Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes.

Major intrinsic polypeptide (MIP), a 28-kDa protein isolated from lens fiber cell membranes, forms large, nonselective channels when reconstituted into lipid bilayers. MIP channels are regulated by voltage, such that these channels close when the potential across the membrane is greater than 30 mV. We have investigated the modulation of the voltage-dependent closure of MIP channels by phosphorylation. In this report, we describe the isolation of two isomers of MIP from lens fiber cell membranes. These isomers differ by a single phosphate at a protein kinase A phosphorylation site. The phosphorylated isomer produces channels that close in response to applied voltages when reconstituted into bilayers. The nonphosphorylated isomer produces voltage-independent channels. Direct phosphorylation with protein kinase A converts voltage-independent channels to voltage-dependent channels in situ. Analyses of macroscopic and single-channel currents suggest that phosphorylation increases the voltage-dependent closure of MIP channels by increasing closed channel lifetimes and the rate of channel closure following the application of voltage.