Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC29
Carbon monoxide inhibition of the cardiac sodium channel, Nav1.5
J. Elies1, A. M. Duke2, M. L. Dallas1, D. S. Steele2, C. Peers1
1. Cardiovascular and Neuronal Remodelling, University of Leeds, Leeds, United Kingdom. 2. Cardiac Muscle and Neuronal Control, University of Leeds, Leeds, United Kingdom.
Carbon monoxide (CO) has been suggested to cause fatal arrhythmias commonly characterized by elongation of the QT interval and early after-depolarizations, reminiscent of long QT syndrome1. In the present study we have used conventional whole-cell patch clamp recordings to investigate the effects of CO on the recombinant human cardiac Nav1.5 channel stably expressed in HEK293 cells2. The CO-releasing molecule CORM-2 inhibited the peak inward current amplitude (Ipeak,Na) without affecting the late current (IL,Na) in a voltage-independent manner (IC50= 1.05 µM). Exposure to 3 µM CORM-2 elicited 71.3 ± 4.7% decrease of basal Ipeak,Na (mean ± S.E.M., n = 21 cells) whilst 3 µM iCORM (the inactive form of the donor) only reduced currents by 4.5 ± 5.2% (n = 9). Inhibition mediated by 3 µM CORM-2 was unaffected by general or mitochondria-targeted antioxidants (0.5 mM ascorbic acid, 3 mM GSH, 0.4 mM Trolox, 0.1 mM MnTMPyP or 0.3 µM MitoQ) and was also unaffected by blockers of mitochondrial electron transport (2 µM rotenone, 1 µM stigmatellin, or 3 µM antimycin A). Inhibitors of protein kinase G (50 µM Rp-8-Br-PET-cGMPS), CaMKII (1 µM KN-93), and p38MAP kinase (10 µM SB203580) were also unable to alter the effects of CO on Ipeak,Na. In contrast, CO-mediated inhibition was dramatically reduced by the nitric oxide synthase (NOS) inhibitor L-NAME (1mM, 1 h incubation) (14.3 ± 9.9% inhibition, n = 8, p < 0.01 compare to % inhibition induced by 3 µM CORM-2 alone). CO raised NO levels in HEK293 cells, as monitored using the nitric oxide (NO)-sensitive fluorescent dye DAF-2. This NO production was blocked by L-NAME, but NO donors (0.2 mM SNAP and 0.1 mM SIN-1) did not mimic CO effects on Ipeak,Na. However, CO-mediated inhibition was reduced or prevented by the reducing agents dithiothreitol (1 mM; 43.7 ± 7.3% inhibition, n = 11, p < 0.01) and L-cysteine (100 µM; 48.6 ± 9.0% inhibition, n = 7, p < 0.05). Our results indicate that the inhibition of Ipeak,Na by CO are largely mediated by a rise of NO produced by localized NOS, and suggest that the channel redox status might be an important determining factor in this effect of CO.
Where applicable, experiments conform with Society ethical requirements