Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel

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.