Role of the major intrinsic protein in lens physiology

The Xenopus oocyte expression system was used to investigate the transport properties of frog lens Major Intrinsic Protein (MIP). SDS-PAGE and immunoblotting demonstrated oocytes injected with MIP mRNA expressed the protein at high levels, and immunolocalization indicated the expressed MIP migrated to the plasma membrane. Both the oocyte water permeability (P{dollar}rmsb{lcub}H2O{rcub}{dollar}) and glycerol permeability (P{dollar}rmsb{lcub}Gly{rcub}{dollar}) were increased in oocytes expressing MIP relative to controls. P{dollar}rmsb{lcub}H2O{rcub}{dollar} was {dollar}3.4times10sp{lcub}-3{rcub} pm0.18times10sp{lcub}-3{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} with MIP vs. {dollar}0.88times10sp{lcub}-3{rcub}pm0.049times10sp{lcub}-1{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} in control oocytes (p {dollar}<{dollar} 0.01). P{dollar}rmsb{lcub}Gly{rcub}{dollar} was {dollar}2.3times10sp{lcub}-6{rcub}pm0.23times10sp{lcub}-6{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} with MIP vs. {dollar}0.92times10sp{lcub}-6{rcub}pm0.086times10sp{lcub}-6{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar} in control oocytes (p {dollar}<{dollar} 0.01). These increases in permeability were specific; the oocyte permeability to sucrose, 2-deoxyglucose, inositol, sorbitol, reduced glutathione and urea was unchanged by expression of MIP. The component of P{dollar}rmsb{lcub}Gly{rcub}{dollar} due to MIP was independent of concentration from {dollar}5times10sp{lcub}-5{rcub}{dollar} to {dollar}5times10sp{lcub}-2{rcub}{dollar} M and had a low temperature dependence, suggesting permeation through MIP occurs by diffusion in a water-filled pore, rather than via a carrier mechanism. The P{dollar}rmsb{lcub}Gly{rcub}{dollar} of MIP was inhibited approximately 90% by 1.0 mM Hg{dollar}sp{lcub}++{rcub}{dollar} whereas the P{dollar}rmsb{lcub}H2O{rcub}{dollar} of MIP was not, suggesting water and glycerol have different permeation pathways through MIP.; Expression of MIP enhanced glycerol phosphorylation, the first step in glycerol metabolism, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on a method we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 {dollar}pm{dollar} 0.014 vs. 0.06 {dollar}pm{dollar} 0.004 pmol min{dollar}sp{lcub}-1{rcub}{dollar} cell{dollar}sp{lcub}-1{rcub}{dollar}; p {dollar}<{dollar} 0.01) without changing the binding of glycerol to the kinase (K{dollar}rmsb{lcub}M{rcub}approx12 mu{dollar}M).; Lastly, we performed experiments to determine the significance of these observations to lens physiology. The water permeability of vesicles prepared from rabbit lens was {dollar}5.0times10sp{lcub}-3{rcub}pm2.3times10sp{lcub}-3{rcub}{dollar} cm s{dollar}sp{lcub}-1{rcub}{dollar}. Given the high content of cholesterol, sphingolipid and long-chained, saturated phospholipid in the lens, this value is probably too high to be mediated by the lipid portion of the plasma membrane, suggesting MIP contributes significantly to the water permeability of the lens. Lenses incubated in the presence of {dollar}sp3{dollar}H-glycerol produced {dollar}sp3{dollar}H-glycerol-3-phosphate in the cortex and nucleus, demonstrating glycerol kinase activity in fiber cells. Thus in the lens, MIP may have a role in the processing of glycerol, either for entry into the glycolytic pathway or in the synthesis of lipid.