Title

Gating kinetics of a calcium-activated potassium channel studied at subzero temperatures

Date of Award

1995

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Physiology and Biophysics

First Committee Member

Wolfgang Nonner, Committee Chair

Abstract

The mechanism(s) by which channels control the flux of ions is still unknown. In single channel recordings at room temperature the transition between closed and open states appears to be instantaneous. A technique to perform single-channel recordings at subzero temperatures was developed in an attempt to slow the conformational changes of the channel-protein enough so that the open/closed transition could be temporally resolved.Single channel recordings of large-conductance Ca-activated K channels were obtained from membrane patches excised from primary cultures of rat fetal skeletal muscle. Temperatures ranged between 25 and $-$35$\sp\circ$C, in the presence of 40% of an antifreeze, ethyleneglycol. Under the experimental conditions used (large KCl concentration gradients, 30-90 mV holding potentials, 3-30 $\mu$M CaCl$\sb2$) channels were preferentially open, closing for only very brief periods (mean 400 $\mu$s at $-$18$\sp\circ$C).Channels conducted and underwent open/closed transitions down to the lowest temperatures explored. Even at subzero temperatures, the average open/closed transition was indistinguishable from a pulse generator transition and hence was not time resolved. On the other hand, the subset of transitions that involved longer closed states exhibited graded changes of current, observed in ensemble-averages as long relaxations lasting ca. 50 ms at $-$10 to $-$20$\sp\circ$C. Individual examples of such open/closed transitions revealed graded, stepwise and oscillatory variations in pore current. Ensemble-average analysis of sojourns below a threshold of approximately 50% of the unit current level indicated that different sojourn durations were associated with different mean current levels, indicating that the typical relaxation of current involves suprathreshold transients. All observed relaxations of pore current appeared as microscopically reversible, and hence were not detectably dependent on the irreversible movements of ions in the pore.In summary, these results suggest that in order to close or open the pore for significant durations, the channel undergoes a substantial structural change that occurs over tens of milliseconds below $-$10$\sp\circ$C. This relaxation is associated with a highly variable pore current that appears to proceed as an overall graded change of current interrupted by brief transients associated with increased pore current.

Keywords

Biology, Animal Physiology; Biophysics, General

Link to Full Text

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