RESUMEN
Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.
Asunto(s)
Proteínas del Ojo/química , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Secuencia de Aminoácidos , Animales , Acuaporinas , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
Wild-type rat lens main intrinsic protein (MIP) was heterologously expressed in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and in mouse erythroid leukaemia cells (MEL C88). Both MEL and Sf21 cell lines expressing wild-type MIP were investigated for the conductance of ions using a whole cell patch clamp technique. An increase in conductance was seen in both expression systems, particularly on lowering the pH to 6.3. In Sf21 cells, addition of antibodies to the NPA1 box resulted in a reduction of current flow. These results suggest that MIP has pH-dependent ion channel activity, which involves the NPA1 box domain.