Abstract
Publisher Summary This chapter discusses the analysis of oxygen-sensitive human cardiac L-type Ca 2+ channel α 1C subunit (hHT isoform). A study is described, which demonstrated the hypoxic inhibition of a recombinant human cardiac L-type Ca 2+ channel α1C subunit (the hHT splice variant). Inhibition was observed following stable α1C expression in HEK293 cells, in the absence of auxiliary subunits, and the effects of hypoxia appeared to mimic perfectly the effects of hypoxia on native L-type Ca 2+ channel activity in smooth muscle cells. In this chapter, the techniques used to generate mutant L-type Ca 2+ channels and to record their responses to hypoxia following expression in HEK293 cells are discussed. Experimental methodology for the construction of mutant L-type Ca 2+ channels, swapping C-tail segments between hHT and rHT cDNA clones, experimental procedures for blue–white colony screening (colorimetric assay) and purification and concentration of DNA are also described.
Published Version
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