Water Channel Properties of Major Intrinsic Protein of Lens

Sabine M Mulders,Gregory M Preston,Peter M.T Deen,William B Guggino,Carel H Van Os,Peter Agre

doi:10.1074/jbc.270.15.9010

Abstract

The functions of major intrinsic protein (MIP) of lens are still unresolved; however the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and target it to the plasma membrane. Control oocytes or oocytes expressing MIP or CHIP exhibited small, equivalent membrane currents that could be reversibly increased by osmotic swelling. When compared with water-injected control oocytes, the coefficient of osmotic water permeability (Pf) of MIP oocytes was increased 4-5-fold with a low Arrhenius activation energy, while the Pf of CHIP oocytes increased > 30-fold. To identify structures responsible for these differences in Pf, recombinant MIP proteins were expressed. Analysis of MIP-CHIP chimeric proteins revealed that the 4-kDa cytoplasmic domain of MIP did not behave as a negative regulator. Individual residues in MIP were replaced by residues conserved among the Aquaporins, and introduction of a proline in the 5th transmembrane domain of MIP raised the Pf by 50%. Thus oocytes expressing MIP failed to exhibit ion channel activity and consistently exhibited water transport by a facilitated pathway that was qualitatively similar to the Aquaporins but of lesser magnitude. We conclude that MIP functions as an Aquaporin in lens, but the protein may also have other essential functions.

Highlights

The eDNA encoding major intrinsic protein (MIP) was isolated from a bovine lens eDNA library, and hydrophobicity plots predicted that MIP is an integral membrane protein with cytoplasmic amino and carboxyl termini and six bilayer-spanning domains (Gorin et al, 1984)
We investigated the water transporting capacities of MIP using the Xenopus oocyte expression system, and water channel properties qualitatively similar to the Aquaporins were observed
In the absence of osmotic swelling, no differences in conductance were measured in oocytes injected with water, MIP cRNA, or channel-forming integral membrane protein (CHIP) cRNA (Fig. 3)

Summary

Water Channel Properties of Major Intrinsic Protein of Lens*

The functions of major intrinsic protein (MIP) of lens are still unresolved; the sequence homology with channel-forming integral membrane protein (CHIP) and other Aquaporins suggests that MIP is a water channel. MIP forms single channels of various conductances (up to 3 nanosiemens) and a weak selectivity for anions (Ehring et al, 1990); Nodulin-26, a homologous protein from soy bean root nodules, was recently shown to behave (Weaver et al, 1994). In spite of these studies with reconstituted MIP protein, studies of MIP in native membranes or expressed in oocytes have failed to confirm a physiologic function for MIP. We investigated the water transporting capacities of MIP using the Xenopus oocyte expression system, and water channel properties qualitatively similar to the Aquaporins were observed

EXPERIMENTAL PROCEDURES

Total amino acids

RESULTS

ORIGINAL VOLUME

CHIP MIP