The Structural Unit of the Thiazide-sensitive NaCl Cotransporter Is a Homodimer*

Abstract

The thiazide-sensitive NaCl cotransporter (NCC) is responsible for the reabsorption of 5% of the filtered load of NaCl in the kidney. Mutations in NCC cause Gitelman syndrome. To gain insight into its regulation, detailed information on the structural composition of its functional unit is essential. Western blot analysis of total membranes of Xenopus laevis oocytes heterologously expressing FLAG-tagged NCC revealed the presence of both complex-(140-kDa) and core (100-kDa)-glycosylated monomers and a broad band of high molecular mass (250–350-kDa) complexes. Chemical cross-linking with dithiobispropionimidate eliminated the low molecular weight bands and increased the intensity of the high molecular weight bands, indicating that NCC is present in multimeric complexes. Co-expression of HA- and FLAG-tagged NCC followed by co-immunoprecipitation demonstrated that these multimers contained at least two complex-glycosylated NCC proteins. The dimeric nature of the multimers was further substantiated by sucrose gradient centrifugation yielding a peak of ∼310 kDa. A concatameric construct of two NCC polyproteins exhibited significant ²²Na⁺ uptake, indicating that the transporter is functional as a homodimer. A concatamer of partially retarded G980R- and wild type (wt)-NCC displayed normal Na⁺ transport. This demonstrates that G980R-NCC, provided that it reaches the surface, is fully active and that wt-NCC is dominant in its association with this mutant. Conversely a concatamer of fully retarded G741R- and wt-NCC did not reach the cell surface, showing that wt-NCC is recessive in its association with this mutant. Oocytes co-expressing G741R- and wt-NCC did not show G741R staining at the plasma membrane, whereas Na⁺ transport was normal, indicating that wt-NCC dimerizes preferentially with itself. The results are discussed in relation to the recessive nature of NCC mutants in Gitelman syndrome.