Publication Date

2009-12-02

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2009-11-19

First Committee Member

Wolfgang Nonner - Committee Chair

Second Committee Member

Charles Luetje - Committee Member

Third Committee Member

Ana Diez-Sampedro - Committee Member

Fourth Committee Member

Karl L. Magleby - Mentor

Fifth Committee Member

Lawrence B. Salkoff - Outside Committee Member

Abstract

Large conductance voltage and Ca2+ activated K+ (BK) channels control electrical excitability in many cell types. BK channels have the largest conductance (~250 pS) of all K+ selective channels. To explore whether a large entrance to the inner cavity of BK channels is required for their large conductance, I examined if changing the size of the entrance alters the single-channel current amplitude. Previous studies suggest that residues E321/E324 in BK channels are located at the entrance to the inner cavity. To test if positions 321/324 are accessible to intracellular ions, I compared single-channel outward current before and after attaching thiol reagents at E321C/E324C. Attachment of MBB and MTSET altered single-channel currents, indicating that positions 321/324 are accessible to the conduction pathway. Decreasing the size of the entrance to the inner cavity by substituting residues with larger side chains, such as tyrosine and tryptophan, at positions 321/324 decreased the conductance, whereas increasing the size of the entrance had little effect on conductance. Increasing [K+]i from 0.15 to 2.5 M negated differences in single-channel outward current associated with side chain volume. Substitutions had less effect on inward currents. Plots of conductance vs. substituted side chain volume could be approximated with a simple model for the conduction pathway described by two resistors in series, R1 and R2. R2 is a variable resistor, with the resistance proportional to the inverse of the volume of the entrance to the inner cavity not occupied by the side chains. R1 is a fixed resistor arising from the other parts of the conduction pathway including the selectivity filter. Fitting the experimental observations indicated that R1+R2 ~5.4 GΩ for glycine substitution, with an R1/R2 ratio of ~17, and an effective radius and length of the entrance to the inner cavity of ~9.0 and 5.4 Å, respectively. The volume of K+ and water were not taken into account. Taken together, the above observations suggest that a large entrance to the inner cavity is needed for the large conductance of BK channels, as my study shows that the entrance is large and that decreasing the entrance size decreases the currents.

Keywords

Single-channel; K Channel; Two-resistor Model

Share

COinS