Selectivity of the Ca binding site in synaptosome Ca channels. Inhibition of Ca influx by multivalent metal cations.

Abstract

K-stimulated (voltage-dependent) influx of "Ca was measured in synaptosomes (isolated presynaptic nerve terminals) from rat brain . Influx was terminated at I s with a rapid-filtration technique, so that most of the Ca uptake was mediated by inactivating ("fast") Ca channels (Nachshen, D . A ., and Blaustein, M . P ., 1980, J. Gen. Physiol ., 76 :709-728) . This influx was blocked by multivalent cations with half-inhibition constants (K I ) that clustered in three distinct groups : (a) K, > I mM (Mg", Sr", and Bat+ ) ; (b) K, = 30-100 AM (Mn 2+, Co t+ , Ni t+ , Cu e+ , Zn2+ , and H g 2+) ; ( c ) K, < I AM (Cd2+, Y s+, Las+ and the trivalent lanthanides, and Ph 2+) . Most of these ions had very little effect on synaptosome steady state membrane potential, which was monitored with a voltage-sensitive fluorescent dye, or on the voltage dependence of Ca influx, which was assessed by measuring voltage-dependent Ca uptake at two levels of depolarization . The blockers inhibited Ca influx by competing with Ca for the channel site that is involved in the transport of divalent cations. Onset of fast channel inhibition by Mg, Co, Ni, Cu, Zn, Cd, La, Hg, and Pb was rapid, occurring within 1 s ; inhibition was similar after 1 s or 30 min of exposure to these ions . The inhibition produced by Co, Cu, Zn, Cd, La, and Pb could be substantially reversed within 1 s by removing the inhibitory cation . The relative efficacies of the lanthanides as fast channel blockers were compared ; there was a decrease in inhibitory potency with decreasing ionic radius . A model of the Ca channel binding site is considered, in which inhibitory polyvalent cation selectivity is determined primarily by coulombic interactions between the binding site and the different cations . The site is envisaged as consisting of two anions (radius 1 A) with a separation of 2 A between them . Small cations are unable to bind effectively to both anions . The selectivity sequences predicted for the alkaline earth cations, lanthanides, and transition metals are in substantial agreement with the selectivity sequences observed for inhibition of the fast Ca channel .Address reprint requests to Dr .

Antibothropic Action of Camellia sinensis Extract Against the Neuromuscular Blockade by Bothrops jararacussu Snake Venom and Its Main Toxin, Bothropstoxin-I

“…The authors suggested that the toxin would act through Ca 2+ channels, since Mn 2+ antagonized both neurotoxic and myotoxic actions of the myotoxin and are related to Ca 2+ fluxes. Mn 2+ is thought to bind to the Ca 2+ -binding region in the pore of Ca 2+ channels, thereby preventing the passage of calcium ions (Nachshen, 1984). The influence of the earlier presynaptic action of BthTX-I is relevant from the pharmacological point of view, as shown here using C. sinensis leaves extract, although clinically the bothropic envenomation shows no signs of neurotoxicity.…”

Effect of calcium and other divalent cations on intracellular pH regulation of frog skeletal muscle.

“…lOD). The order of potency of divalent cations in inhibiting Na-H exchange is similar to their order of blocking Ca channels in synaptosomes (Nachshen, 1984). Since La is particularly effective in synaptosomes, we examined its effect on pHi recovery in frog muscle.…”

“…Modifier binding sites have been proposed for (among others) internal protons (Aronson, Nee & Suhm, 1982), external quinidine (Mahnensmith & Aronson, 1985), external Li, and amiloride (Ives, Mircheff, Yee & Warnock, 1984) on the Na-H exchanger of renal brush-border membrane vesicles, Ba on K channels in Necturus gall-bladder (Reuss, Cheung & Grady, 1981), and multivalent cations on fast Ca channels of rat brain synaptosomes (Nachshen, 1984). The latter study is noteworthy in that the multivalent cations fell into three groups based on their potency in inhibiting Ca influx.…”

“…The selectivity of the site for various cations can be calculated from the change in free energy (AG) in the exchange reaction involving replacement of Ca2+ at a binding site, X, by another cation, M2+, and is given by: AGM2+-Ca2+ = (AGCa2+-H20 A Uca+x) -(AGM2+-HO -A UM2+_X) This corresponds to eqn. (6) of Nachshen (1984) after correction for an error in sign. AGCa2+-H20 and AGM2+-H.0 are the free energies of hydration for Ca2+ and M2+, respectively (both negative), and AUca2+-x and AUM2+-x are the changes in internal energy for Ca2+ and M2+ binding to the site (also both negative).…”

“…Binding occurs at constant pressure and involves no change in volume or entropy; thus, changes in internal energy and free energy are the same. Since non-coulombic interactions may be important in determining selectivity in chemically dissimilar elements (Nachshen, 1984), we analysed only the alkali earth metals in detail. The values for free energy of hydration of Marcus & Kertes (1969), or the lower values of Friedman & Krishnan (1973), yield the same selectivity sequences for the alkali earth metals as given by Nachshen (1984 , Table VI).…”