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MIP 28 forms channels in planar lipid bilayers
E Modesto1, L Barcellos, A C Campos-de-Carvalho
1Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
Abstract:
Fiber cells, which constitute most of the lens tissue, have large amounts of a protein named the main intrinsic protein (MIP) in their plasma membrane. MIP seems to vary among species. On SDS-PAGE, MIP from bovine lens (MIP 26) migrates faster than MIP from chicken lens (MIP 28), which runs as a 28-kDa protein. Recently a number of laboratories have shown that MIP 26 forms channels in lipid bilayers. We have isolated membrane fractions highly enriched in MIP 28 from chicken lens and incorporated channel activity into planar bilayers from these membrane fractions before and after treatment with the detergent Triton X-100. Detergent treatment does not seem to affect channel properties. We have attempted to block channel activity with polyclonal antibodies against bovine and chicken MIP but failed to detect blockade using either detergent-free or detergent-treated membranes. Single channel size in symmetric solutions of 300 mM K2SO4 (300-400 pS) agrees well with published results if one allows for corrections in ionic strength. Preliminary experiments indicate that the incorporated channels display voltage dependence. The channel activity recorded from MIP 28-enriched membrane fractions is qualitatively similar to that described for MIP 26 membrane fractions incorporated into bilayers. In contrast to previous reports, we do not find it necessary to add the membrane fractions to both sides of the bilayer to obtain channel incorporation. This may reflect the fact that MIP does not span two bilayers.
Insights
Chicken lens main intrinsic protein (MIP 28) forms channels in lipid bilayers, similar to bovine MIP 26. These channels exhibit voltage dependence and their properties are unaffected by detergent treatment.
Area of Science:
- Ophthalmology
- Biophysics
- Membrane Biology
Background:
- Main intrinsic protein (MIP) is abundant in lens fiber cell membranes.
- MIP exhibits species-specific variations, such as MIP 26 (bovine) and MIP 28 (chicken).
- Previous studies suggest MIP 26 forms channels in lipid bilayers.
Purpose of the Study:
- To isolate and characterize channel activity of chicken lens MIP 28.
- To investigate the effect of detergent treatment on MIP 28 channel properties.
- To compare MIP 28 channel characteristics with those of MIP 26.
Main Methods:
- Isolation of membrane fractions enriched in chicken MIP 28.
- Incorporation of membrane fractions into planar lipid bilayers.
- Electrophysiological recordings of channel activity.
- Functional assays using antibodies and detergent treatment.
Main Results:
- MIP 28-enriched fractions exhibited channel activity in lipid bilayers.
- Detergent treatment (Triton X-100) did not alter channel properties.
- Antibody blockade attempts against MIP were unsuccessful.
- Single channel conductance was measured (300-400 pS in 300 mM K2SO4).
- Channels displayed voltage dependence and qualitative similarity to MIP 26 channels.
Conclusions:
- Chicken lens MIP 28 forms functional ion channels in lipid bilayers.
- MIP 28 channel properties are robust to detergent treatment.
- The incorporation method suggests MIP may not span the entire bilayer.
- Further research is needed to elucidate the precise structure and function of MIP channels.