Methods Enzymol 2021 1;654:203-224. Epub 2021 Apr 1.
Departments of Pediatrics, Physiology & Biophysics, and Pharmaceutical Sciences, Susan and Henry Samueli College of Health Sciences, University of California, Irvine, CA, United States. Electronic address:
In this method paper, we describe protocols for using membrane-tethered peptide toxins (T-toxins) to study the structure/function and biophysics of toxin-channel interactions with two-electrode voltage clamp (TEVC). Here, we show how T-toxins can be used to determine toxin equilibrium affinity, to quantify toxin surface level by enzyme-linked immunosorbent assay (ELISA) and/or single-molecule total internal reflection fluorescence (smTIRF) microscopy, to assess toxin association and dissociations rate, to identify toxin residues critical to binding via scanning mutagenesis, and to study of toxin blocking mechanism. The sea anemone type I (SAK1) toxin HmK and a potassium channel are used to demonstrate the strategies. Read More