This chapter focuses on the purification and reconstitution of epithelial chloride channels. To purify a protein requires a functional assay and for a channel the obvious function is ion transport. However, membrane proteins must be solubilized for purification and to assay from this condition requires either reconstitution or the development of a ligands through which to construct a binding assay. This chapter has applied the methods to successfully purify and reconstitute C1- channels from a variety of sources, including apical membranes from bovine tracheal mucosa, sarcolemma from rabbit striated muscle, and bovine thyroid. This chapter also observed electro-physiologically distinct channels from each source. That apparently different C1- channels from these diverse sources would bind to an indanyloxyacetic acids (IAA) affinity column was unexpected. As a positively charged analog to the IAAs also inhibits C1- transport, IAA compounds do not merely compete for a C1-binding site.
ASJC Scopus subject areas
- Molecular Biology