Modulation of Calcium Sensitivity by the Beta Subunits of BK Channels
Date of Award
Doctor of Philosophy (PhD)
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 subunits, which have tissue-specific distributions. There are four types of β subunits (β1- β4) 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 β1 and β2 subunits both increase Ca2+ sensitivity, so the goal in this dissertation was to 1) understand how perturbations in the Slo1 subunit affect β subunit’s modulation of Ca2+ sensitivity and 2) for the β2 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 β 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 β subunits, with the exception of β3b 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 2 modulation on two BK channel orthologs, different chimeras were designed and coexpressed with β2 subunits. The results identified the importance of N-terminus to S0 and cytoplasmic linker to AC region (N-terminal region of RCK1) for β2 dependent increase in Ca2+ sensitivity. Furthermore, the Linker/AC region was specifically involved in β2 modulation but not in β1, even though both β subunits increase Ca2+ sensitivity. However, modulation by both β1 and β2 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 β3 and β4 subunits in BK channels. In the case of the β2 subunit, it will be interesting to see how these regions affect Ca2+ sensitivity without directly affecting Ca2+ binding.
Nichols, Kurt Thoroughman, Ralf Wessel