Single point mutations affect fatty acid block of human myocardial sodium channel α subunit Na ⁺ channels

Abstract

Suppression of cardiac voltage-gated Na ؉ currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The ␣ subunit of the human cardiac Na ؉ channel (hH1␣) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1 ␣ Na ؉ current (INa) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I Na in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1␣ Na ؉ channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of INa. In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 M EPA were significantly smaller for N406K than for the wild type. Coexpression of the ␤1 subunit and N406K further decreased the inhibitory effects of EPA on I Na in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V1/2 of the steady-state inactivation in HEK293t cells coexpressing the ␤1 subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on I Na, and coexpression with ␤1 decreased this effect even more. Therefore, asparagine at the 406 site in hH1␣ may be important for the inhibition by the PUFAs of cardiac voltage-gated Na ؉ currents, which play a significant role in the antiarrhythmic actions of PUFAs.human cardiac Na ϩ channel ͉ ␤1 subunit ͉ polyunsaturated fatty acids

Potent block of inactivation-deficient Na+ channels by n-3 polyunsaturated fatty acids

“…Certain mutations (F1760K and Y1767K) in this region of hH1 ␣ Na ϩ channels were found eliminate the inhibitory effects of lidocaine and cocaine on cardiac I Na in HEK293t cells transfected with these mutants, but they did not alter the inhibition of I Na by n-3 PUFAs. In contrast, the mutant N406K in the D1-S6 region greatly attenuated the effects of the n-3 PUFAs on cardiac I Na (21). These results indicate that EPA may bind to a region (D1-S6) different from the one to which local anesthetics bind (D4 -S6).…”

“…Cells were split twice per week. When HEK293t cells were grown to ϳ50% confluence, transfection of the wild-type cardiac Na ϩ channel (hNav1.5; 4 g) or a mutant (3 g) of the ␣-subunit of the human cardiac Na ϩ channel (hH1) plus the rat Na ϩ channel ␤1-subunit (20 g) and CD8 cDNA (1 g) was performed using a calcium phosphate precipitation method (20,21). Expression of Na ϩ channels was adequate for current recording.…”

Omega-3 Fatty Acids and Cardiac Arrhythmias: Prior Studies and Recommendations for Future Research

“…95,96 The effects of PUFAs on cardiac Na ϩ channels have also been studied with the use of heterologous expression systems based on either Xenopus oocytes or mammalian HEK cells. [97][98][99] Collectively, these findings show significant dose-dependent inhibition of Na ϩ current at PUFA concentrations similar to those that have been measured in plasma in human trials or animal studies that demonstrate protection against arrhythmias or sudden death. 46,93 Coexpression of sodium channels with the appropriate ␤-subunits can modify the effects of PUFAs on the Na ϩ current, 99 and the so-called persistent or noninactivating component of the Na ϩ current may be preferentially altered.…”

“…95 In addition, heterologous expression experiments using Na ϩ channels with single-point mutations have identified regions of interactions of DHA with Na v 1.5. 97 The possibility that some of these PUFA effects in mammalian heart could be targeted to a specific component of the Na ϩ channel kinetic scheme (ie, slow inactivation) is of interest. Additional examination of this possibility requires knowledge of the expression levels of the ␤-subunits that interact with Na v 1.5 in the mammalian ventricle.…”

Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

“…These LC-PUFA-containing phospholipids mediate membrane fl uidity, thereby modulating activities of ion channels and membrane-associated proteins or enzymes that require specifi c biophysical properties of lipid membranes for their normal function (190)(191)(192). For example, the whole phototransduction cascade from rhodopsin activation to opening and closing of nucleotide gated channels has been shown to be modulated by membrane LC-PUFA ( 43,(193)(194)(195)(196)(197).…”