the recently resolved Cav1.1 cryo-EM structure, is proposed here to explain how turrets could influence ion selectivity.enCalcium ChannelsT-type Calcium ChannelGiant Pond SnailElectrophysiologyMembrane BiophysicsIon SelectivityIon PermeabilityCalcium PermeabilitySodium PermeabilityMaster Thesis