UBC Theses and Dissertations
Characterization of the +SSTR and ΔSSTR splice variants of the Cav2.1 P/Q-type voltage-gated calcium channel Waheed, Zeina
Cav2.1 P/Q-type voltage-gated calcium channels are essential for neurotransmission in many regions of the mammalian central nervous system (CNS). Alternative splicing generates functional diversity between Cav2.1 splice isoforms and is thought to be a mechanism by which fine-tuning and complexity of Cav2.1-mediated activities occur. The Cav2.1 +SSTR splice variant, located in the S3-S4 linker of domain III, has been identified in rodent brain although its effects on the biophysical and pharmacological properties of Cav2.1 have not been previously studied. Here, by performing splice variant-specific quantitative real-time PCR on selected regions of the rat CNS I demonstrate that +SSTR variant channels are differentially expressed spatially with predominant expression in the brainstem, reticular thalamus and spinal cord. Using whole-cell patch-clamp electrophysiology performed on transfected HEK 293 cells I have shown that compared to ΔSSTR channels, +SSTR variants exhibit faster activation kinetics and a hyperpolarizing shift in the voltage-dependence of activation and inactivation. Additionally, the +SSTR and ΔSSTR variants respond differently to increasing durations of action potential waveforms (APWs) with the charge transference through +SSTR channels being significantly less sensitive to APW broadening than ΔSSTR channels. Together, these data suggest that the unique biophysical properties of the Cav2.1 splice variants contribute to distinct roles in CNS synaptic physiology by relaying different types of action potential-encoding synaptic information. Lastly, I examined whether the +SSTR variant affected the sensitivity of Cav2.1 to the gating modifier peptide toxin ω-Agatoxin-IVA. Using whole-cell patch-clamp electrophysiology I found that the effects of ω-Agatoxin-IVA on current block did not significantly differ between the +SSTR and ΔSSTR splice variants suggesting that SSTR insertion does not affect the binding of ω-Agatoxin-IVA to Cav2.1 channels. The differential expression of Cav2.1 splice variants and their unique channel properties provides insight into the mechanisms by which complexity of P/Q-type calcium channel-mediated signaling contributes to CNS physiology.
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