K‐CL COTRANSPORTER‐2 (KCC2) EXPRESSION IN CHICKEN CARDIOMYOCYTES

Abstract

K‐Cl cotransporter‐2 (KCC2) is widely considered to be expressed only in mature central neurons. Here, we report the novel finding that KCC2 is expressed in chicken cardiomyocytes. Previously, we prepared antibodies against a fusion protein encoded by exons 22–24 of rat KCC2 that broadly recognized KCC2 in vertebrate brains. While these antibodies have never shown reactivity with protein outside the CNS of therian mammals, they have never been tested in other tissues of lower vertebrates. We confirmed in rat that KCC2 was exclusively expressed in brain, but in chicken, we observed expression in both brain and heart. In silico analysis showed that while exon 22 is unique to the KCC2 isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. A blocking peptide was used to eliminate cross‐reactivity with chicken KCC4 protein, but it did not prevent reactivity with chicken heart. Furthermore, real time PCR revealed robust KCC2 transcript expression in chicken brain and heart, but nearly 10 times less KCC4 transcript in both tissues. These data confirm that our KCC2 antibodies are indeed recognizing KCC2 in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular myocytes. We hypothesize that KCC is critical for cardiomyocyte Cl− homeostasis, especially under conditions of elevated heart rates.