The long-term goal of this research is to probe the structure and function of the potassium channel outer vestibule with peptide neurotoxins (a-KTx). Potassium channels regulate numerous cellular processes such as muscle contraction, nerve transmission, hormone release and cell growth. The essence of potassium channel function in all of these processes relies on regulation or gating of transmembrane K+ flux through the channel pore. The a-KTx peptides selectively inhibit potassium channels with high affinity by binding to and occluding the outer pore. This region is important since it is analogous to the active site of an enzyme in that it allows the exquisite selection of K+ ions over other ions in solution. In addition, dynamic changes in the outer vestibule may contribute to gating of K+ flux through the pore. With their rigid, known 3D structure, a-KTx peptides are valuable molecular probes for identifying cationic- and gating- dependent changes in the outer vestibule. Large-conductance calcium-activated (maxi-K) and voltage-gated (KV1) potassium channels exhibit striking differences in their a-KTx specificity that may correlate with structural and functional differences in their outer vestibules. Our first goal is to understand how a-KTx peptides discriminate between maxi-K and KV1 outer vestibules at the molecular level. Using site-directed mutagenesis, we will identify pairwise interactions between toxin and channel amino acids that underlie a-KTx specificity. Guided by 3D channel models, we will examine how these pairwise interactions change with gating. These findings will provide insight into the dynamic relationship between gating and permeation in the channel outer vestibule.
