Cytoskeletal Protein 4.1R Affects Sodium Current in Cardiomyocytes from Transgenic Mice with Prolonged QT Interval

The 4.1 proteins are part of the spectrin-associated cytoskeleton, promote mechanical stability of plasma membranes and are required for cell surface expression of several ion transporters. Protein 4.1R is expressed in the heart and upregulated in human heart failure but its functional role in the myocardium is unknown. 4.1R deficient mice (4.1RKO) have prolonged QT interval and, at cardiomyocyte level, prolonged action potential duration and Ca2+ regulation abnormalities. The causes for these changes remain elusive. We have measured, using whole-cell patch clamping of isolated cardiomyocytes from 4.1RKO, the persistent Na+ current (IpNa), implicated in long QT interval in patients. From a holding potential of -100 mV increasing voltage (20 mV) steps from –80mV to +20 mV for 1 s were applied. The protocol was repeated in the presence of 30 µM tetradotoxin (TTX), and the TTX-sensitive current was analysed. IpNa density was increased in 4.1RKO myocytes compared with wild type (WT) (average IpNa density between 50 and 100 ms after depolarisation for steps to –20 mV (pA/pF): WT = –0.53 ± 0.03 (16); 4.1R KO = –0.36 ± 0.05 (25) (Mean ± SEM (n)); p<0.01; IpNa integral between 50 and 300 ms after depolarisation (pC/pF): WT = –57 ± 9 (16); 4.1R KO = –98 ± 9 (25); p<0.01). To evaluate possible direct binding between SCN5a and 4.1R, a peptide biotin-EPITTTLRRKHEEVSA was synthesized (SCN5a-pep: corresponding to residues 1893–1909 of SCN5a and similar to a 4.1R binding sequence of anion exchanger AE1). A scrambled version of the wild type peptide was used as control. The peptides were mixed with extracts from COS7 cells that had been transfected with a mouse 120kDa cardiac 4.1R tagged with the HA epitope, or extract from cells transfected with vector only. After incubation, bound complexes were recovered on streptavidin beads, and analysed by Western blotting using anti-HA antibody. The 120kDa band corresponding to 4.1R was found only in the SCN5a-pep and not in the control peptide pull-down suggesting that SCN5a can bind 4.1R. Our data indicate a novel function for protein 4.1R in modulating the properties of the Na+ channel, possibly by direct interaction. This may underlie a significant role of proteins 4.1 on the electrophysiology of the heart in normal conditions and in disease.