Regulation of the water channel aquaporin‐1: isolation and reconstitution of the regulatory complex

Abstract

Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP-1. AQP-1 and G i3 have been implicated in GTP-induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, G i3 , potassium channel IRK-8, and the chloride channel ClC-2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocked GTP-induced ZG swelling. RBC known to possess AQP-1 at the plasma membrane, swell on exposure to the G i-agonist mastoparan, and respond similarly to ONO-RS-082 and glyburide, as ZGs. Liposomes reconstituted with the AQP-1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO-RS-082 abolished the GTP effect. Immunoisolate-reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. Our results demonstrate a G i3 -PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation.

Secretion machinery at the cell plasma membrane

“…AQP-1 immunoisolate-reconstituted planar lipid membrane demonstrate conductance, which is sensitive to glyburide and an AQP-1 specific antibody. These results demonstrate a G αi3 -PLA2 mediated pathway and potassium channel involvement in AQP-1 regulation at the ZG membrane [49], contributing to ZG swelling. Similarly, AQP-6 involvement has been demonstrated in GTP-induced and G omediated synaptic vesicle swelling in neurons [22].…”

“…Isolated secretory vesicles and reconstituted swelling-competent proteoliposomes have been utilized [13,[20][21][22]49] to determine the mechanism and regulation of vesicle swelling. As previously discussed, isolated ZGs from the exocrine pancreas swell rapidly in response to GTP [20], suggesting rapid water gating into ZGs following exposure to GTP.…”

“…Results from studies demonstrate the presence of the water channel aquaporin-1 (AQP1) at the ZG membrane [21] and its participation in GTP-mediated water entry and vesicle swelling. Further, the molecular regulation of AQP1 at the ZG membrane has been studied [49], providing a general mechanism of secretory vesicle swelling. Detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, G αi3 , potassium channel IRK-8, and the chloride channel ClC-2 [49].…”

“…Further, the molecular regulation of AQP1 at the ZG membrane has been studied [49], providing a general mechanism of secretory vesicle swelling. Detergent-solubilized ZGs immunoprecipitated with monoclonal AQP-1 antibody, co-isolates AQP-1, PLA2, G αi3 , potassium channel IRK-8, and the chloride channel ClC-2 [49]. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO-RS-082, blocks GTPinduced ZG swelling.…”

Addendum to “Regulation of the water channel aquaporin‐1: isolation and reconstitution of the regulatory complex” [Cell Biol. Int. 28(1) (2004) 7–17]*

“…Therefore, the little if any detectable increase in RBC volume in hypotonic media observed in our light-scattering studies ( Fig. 5B; Abu-Hamdah et al, 2004) may be due to the low sensitivity of our light-scattering measurements compared to AFM analysis.…”