title: Isoforms of protein 4.1 are differentially distributed in heart muscle cells: relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system.
abstract: The 4.1 proteins are cytoskeletal adaptor proteins that are linked to the control of mechanical stability of certain membranes and to the cellular accumulation and cell surface display of diverse transmembrane proteins. One of the four mammalian 4.1 proteins, 4.1R (80 kDa/120 kDa isoforms), has recently been shown to be required for the normal operation of several ion transporters in the heart (Stagg MA et al. Circ Res, 2008; 103: 855-863). The other three (4.1G, 4.1N and 4.1B) are largely uncharacterised in the heart. Here, we use specific antibodies to characterise their expression, distribution and novel activities in the left ventricle. We detected 4.1R, 4.1G and 4.1N by immunofluorescence and immunoblotting, but not 4.1B. Only one splice variant of 4.1N and 4.1G was seen whereas there are several forms of 4.1R. 4.1N, like 4.1R, was present in intercalated discs, but unlike 4.1R, it was not localised at the lateral plasma membrane. Both 4.1R and 4.1N were in internal structures that, at the level of resolution of the light microscope, were close to the Z-disc (possibly T-tubules). 4.1G was also in intracellular structures, some of which were coincident with sarcoplasmic reticulum. 4.1G existed in an immunoprecipitable complex with spectrin and SERCA2. 80 kDa 4.1R was present in subcellular fractions enriched in intercalated discs, in a complex resistant to solubilization under non-denaturing conditions. At the intercalated disc 4.1R does not colocalise with the adherens junction protein, β-catenin, but does overlap with the other plasma membrane signalling proteins, the Na/K-ATPase and the Na/Ca exchanger NCX1. We conclude that isoforms of 4.1 proteins are differentially compartmentalised in the heart, and that they form specific complexes with proteins central to cardiomyocyte Ca(2+) metabolism.
authors.affiliation: King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London SE1 1UL, UK.
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: Pinder
authors.forename: Pamela M
authors.initials: PM
authors.lastname: Taylor-Harris
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Bennett
authors.forename: Edward
authors.initials: E
authors.lastname: Carter
authors.forename: Nandini V L
authors.initials: NV
authors.lastname: Hayes
authors.forename: Mikayala D A
authors.initials: MD
authors.lastname: King
authors.forename: Mark R
authors.initials: MR
authors.lastname: Holt
authors.forename: Alison M
authors.initials: AM
authors.lastname: Maggs
authors.forename: Philippe
authors.initials: P
authors.lastname: Gascard
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Baines
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D006706:Homeostasis; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D013049:Spectrin / Q000737:chemistry / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; C540651:Epb4.1l2 protein, mouse; C514956:Epb41 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D009479:Neuropeptides; C120557:erythrocyte membrane protein band 4.1-like 1; D013049:Spectrin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506947:Atp2a2 protein, mouse; D002118:Calcium