K-Cl co-transport: immunocytochemical and functional evidence for more than one KCC isoform in high K and low K sheep erythrocytes

K-Cl co-transport (COT) is significantly higher in low K (LK), L-antigen (L ) positive, than in high K (HK), M-antigen (M) positive, sheep red blood ce lls (SRBCs) and is inhibited by sheep allo-anti-L, antibody. To answer the question of whether this difference in K-Cl co-transport activity resides a t the level of the transporter or its regulation, a combined immunocytochem ical and functional approach was taken. At least four isoforms of K-CI COT encoded by different KCC genes are known, with 12 transmembrane domains and cytoplasmic C- and N-terminal domains (Ctd and Ntd, respectively). Polyclo nal anti-rat (rt)KCC1 antibodies against a fusion peptide with 77 amino aci ds from the Ctd of rtKCC1 and anti-human (h)KCC3 against an 18-aa peptide f rom the Ntd of hKCC3, were prepared in rabbits (rb). Two distinctly separat e protein bands of 180 and 145 kDa molecular mass were detected in hemoglob in-free ghosts from RBCs of two LK (one homozygous LL and one heterozygous LM) and one HK (homozygous MM) sheep by Western blots with rb anti-rtKCC1 a nd rb anti-hKCC3. Confocal microscopy showed specific immunostaining of KCC 1 with rb anti-rtKCC1, and of KCC3 with rb anti-hKCC3, in white ghosts from both LK and HK SRBCs. To test the functional heterogeneity of K-Cl COT, th e effect of the anti-L, antibody was assessed on K-Cl COT activated by the kinase inhibitor staurosporine. Incubation of LK SRBCs with anti-Li serum i nhibited by 30% staurosporine-stimulated K-Cl COT suggesting that approxima tely two-thirds of the transport activity is independent of the L, antigen. That staurosporine altered the L, antigen/antibody reaction is unlikely si nce the action of another antibody, anti-Lp, stimulating the Na/K pump flux , was not modified. The present results, in conjunction with earlier work, lead to the hypothesis that the partial anti-Lt inhibition of K-Cl COT may be related to the molecular KCC dimorphism, seen in these cells with anti-K CC1 and anti-KCC3 antibodies. ((C)) 2001 Elsevier Science Inc. All rights r eserved.