K+-Cl- cotransporter-2 KCC2 in chicken cardiomyocytes

Abstract

Using antibodies prepared against a unique region (exon 22-24) of rat K(+)-Cl(-) cotransporter-2 (KCC2), we confirmed that the ~140-kDa KCC2 protein is exclusively expressed in rat brain, but in chicken, we observed strong reactivity not only with the ~140-kDa KCC2 protein in brain but also a slightly larger ~145-kDa protein in heart. In silico analysis showed that while exon 22 of KCC2 is unique to this isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. To eliminate potential cross-reactivity with chicken KCC4, the antibodies were preadsorbed with blocking peptides prepared over the only two regions showing significant sequence identity to chicken KCC4. This completely eliminated antibody recognition of exogenously expressed chicken KCC4 but not of the ~145-kDa protein in chicken heart, indicating that chicken heart expresses KCC2. Real-time PCR confirmed robust KCC2 transcript expression in both chicken brain and heart. Chicken heart expressed predominantly the longer KCC2a splice variant consistent with the larger ~145-kDa protein in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular cardiomyocytes. We hypothesize that KCC2 is an important Cl(-) extrusion pathway in avian cardiomyocytes that counters channel-mediated Cl(-) loading during high heart rates with β-adrenergic stimulation. While KCC2 is absent from mammalian cardiomyocytes, understanding the role that the other KCC isoforms play in Cl(-) homeostasis of these cells represents a nascent area of research.

Fig. 1.

Structure of K⁺-Cl⁻ cotransporters. A: hypothetical model of human K⁺-Cl⁻ cotransporter-2 (KCC2) where branched lines are potential N-linked glycosylation sites and secondary structural elements are displayed as helices (α-helical) and wavy lines (β-sheet). Color of individual amino acid residues indicate degree of similarity on a per residue basis between human KCC2 and human KCC4: red residues are identical and black residues are absent from human KCC4 (figure was kindly provided by Bliss Forbush using DNAPLOT). B: amino acid alignment of vertebrate KCC2 and KCC4 isoforms over exon 22 of KCC2.

Fig. 2.

Avian and mammalian gene structure of exons (KCC2 equivalent of exons 21–26) encoding the intracellular carboxyl-terminus of the K⁺-Cl⁻ cotransporters (Slc12a4–7).

Fig. 3.

Western blot analysis of membranes prepared from rat (A) and chicken (B) tissues. Rat and chicken brain membranes were loaded at 5 and 10 μg, respectively, whereas membranes from all other tissues were loaded at 100 μg. Western blot was probed with rb-B22-KCC2 antibodies (1:2,000).

Fig. 4.

Deglycosylation of membranes from rat brain, chicken brain, and chicken heart. Membranes from whole rat brain, chicken brain, and chicken heart were incubated with (+) or without (−) N-glycosidase F for 4 h at 37°C. Western blot was probed with rb-B22-KCC2 antibodies (1:2,000).

Fig. 5.

Cross-reactivity of the rb-B22-KCC2 antibodies with chicken KCC4. Membranes were prepared from untransfected HEK-293 cells (control) and HEK-293 cells stably expressing either the rat KCC2 protein, chicken KCC4-S1, or chicken KCC4-S2 protein. Each of the expression constructs for the rat KCC2, chicken KCC4-S1, and chicken KCC4-S2 proteins were epitope tagged with the 10-amino acid c-myc peptide. Western blots panels were probed with either the rb-B22-KCC2 antibodies (left; 1:2,000) or the c-myc peptide monoclonal antibody (right; 1:2,000).

Fig. 6.

Immunoadsorption of the rb-B22-KCC2 antibodies. A: structural models of the carboxy-terminus of rat KCC2 encoded by exons 22–26; top model: degree of similarity on a per residue basis between rat KCC2 and chicken KCC4; bottom model: 9- and 13-mer peptides used in preadsorption experiments (purple residues). B: rb-B22-KCC2 antibodies (1:1,000) were first preadsorbed with increasing amounts of the 13-mer peptide (0- 50 μg) in PBS/milk overnight at 4°C. Strip blots of membranes from HEK-293 cells expressing either rat KCC2 or chicken KCC4-S1 were then probed with each of the preadsorbed fractions (final antibody concentration 1:2,000). C–F: immunoreactivity of rb-B22-KCC2 antibodies with chicken brain and chicken heart following preadsorption with 10 μg 13-mer and/or 9-mer peptides. rb-B22-KCC2 antibodies were preadsorbed with no peptide (C: control), 10 μg 13-mer peptide (D), 10 μg 9-mer peptide (E), or 10 μg both peptides (F) in PBS/milk overnight at 4°C. Strip blots of membranes from HEK-293 cells expressing either rat KCC2 or chicken KCC4-S1, chicken brain, or chicken heart were probed with each of the preadsorbed fractions (final antibody concentration 1:2,000).

Fig. 7.

KCC2 mRNA expression in chicken tissues. A: RT-PCR of KCC2 in chicken tissues. B: RT-PCR demonstrating the specificity and single amplicon production of each primer set for KCC1, KCC2, KCC4, KCC2a, and KCC2b. C: semiquantitative real-time PCR analysis of KCC1, KCC2, KCC4, KCC2a, and KCC2b in chicken brain and chicken heart. Mean normalized expression level of each transcript was determined using the equation: (E_ref)^Ct−ref/(E_target)^Ct−target with GAPDH used as reference and where C_t = crossing threshold (see materials and methods). Values are means ± SE of 8 runs from four separate RNA extractions (2 animals). Brain and heart means are statistically different for each isoform or splice variant tested (P < 0.05 using two sample t-test).

Fig. 8.

Amino acid alignment of exon 1a (A) and exon 1b (B) for avian and mammalian KCC2. Amino acid similarity is shown as grayscale color with black (100% similar), gray (60–99% similar), and white (<60% similar). Scoring matrix was Blosum85 with a threshold of 2.

Fig. 9.

Immunolocalization of KCC2 in adult chicken heart. Left: left ventricle was labeled with a guinea pig antibody against KCC2 (green), a rabbit antibody against cardiac troponin-I (red), and the nuclear stain ToPro3 (blue); prominent KCC2 labeling is observed along the margin of red-stained cardiomyocytes. Cardiac troponin-I antibodies were kindly provided by Aldrin Gomes (University of California, Davis). Right: left ventricle was labeled with either a guinea pig antibody (top) or a rabbit antibody (bottom) against KCC2 (green), a mouse antibody against Na,K-ATPase-α (α6; red) and the nuclear stain ToPro3 (blue); note colocalization of KCC2 and Na,K-ATPase-α along the plasma membrane of cardiomyocytes. Bars = 20 μm.