State-dependent action of grayanotoxin I on Na+ channels in frog ventricular myocytes

Distinct properties of grayanotoxin (GTX) among other lipid-soluble toxins were elucidated by quantitative analysis made on the Na+ channel in frog ventricular myocytes.GTX-modified current (IGTX) was induced strictly in proportion to the open probability of Na+ channels during preconditioning pulses irrespective of its duration, amplitude or partial removal of inactivation by chloramine-T. This confirms that GTX binds to the Na+ channel exclusively in its open state, while batrachotoxin (BTX) was reported to be capable of modifying slow-inactivated Na+ channels, and veratridine exhibited voltage-dependent modification.The GTX-modified channel did not show any inactivation property, which is different from reported results with veratridine and BTX.Estimated unbinding rates of GTX were in reverse proportion to the activation curve of GTX-modified Na+ channels. This was not the previously reported case with veratridine.A model including unbinding kinetics of GTX and slow inactivation of unmodified Na+ channels in which GTX was permitted to bind only to the open state of Na+ channels indicated that unbinding reactions of GTX occur only in the closed state

Monovalent cation (MC) current in cardiac and smooth muscle cells: regulation by intracellular Mg(2+) and inhibition by polycations

1. Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but specific and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2. Here we show that small MC current is present in intact cells and could be dramatically up-regulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca(2+) release-activated Ca(2+)-selective (CRAC) channels, but its activation did not require depletion of Ca(2+) stores, and was observed when the cells were dialyzed with, or without BAPTA. 3. Intracellular free Mg(2+) inhibits MC current with K(d)=250 μM. 4. Extracellular (but not intracellular) spermine effectively blocked MC current with K(d) =3–10 μM, while store-operated cations (SOC) channels and capacitative Ca(2+) influx were not affected. 5. Spermine effectively inhibited MC current-induced SMC depolarization, and prevented Ca(2+) paradox-induced vascular contracture. 6. Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7. It is concluded that MC current could be regulated by intracellular Mg(2+), and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function