A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function

Abstract

Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart Na V 1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the Na V 1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na ؉ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.Channelopathies, so named because they represent a set of diseases caused by mutations in genes coding for ion channels, are a new and growing class of human disorders that include but are not limited to diabetes, muscle disorders, neurological disease, and cardiac arrhythmias (1). A surprising number of channelopathies associated with a wide diversity of human disease are caused by similar mutation-induced changes in ion channel function. Inactivation of voltage-dependent Na ϩ channels is an example of a physiological process critically important in many tissues that, when altered by mutation, can result in muscle weakness, inherited epilepsies, autism, or cardiac arrhythmia (2-4). Clinical consequences of inherited mutations that disrupt Na ϩ channel inactivation provide the most direct link between ion channel biophysics and human physiology and pathophysiology. Conversely, investigation into the consequences of inherited mutations on ion channel function has, in many cases, provided insight into the physiological importance of novel regions and/or structures of ion channel proteins.In the heart, Na ϩ channels (Na V 1.5) 4 primarily underlie action potential initiation and propagation but more recently have been shown to be critical determinants of action potential duration, particularly in the setting of certain inherited channelopathies. Inherited mutations in SCN5A, the gene coding for Na V 1.5, are now known to underlie multiple inherited cardiac arrhythmias, including the congenital long QT syndrome variant 3, Brugada syndrome, and isolated conduction disease (5), and in most cases, these inherited mutations disrupt channel inactivation.Fast inactivation of Na ϩ channels is due to rapid block of the inner mouth of the channel pore by the cytoplasmic linker between domains III and IV that occurs within milliseconds of membrane depolarization...

Modulatory Proteins Can Rescue a Trafficking Defective Epileptogenic Nav1.1 Na+Channel Mutant

“…We have shown that molecular interactions taking place in the C-terminal cytoplasmic domain of Na ϩ channel ␣ subunit, where M1841T is located, can effectively rescue the mutant channel. This domain is functionally important, because it modulates inactivation properties and contains several binding sites for modulatory proteins (Mantegazza et al, 2001;Abriel and Kass, 2005;Glaaser et al, 2006). M1841T lies in an area that includes a ␤1/␤3 binding site (Spampanato et al, 2004), and we have shown that the interaction between the C-terminal domains of ␣ and mutant ␤1-C121W subunits is sufficient to induce ϳ60% of the rescue observed with wild-type ␤1.…”

Solution NMR Structure of the C-terminal EF-hand Domain of Human Cardiac Sodium Channel NaV1.5

“…2C). Mutations of hydrophobic residues in Na V 1.5 have also been shown to lead to perturbation of channel function (49). Such mutations would be expected to significantly destabilize the domain and may perturb its structure leading to alteration of channel function.…”

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

“…However, this position is within the predicted fifth helix and thus not the region involved in formation of the EF-hand motifs (44 -47, 50 -53). Therefore, alterations at this position are unlikely to directly disrupt the overall secondary structure of the pair of EF-hands that are formed by intermolecular interactions between side chains in the interface of helices I with IV and II with III (44,49,54). Furthermore, the Na v 1.3 K1826E and Na v 1.2 E1880K mutations are unlikely to function as helix breakers because the replacement amino acids have higher helical propensities than the original amino acids (55).…”