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Am J Physiol Renal Physiol 296: F1396-F1404, 2009. First published March 18, 2009; doi:10.1152/ajprenal.00018.2009
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Vasopressin-induced differential stimulation of AQP4 splice variants regulates the in-membrane assembly of orthogonal arrays

Alfred N. Van Hoek,1 Richard Bouley,1 YingXian Lu,1 Claudia Silberstein,2 Dennis Brown,1 Martin B. Wax,3 and Rajkumar V. Patil3

1Center for Systems Biology and Program in Membrane Biology and Nephrology Division, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; 2Laboratorio de Fisiopatogenia, Departamento de Fisiología, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina; and 3Ophthalmology Discovery Research, Alcon Laboratories, Fort Worth, Texas

Submitted 13 January 2009 ; accepted in final form 14 March 2009

Aquaporin-4 (AQP4) is a basolateral water channel in collecting duct principal cells and assembles into orthogonal array particles (OAPs), the size of which appears to depend on relative expression levels of AQP4 splice variants. Because the higher-order organization of AQP4 was perturbed by vasopressin in Brattleboro rats and phosphorylation sites have been identified on AQP4, we investigated whether vasopressin and forskolin (Fk) affect AQP4 assembly and/or expression in LLC-PK1 cells stably transfected with the AQP4 splice variant M23, which is responsible for formation of OAPs, and/or the splice variant M1, which does not form OAPs. Our data show that [lys8]-vasopressin (LVP) and Fk treatment led to differential increases in expression levels of M23-AQP4 and M1-AQP4 that varied as a function of incubation time. At early time points (day 1) expression of M1 was significantly stimulated (4.5-fold), over that of M23 (1.6-fold), but after 3 days the expression of M23 became predominant (4.1-fold) over that of M1 (1.9-fold). This pattern of stimulation was dependent on an intact AQP4 residue serine 111 and required protein synthesis. In cells expressing both M1 and M23 (M1/M23 ~ 1), with small sized OAPs at the membrane, the LVP/Fk-induced stimulation of M23 was modified and mimicked that of M1 when expressed alone, suggesting a dominant role for M1. In Brattleboro kidney inner medulla, an 8-day chronic exposure to the vasopressin agonist (dDAVP) led to reduction in M1 and a significant increase in M23 immunoblot staining (M1/M23 = 2/3 -> 1/4). These results indicate that AQP4 organization and expression are regulated by vasopressin in vivo and in vitro and demonstrate that the dominant role for M1 is restricted to a one-to-one interaction between AQP4 splice variants that regulates the membrane expression of OAPs.

hydrophobic plasma membrane protein; osmotic water transport


Address for reprint requests and other correspondence: A. N. van Hoek, Simches Research Center, 8.208, 185 Cambridge St., Boston, MA 02114 (e-mail: vanhoek{at}receptor.mgh.harvard.edu and/or vanhoek.alfred{at}mgh.harvard.edu)






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