Transmembrane helix 5 is critical for the high water permeability of aquaporin

Aquaporin-2 (AQP2), a vasopressin-regulated water channel, plays a major ro le in urinary concentration. AQP2 and the major intrinsic protein (MIP) of lens fiber are highly homologous (58% amino acid identity) and share a topo logy of six transmembrane helices connected by five loops (loops A-E). Desp ite the similarities of these proteins, however, the water channel activity of AQP2 is much higher than that of MIP. To determine the site responsible for this gain of activity in AQP2, several parts of MIP were replaced with the corresponding parts of AQP2, When expressed in Xenopus oocytes, the os motic water permeability (P-f) of MIP and AQP2 was 48 and 245 x 10(-4) cm/s , respectively. Substitutions in loops B-D failed to increase P-f, whereas substitution of loop E significantly increased P-f 1.5-fold. A similar incr ease in P-f was observed with the substitution of the front half of loop E, P-f measurements taken in a yeast vesicle expression system also confirmed that loop E had a complementary effect, whereas loops B-D did not. However , P-f values of the loop E chimeras were only similar to 30% of that of AQP 2, Simultaneous exchanges of loop E and a distal half of transmembrane heli x 5 just proximal to loop E increased P-f to the level of that of AQP2. Rep lacement of helix 5 alone stimulated P-f 2.7-fold. Conversely, P-f was decr eased by 73% when helix 5 of AQP2 was replaced with that of MIP. Moreover, P-f was stimulated 2.6- and 3.3-fold after helix 5 of AQP1 and AQP4 was spl iced into MIP, respectively. Our findings suggested that the distal half of helix 5 is necessary for maximum water channel activity in AQP. We specula te that this portion contributes to the formation of the aqueous pore and t he determination of the flux rate.