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

TY - JOUR

T1 - Isoforms of protein 4.1 are differentially distributed in heart muscle cells

T2 - Relation of 4.1R and 4.1G to components of the Ca2+ homeostasis system

AU - Pinder, Jennifer C.

AU - Taylor-Harris, Pamela M.

AU - Bennett, Pauline M.

AU - Carter, Edward

AU - Hayes, Nandini V. L.

AU - King, Mikayala D. A.

AU - Holt, Mark R.

AU - Maggs, Alison M.

AU - Gascard, Philippe

AU - Baines, Anthony J.

PY - 2012/8/1

Y1 - 2012/8/1

N2 - 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, beta-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 Ca2+ metabolism.

AB - 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, beta-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 Ca2+ metabolism.

KW - Cardiac myocytes

KW - Sarcoplasmic reticulum

KW - Intercalated disc

KW - Cytoskeleton

KW - Protein 4.1

KW - Spectrin

KW - SERCA2

KW - ALPHA-II-SPECTRIN

KW - ACTIN-BINDING DOMAIN

KW - C-TERMINAL DOMAIN

KW - FUNCTIONAL-CHARACTERIZATION

KW - CYTOSKELETAL PROTEIN-4.1

KW - INTERMEDIATE FILAMENT

KW - MEMBRANE STABILITY

KW - SEPTATE JUNCTIONS

KW - MAMMALIAN HEART

KW - GENE FAMILY

U2 - 10.1016/j.yexcr.2012.03.003

DO - 10.1016/j.yexcr.2012.03.003

M3 - Article

SN - 0014-4827

VL - 318

SP - 1467

EP - 1479

JO - Experimental Cell Research

JF - Experimental Cell Research

IS - 13

ER -