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Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel
1Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Abstract:
Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.
Insights
Charybdotoxin (CTX) blocks calcium-activated potassium channels. Lysine-27 on CTX is crucial for potassium ions to destabilize toxin binding, revealing a key interaction site.
Area of Science:
- Molecular biology
- Biophysics
- Ion channel pharmacology
Background:
- Charybdotoxin (CTX) is a potent blocker of high-conductance calcium-activated potassium channels (KCa).
- The interaction site for CTX is located at the external mouth of the channel.
- Potassium ions (K+) from the internal solution can destabilize bound CTX.
Purpose of the Study:
- To identify the specific residue(s) on CTX responsible for mediating the destabilization effect by internal K+.
- To elucidate the mechanism by which K+ interacts with CTX bound to the channel.
Main Methods:
- Site-directed mutagenesis of Charybdotoxin (CTX) to create specific point mutants.
- Expression of mutant CTX in E. coli.
- Reconstitution of purified Ca2+-activated K+ channels into planar lipid bilayers.
- Electrophysiological recordings using single-channel analysis to measure CTX dissociation rates.
Main Results:
- A single positively charged residue, Lysine-27 (Lys-27), was identified as the sole mediator of K+-induced CTX dissociation.
- Internal K+ accelerated CTX dissociation in a voltage-dependent manner only when position 27 carried a positive charge.
- Substitution of Lys-27 with neutral residues (Asn or Gln) rendered CTX dissociation insensitive to internal K+ and voltage.
- Neutral substitutions at other positions did not eliminate the K+ destabilization effect.
Conclusions:
- Lys-27 on CTX is positioned near a K+-specific site at the external end of the channel pore.
- A K+ ion binding to this site electrostatically repels the epsilon-amino group of Lys-27, leading to CTX destabilization.
- This finding provides critical insights into the molecular interactions governing ion channel block and toxin binding dynamics.