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Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells

Renal Unit and Program in Membrane Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Submitted 11 December 2003 ; accepted in final form 17 May 2004

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK₁ cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK₁ cells using videomicroscopy, gave osmotic water permeability coefficient (P_f) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p_f (cm³/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P_f = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios (1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p_f for the M23 variant. Expression of an M23-AQP4-Ser^111E mutant produced 1.5-fold greater single-channel p_f and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser¹¹¹ is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

water transport; freeze-fracture; LLC-PK₁ cells; orthogonal arrays; intramembraneous particles

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