Mechanism by which the beta subunit modulates the gating of mSLO, a large -conductance calcium -activated potassium channel

Abstract

Large-conductance calcium-activated potassium (BK) channels are characterized by their activation by both calcium and membrane depolarization, and play important roles in diverse physiological processes. The pore-forming α subunit of BK channels has been cloned from a number of species and has been shown to form functional channels when expressed in various expression systems. The co-expression of an auxiliary BK channel β subunit along with the α subunit leads to an increase in Ca²⁺ sensitivity of approximately ten-fold, as compared with expression of the α subunit alone.

The first section of this dissertation investigates the mechanism for this β subunit-induced increase in Ca²⁺ sensitivity in the BK channel cloned from mouse (mSlo). The patch-clamp recording technique was used to examine currents flowing through single BK channels expressed in HEK 293 cells with and without the β subunit. I found that the β subunit increased channel open probability (Po) by increasing burst duration 20–100 fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising Ca²⁺_i in the absence of the β subunit.

The second section of this dissertation examines the gating of BK channels with and without the β subunit in the absence of Ca²⁺ _i. I found that both α and α+β channels typically gate among 2–3 open and 3–4 closed states in the absence of Ca ²⁺_i, thus rejecting the Monod-Wyman-Changeux (MWC) model for tetrameric allosteric proteins which predicts that the gating will be confined to only one closed and one open state in ∼0 μM Ca²⁺ .

I also tested whether the β subunit requires Ca²⁺ _i to exert its effect by examining the differences in the behavior of the channel induced by the β subunit in the absence of Ca²⁺ _i. I found that in ∼0 μM Ca²⁺_i and at +30 mV membrane potential, the β subunit increases Po (∼10-fold), mean open time (∼10 fold), mean burst duration (∼20-fold) and mean number of openings per burst (∼2-fold). Mean shut interval durations remained unchanged. The β subunit exerts its effect on the above indicated parameters from ∼0 to higher Ca²⁺_i and over a range of examined voltages (+30 to +70 mV for α channels and +30 to +90 mV for α+β channels). The β subunit effect was not equivalent to an increase in membrane potential as increasing Po with depolarization gave different channel kinetics than increasing Po with β subunit. (Abstract shortened by UMI.)