Heterotetrameric composition of aquaporin-4 water channels

Aquaporin (AQP) water channel proteins are tetrameric assemblies of individ ually active similar to 30 kDa subunits, AQP4 is the predominant water chan nel protein in blain, but immunoblotting of native tissues has previously y ielded multiple poorly resolved bands. AQP4 is known to encode two distinct mRNAs with different translation initiating methionines, M1 or M23. Using SDS-PAGE urea gels and immunoblotting with anti-peptide antibodies, four po lypeptides were identified in brain and multiple other rat tissues with the following levels of expression: 32 kDa > 34 kDa > 36 kDa > 38 kDa, The 34 and 38 kDa polypeptides react with an antibody specific for the N-terminus of the hi I isoform, and 32 and 34 kDa correspond to the shorter M23 isofor m. Immunogold electron microscopic studies with rat cerebellum cryosections demonstrated that the 34 kDa polypeptide colocalizes in perivascular astro cyte endfeet where the 32 kDa polypeptide is abundantly expressed. Velocity sedimentation, cross-linking, and immunoprecipitation analyses of detergen t-solubilized rat brain revealed that the 32 and 34 kDa polypeptides reside within heterotetramers. Immunoprecipitation of AQP4 expressed in Xenopus l aevis oocytes demonstrated that heterotetramer formation reflects the relat ive expression levels of the 32 and 34 kDa polypeptides; however, tetramers containing different compositions of the two polypeptides exhibit similar water permeabilities. These studies demonstrate that AQP4 heterotetramers a re formed from two overlapping polypeptides and indicate that the 22-amino acid sequence at the N-terminus of the 34 kDa polypeptide does not influenc e water permeability but may contribute to membrane trafficking or assembly of arrays.