Title

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

Date of Award

1999

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Physiology and Biophysics

First Committee Member

Karl L. Magleby, Committee Chair

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 alpha 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 beta subunit along with the alpha subunit leads to an increase in Ca2+ sensitivity of approximately ten-fold, as compared with expression of the alpha subunit alone.The first section of this dissertation investigates the mechanism for this beta 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 beta subunit. I found that the beta 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 beta subunit was not equivalent to raising Ca2+i in the absence of the beta subunit.The second section of this dissertation examines the gating of BK channels with and without the beta subunit in the absence of Ca2+ i. I found that both alpha and alpha+beta 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 muM Ca2+ .I also tested whether the beta subunit requires Ca2+ i to exert its effect by examining the differences in the behavior of the channel induced by the beta subunit in the absence of Ca2+ i. I found that in ∼0 muM Ca2+i and at +30 mV membrane potential, the beta 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 beta 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 alpha channels and +30 to +90 mV for alpha+beta channels). The beta subunit effect was not equivalent to an increase in membrane potential as increasing Po with depolarization gave different channel kinetics than increasing Po with beta subunit. (Abstract shortened by UMI.)

Keywords

Biology, Neuroscience; Biophysics, Medical; Biophysics, General

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:9961249