Two missense mutations (L111R and N122D) were engineered into exon 4. A floxed neo cassette included in the targeting vector was excised from intron 3 prior to germline transmission. The mutations, which manifest in the first extracellular domain of the alpha 2 isoform, impair cardiac glycoside affinity. Western blot analysis of mutant mice indicated that the mutations did not affect either the level protein expression or the distribution of the alpha 1 and alpha 3 isoforms.