Modulation of calcium sensitivity by the beta subunits of BK channels.

摘要

Synergistic activation by membrane voltage and intracellular Ca2+ is a unique property of large conductance Ca2+ activated K+ (BK) channels, which are found in many cell types including smooth muscles, neurons and endocrine cells. Functional variation of BK channels in different tissues is largely accounted by the association with accessory beta subunits, which have tissue-specific distributions. There are four types of beta subunits (beta1-beta4) that can associate with the pore forming Slo1 subunit to uniquely modulate the Ca2+ and voltage sensitive properties in BK channels. Studies in the past have shown beta1 and beta2 subunits both increase Ca2+ sensitivity, so the goal in this dissertation was to (1) understand how perturbations in the Slo1 subunit affect beta subunit's modulation of Ca2+ sensitivity and (2) for the beta2 subunit specifically, to identify the regions on the Slo1 subunit required in its modulation of Ca2+ sensitivity. In both studies, constructs were coexpressed with and without beta subunits where their Ca2+ sensitivities were compared and their conductance-voltage relations were fitted with an allosteric gating model. In the first study, using a mutation that alters Ca2+ dependent activation, I show that in the presence of the beta subunits, with the exception of beta3b subunit, the mutation generally increased Ca2+ sensitivity to the same extent as in Slo1-only channels. In the second study, taking advantage of differential effect of beta2 modulation on two BK channel orthologs, different chimeras were designed and coexpressed with beta2 subunits. The results identified the importance of N-terminus to S0 and cytoplasmic linker to AC region (N-terminal region of RCK1) for beta2 dependent increase in Ca2+ sensitivity. Furthermore, the Linker/AC region was specifically involved in beta2 modulation but not in beta1, even though both beta subunits increase Ca2+ sensitivity. However, modulation by both beta1 and beta2 subunits was unaffected by Ca2+ binding site mutations as long as there was one intact binding site. Collectively, these results provide a basis for future studies identifying the molecular basis for modulation by the beta3 and beta4 subunits in BK channels. In the case of the beta2 subunit, it will be interesting to see how these regions affect Ca2+ sensitivity without directly affecting Ca 2+ binding.