Abstract
Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Overall, this protocol will enable future APC-based studies of ncAA-containing ion channels in mammalian cells.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Additional discussion on the rationale and limitations of the control experiments.
