Oger Rougier

The action of acetylcholine on background conductance in frog atrial trabeculae.

The action of acetylcholine (ACh) on membrane potential and currents in frog atrial muscle has been studied with a double sucrose gap technique. The results show the following. 1. ACh induces the development of an extra current, outward at the resting potential, which is dependent on the ACh concentration. 2. The preparation does not show any sign of desensitization. 3. The reversal potential of the current induced by ACh is between 0 and 20 mV more negative than the resting potential and behaves as a K electrode. 4. The mechanism of ACh‐induced K conductance presents inward going rectification properties. 5. The delayed outward current is not affected by ACh. However the evolution of its tail current seems to indicate a process of K accumulation related to the ACh‐induced current.

Tityusγ toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane

Toxin γ from the venom ofTityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3×10−10 M. Hyperpolarization to potentials more negative than the resting potential (E=−90 mV) reduces or abolishes the effect of the toxin.Radioiodinated toxin γ binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1×10−11 M. Data obtained with both rabbit and frog muscle indicate that toxin γ is specific for Na+ channels in surface membranes. Toxin γ does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is oneTityus γ toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed thatTityus γ toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II fromCentruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel thanTityus γ toxin.

Excitation contraction coupling in skeletal muscle: Evidence for a role of slow Ca2+ channels using Ca2+ channel activators and inhibitors in the dihydropyridine series

Ca2+ current and tension have been simultaneously recorded from single twitch fibres of the semi-tendinosus of Rana esculenta in a medium containing a physiological Ca2+ concentration (1.8 mM). Under appropriate conditions it can be shown that tension develops in two phases. The first is rapid and reaches its maximum before activation of the inward Ca2+ current. The second phase is slower and with a time course which appears to be correlated with that of the inward current. Nifedipine, a specific Ca2+ channel inhibitor greatly reduced ICa2+ and the slower component of tension. Bay K8644 a Ca2+ channel activator, which has receptors on T-tubule, increased ICa2+ and the slow component of tension. These results indicate that a slow component of skeletal muscle contraction is related to the inward Ca2+ current flowing through dihydropyridine sensitive voltage-dependent Ca2+ channels.

Analyse électrophysiologique du plateau des réponses myocardiques, mise en évidence d'un courant lent entrant en absence d'ions bivalents

SummaryIn isolated perfused rat heart, electrophysiological analysis of myocard membrane properties in the absence of divalent ions during the plateau of the action potential, shows that: 1.The maintenance of the membrane potential at plateau level (nearE=0 mV) does not depend on the action of an active cellular potassium absorption mechanism dependent on metabolism.2.The plateau potential depends on the external sodium concentration, the membrane behaves like sodium electrode.3.The plateau potential is little or not at all modified by tetrodotoxine, but totally abolished by manganese ions.A Voltage clamp of frog atrial muscle shows, under identical conditions, the existence of a slow inward sodium current responsible for long duration responses. This current is not modified by tetrodotoxine, but suppressed by manganese ions.So, under similar experimental conditions (Ca++, Mg++, free solution) the existence of long duration sodium responses in the rat proves the existence of a slow channel identical to that previously described in the frog.ZusammenfassungAm durchströmten isolierten Herzen der Ratte ergibt die elektrophysiologische Untersuchung der Eigenschaften der Myokardmembran während des Aktionspotentialplateaus und in Abwesenheit zweiwertiger Ionen folgende Resultate: 1.Die Blockierung der Membranpolarisation am Plateau, in der Nähe vonE=0 mV, beruht nicht auf der Beteiligung eines stoffwechselabhängigen aktiven Kaliumresorptionssystems der Zellen.2.Die Höhe des Plateaus ist von der Natriumaußenkonzentration abhängig, wobei die Membran sich wie eine Natriumelektrode verhält.3.Dieses Plateau wird durch Tetrodotoxin wenig oder nicht beeinflußt aber durch Mangan unterdrückt.Die unter den gleichen Versuchsbedingungen am Frosch durchgeführten voltageclamp Versuche zeigen, daβ ein langsamer, für die langdauernden Aktionspotentiale verantwortlicher Natriumeinwärtsstrom vorhanden ist, der durch TTX nicht beeinfluβt, durch Mangan jedoch inhibiert werden kann. Ebenso zeigen die bei der Ratte auftretenden langdauernden Natriumpotentiale, die durch TTX nicht beeinfluβt, durch Mangan jedoch inhibiert werden, daβ ein slow-channel ähnlich dem bereits am Frosch beschriebenen vorhanden ist.

Activation of ATP-dependent K+ channels by metabolic poisoning in adult mouse skeletal muscle: role of intracellular Mg(2+) and pH.

1. The effects of metabolic poisoning, intracellular Mg(2+) and pH on ATP‐dependent K+ (K+ATP) channels were examined in adult mouse isolated skeletal muscle fibres using the patch clamp technique. 2. In cell‐attached membrane patches, while openings of one kind of channel could only rarely be detected under control conditions, cell poisoning with fluorodinitrobenzene (FDNB), dinitrophenol (DNP) and cyanide (CN) induced a strong and partially reversible increase in channel activity. 3. Slope conductance and glibenclamide sensitivity of this outward current indicated that the channel activated during poisoning was the K+ATP channel. 4. Single channel current amplitude was reduced during poisoning, but remained unchanged when activation of the K+ATP channel was induced by cromakalim. 5. In inside‐out membrane patches, in the absence of intracellular ATP, intracellular application of Mg2+ decreased channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 6. In the absence of intracellular ATP, a decrease in intracellular pH induced a reduction in channel activity and single channel current amplitude. Inhibition of K+ATP channels by ATP was also reduced. 7. The reduction of single channel current amplitude during poisoning was attributed to an increase in intracellular Mg2+ concentration caused by a fall in intracellular ATP concentration. These results also show that metabolic poisoning causes direct activation of K+ATP channels in skeletal muscle, and that is activation is at least partially mediated through an increase in intracellular Mg(2+) concentration and a decrease in intracellular pH.

Kinetic analysis of the delayed outward currents in frog atrium. Existence of two types of preparation

1. The outward currents underlying delayed rectification have been studied in frog auricular trabeculae with the double sucrose gap method.

Intracellular Ca2+ changes and Ca2+‐activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.

1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo‐1 and single channel activity using the patch clamp technique. 2. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/‐ 30 nM from a mean resting value of 72 +/‐ 7 nM (n = 13). In cells bathed with a K(+)‐rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]i from a mean resting value of 53 +/‐ 7 nM to a maximum of 222 +/‐ 24 nM (n = 33). 3. In cell‐attached membrane patches at the endplate membrane of muscle fibres bathed in a K(+)‐rich external solution, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward current of an average amplitude of 4.6 +/‐ 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2(+)‐activated K+ channels. At 0 mV, in inside‐out patches excised from the endplate membrane area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)‐rich solution in the pipette, respectively. Half‐maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside‐out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh‐induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+‐activated K+ channels in the adjacent surface membrane.

Block by Cs of K currentiK1 and of carbachol induced K currentiCch in frog atrium

Abstract1. In frog atrium, Cs ions block both the inward rectifieriK1 and the carbachol induced K currentiCch. 2. BothiK1 andiCch display a high affinity for Cs with a K0.5 of 4×10−5 M forik1 and of 8×10−5 M foriCch atV=−50 mV. 3. Block of bothiK1 andiCch is strongly voltage dependent. When fitted by the block model of Woodhull (1973), δ is >1 for the two currents. 4. From these similarities, action of Cch on frog atrium K permeability could be interpreted as a modification ofiK1.

Role of the Na+−Ca2+ exchange in the calcium paradox in frog auricular trabeculae

After a Ca2+-free ouabain perfusion of 8-10 min duration, reperfusion of isolated stimulated frog auricular trabeculae with Ca2+ ouabain containing medium resulted in a large and transient contracture. The contracture was weaker in a quiescent preparation or in the absence of ouabain. In sodium-free (Li+ substitute) ouabain containing medium, the amplitude of the contracture was largely decreased while it disappeared completely in Na+-free medium without ouabain. Moreover this contracture was suppressed by 15 mM Mn2+. Although the approach was only indirect, these results suggest that the contracture is due to an entry of Ca2+ ions through the Na+-Ca2+ exchange mechanism and that this could be the unique route of calcium entry during calcium repletion.

Nifedipine and bay K inhibit contraction independently from their action on calcium channels

Voltage-clamp experiments were performed on twitch skeletal muscle fibres in a double sucrose-gap device in order to investigate the effect of 1,4-dihydropyridines (DHP) on excitation-contraction coupling during 50 ms step depolarizations. External solution used was free of permeant anions and contained only Ca++ as permeant cation. It is shown that in these conditions Nifedipine (a Ca++ channel antagonist) and BAY K 8644 (a Ca++ channel agonist) inhibit contraction in a way independent of their action on the gating of tubular calcium channels. These results indicate also that a close relation between DHP receptors and calcium channels must be taken with caution.