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 alpha subunit of the human cardiac Na(+) channel (hH1(alpha)) 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(alpha) Na(+) current (I(Na)) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I(Na) in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1(alpha) Na(+) channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of I(Na). 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 microM EPA were significantly smaller for N406K than for the wild type. Coexpression of the beta(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 V(1/2) of the steady-state inactivation in HEK293t cells coexpressing the beta(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 beta(1) decreased this effect even more. Therefore, asparagine at the 406 site in hH1(alpha) 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.
Subject(s)
Fatty Acids/metabolism , Sodium Channels/metabolism , Animals , Cell Line, Transformed , Docosahexaenoic Acids/metabolism , Docosahexaenoic Acids/pharmacology , Fatty Acids/pharmacology , Gene Expression , Humans , Mutagenesis, Site-Directed , Myocardium/metabolism , Oleic Acids/metabolism , Oleic Acids/pharmacology , Oxidation-Reduction , Rats , Sodium Channels/genetics , Sodium Channels/physiology , Stearic Acids/metabolism , Stearic Acids/pharmacologyABSTRACT
Pulmonary surfactant is secreted via exocytosis of lamellar bodies (LBs) by alveolar type II cells. Here we analyzed the dependence of LB exocytosis on intracellular Ca(2+) concentration ([Ca(2+)](i)). In fura 2-loaded cells, [Ca(2+)](i) was selectively elevated by flash photolysis of a cell-permeant caged Ca(2+) compound (o-nitrophenyl EGTA-AM) or by gradually enhancing cellular Ca(2+) influx. Simultaneously, surfactant secretion by single cells was analyzed with the fluorescent dye FM 1-43, enabling detection of exocytotic events with a high temporal resolution (T. Haller, J. Ortmayr, F. Friedrich, H. Volkl, and P. Dietl. Proc. Natl. Acad. Sci. USA 95: 1579-1584, 1998). Exocytosis was initiated at a threshold concentration near 320 nmol/l with both instantaneous or gradual [Ca(2+)](i) elevations. The exocytotic response to flash photolysis was highest during the first minute after the rise in [Ca(2+)](i) and thus almost identical to purinoceptor stimulation by ATP. Correspondingly, the effects of ATP on initial secretion could be sufficiently explained by its ability to mobilize Ca(2+). This was further demonstrated by the fact that exocytosis is significantly blocked by suppression of the ATP-induced Ca(2+) signal below approximately 300 nmol/l. Our results suggest a highly Ca(2+)-sensitive step in LB exocytosis.
