Ion channels mediate a wide variety of physiological processes by forming small pores across the membranes that allow regulated flow of ions into or out of the cell. The primary linear sequences of ion channel proteins, like any proteins, are composed by 20 different amino acids, each of which is determined by specific triplet codon in their genes. Site-directed mutagenesis is a widely used molecular biology method to change the triplet in the coding sequence and thereby the amino acid residue in the protein sequence. Functional characterization of the ion channels carrying point mutations allows us to interrogate the structure-function relationships of the ion channels. Here, we will describe the site-directed mutagenesis procedures, in which the wide-type cDNA or plasmid is used as a template to synthesize the complementary mutation-containing DNAs from two mutagenic primers in the polymerase chain reaction.