Tethered peptide toxins for ion channels.
Methods Enzymol
; 654: 203-224, 2021.
Article
em En
| MEDLINE
| ID: mdl-34120714
ABSTRACT
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. T-toxins offer experimental flexibility that facilitates studies of toxin variants by mutation of the expression plasmid, avoiding the need to synthesize and purify individual peptides, speeding and reducing the cost of studies. T-toxins can be applied to peptide toxins that target pores or regulatory domains, that inhibit or activate, that are derived from different species, and that bind to different types of ion channels.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Anêmonas-do-Mar
Limite:
Animals
Idioma:
En
Revista:
Methods Enzymol
Ano de publicação:
2021
Tipo de documento:
Article
País de afiliação:
Estados Unidos