Mechanism by which the beta1 subunit modulates the gating of BK channels

In the first part of my dissertation I examine the mechanism by which the beta1 subunit modulates the gating of the BK channel by exploring which structure features of the alpha subunit are involved in the major action of the beta1 subunit of increasing the apparent Ca2+ sensitivity and the Po of the channel. I found that the beta1 subunit increased the apparent Ca2+-sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0--S8) and tails (S9--S10) or full length, and this increase was still observed after the Ca2+ bowl was inactivated by mutation. These findings indicate that the increase in apparent Ca2+ sensitivity induced by the beta1 subunit does not require either the Ca2+ bowl or the linker between the core and the tail, and that Slo3 tails cannot substitute for Slol tails.I also observed that the beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in the apparent Ca2+ sensitivity required Slo1 tails and was absent with Slo3 tails. This differential effect of the beta1 subunit on voltage and Ca2+ activation of the channel suggests that voltage and Ca2+ may be acting through separate pathways.The second part of my dissertation explores further the mechanism by which the beta1 subunit modulates the gating of BK channel by examining the action of the beta1 subunit on a triply mutated channel in which the Ca2+- and Mg2+-dependent activating mechanisms were completely disrupted by mutating three sites to remove the Ca 2+ and Mg2+ sensitivity. I found that the presence of the beta1 subunit partially restored Ca2+ sensitivity to the triply mutated channels, but not Mg2+ sensitivity. These observations suggest that the beta1 subunit increases Po by working through the Ca2+-, rather than the Mg2+-, dependent activating mechanisms, and that the action of the beta1 subunit is downstream from the Ca2+ binding sites.Finally, I also showed that Mg2+i can inhibit, rather than activate, BK channels in the presence of the beta1 subunit for intermediate levels of Ca2+i. This is in contrast to the activating effect of Mg2+ in the absence of the beta1 subunit, reported previously. This differential effect of Mg2+, which is controlled by the presence or absence of the beta1 subunit, provides a novel regulatory mechanism of BK channel activity. (Abstract shortened by UMI.)