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Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics

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Abstract

We used the whole cell patch clamp technique to investigate the characteristics of modification of cardiac Na+ channel gating by the sea anemone polypeptide toxin anthopleurin-A (AP-A). Guinea pig ventricular myocytes were isolated enzymatically using a retrograde perfusion apparatus. Holding potential was −140 mV and test potentials ranged from −100 to + 40 mV (pulse duration 100 or 1000 ms). AP-A (50–100 nM) markedly slowed the rate of decay of Na+ current (I Na) and increased peak I Na conductance (g Na) by 38±5.5% (mean±SEM, P < 0.001, n = 12) with little change in slope factor (n = 12) or voltage midpoint of the g Na/V relationship after correction for spontaneous shifts. The voltage dependence of steady-state I Na availability (h ∞) demonstrated an increase in slope factor from 5.9±0.8 mV in control to 8.0±0.7 mV after modification by AP-A (P < 0.01, n = 14) whereas any shift in the voltage midpoint of this relationship could be accounted for by a spontaneous time-dependent shift. AP-A-modified I Na showed a use-dependent decrease in peak current amplitude (interpulse interval 500 ms) when pulse duration was 100 ms (−15±2%, P < 0.01, n = 17) but showed no decline when pulse duration was 100 ms (−3±1%). This use-dependent effect was probably the result of a decrease in the rate of recovery from inactivation caused by AP-A which had a small effect on the fast time constant of recovery (from 4.1±0.3 ms in control to 6.0±1.1 ms after AP-A, P < 0.05) but increased the slow time constant from 66.2±6.5 ms in control to 188.9±36.4 ms (P< 0.002, n = 19) after exposure to AP-A. Increasing external divalent cation concentration (either Ca2+ or Mg2+) to 10 mM abolished the effects of AP-A on the rate of I Na decay. These results demonstrate that modification of cardiac Na+ channels by AP-A markedly slowed I Na inactivation and altered the voltage dependence of activation; these alterations in gating characteristics, in turn, caused an increase in g Na presumably by increasing the number of channels open at peak I Na. AP-A slows the rate of recovery of I Na from inactivation which is probably the basis for a use-dependent decrease in peak amplitude. Finally, AP-A binding is sensitive to external divalent cation concentrations. Thus, increasing [Mg2+]o or [Ca2+]o displaces AP-A from binding, suggesting that they share related binding sites on the external surface of the Na+ channel.

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  1. Reingold ECG Center, Department of Medicine (Cardiology) and the Feinberg Cardiovascular Research Institute, Northwestern University Medical School, 310 E. Superior Street, 60611, Chicago, IL, USA

    J. Andrew Wasserstrom, James E. Kelly & Kristine N. Liberty

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  1. J. Andrew Wasserstrom
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  2. James E. Kelly
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  3. Kristine N. Liberty
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Wasserstrom, J.A., Kelly, J.E. & Liberty, K.N. Modification of cardiac Na+ channels by anthopleurin-A: effects on gating and kinetics. Pflügers Arch. 424, 15–24 (1993). https://doi.org/10.1007/BF00375097

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  • DOI: https://doi.org/10.1007/BF00375097

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