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A voltage‐clamp study was made of some properties of the non‐synaptic hyperpolarization‐activated Cl‐ conductance recently described in Aplysia neurones loaded with Cl‐ ions (Chesnoy‐Marchais, 1982). The experiments were performed on an identified family of neurones, which present cholinergic responses allowing an easy measurement of the equilibrium potentials of Cl‐ (ECl) and K+ ions (EK). The Cl‐ selectivity of the hyperpolarization‐activated conductance was deduced from four observations: (1) the extrapolated reversal potential of the hyperpolarization‐activated current, Er, was close to the reversal potential of the cholinergic Cl‐ response, which is the equilibrium potential for Cl‐ ions, ECl. (2) Modifications of the intracellular or extracellular Cl‐ concentration induced changes of the reversal potential Er. (3) A prolonged and intense activation of the current lowered the intracellular Cl‐ concentration. (4) The current persisted after complete substitution of intracellular and extracellular cations by CS+ ions, as well as after replacement of extracellular Na+ ions by Tris. The steady‐state Cl‐ conductance (gss) increases steeply with hyperpolarization. The kinetics of activation and deactivation are exponential and are characterized by the same voltage‐dependent time constant (tau), of the order of a few seconds or fractions of seconds. The curves gss(V) and tau (V) can both be fitted by a two‐state model in which the rate constants are exponential functions of the membrane potential (e‐fold change for 12‐16 mV). The Cl‐ current is much more affected by changes of the intracellular Cl‐ concentration than predicted simply from the change in Cl‐ driving force. Both the conductance and the time constant of activation are strongly modified. Modifications of the extracellular Cl‐ concentration do not always alter the amplitude of the hyperpolarization‐activated Cl‐ current, but systematically affect its kinetics. The hyperpolarization‐activated current is abolished after prolonged exposure of the cell to an artificial sea water where NO3‐ ions replace Cl‐ ions, as well as after intracellular injections of NO3‐ ions. Increasing the external pH shifts the gss(V) and tau (V) curves to the left. Lowering the external pH has reverse but less pronounced effects. In cells which were not loaded with Cl‐ ions and did not present the hyperpolarization‐activated Cl‐ current, this current could be detected if the hyperpolarizing jump was preceded by short depolarizing pulses. In cells which were loaded with Cl‐ ions, the Cl‐ current became larger after a short depolarizing pulse. In the presence of extracellular Co2+ ions, depolarizing pulses no longer increased the Cl‐ current.(ABSTRACT TRUNCATED AT 400 WORDS)

Two kinds of single channels, carrying inward currents even above the Na and Cl ion equilibrium potentials, were observed in outside‐out patches from Aplysia neurones bathed in K‐free internal and external solutions. The channel carrying the larger elementary current has been studied in detail. When the internal solution contained mainly CsCl, this channel usually inactivated during the first minutes following isolation of the membrane patch. However, when the internal solution contained NaCl instead of CsCl, the channel remained functional during several hours, thus allowing the present study. Na‐Tris, NaCl‐mannitol and Ca‐Ba external substitution experiments showed that the channel studied is much more permeable to divalent cations than to sodium ions. Mono‐exponential open‐time distributions obtained under identical conditions from different membrane patches indicated either slow (in the order of 100 ms at 0 mV) or rapid (a few milliseconds at 0 mV) mean open‐times. Biphasic open‐time distributions could be obtained from other membrane patches under the same conditions. These results suggest the existence of two different gating modes. Both the open‐time distribution and the closed‐time distribution are voltage sensitive: membrane depolarization activates the channel by lengthening the openings and shortening the closures. The threshold of activation if any, is very low and the inactivation, if present, is never complete. Ca‐Ba, Ca‐Sr and Ca‐Mg external substitution experiments showed that the elementary current amplitude is not very sensitive to the nature of the external divalent cation. The elementary current can be slightly larger when carried by Ba ions rather than by Ca ions, but is nearly identical whether carried by Ca, Sr or even Mg ions, which leads to the elementary conductance sequence: Ba greater than or equal to Ca = Sr congruent to Mg. In contrast, the mean open‐time of the channel is very sensitive to the nature of the external permeant ion. The longest mean open‐time is observed in the presence of Ca ions, and the mean open‐time sequence is: Ca greater than Sr greater than Ba greater than Mg. The closed‐time distribution is also affected by the nature of the external divalent cation. The above results show that the nature of the permeant ion affects the kinetic properties of the channel much more than its elementary current.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The whole‐cell voltage‐clamp mode of the patch‐clamp technique was used to investigate the presence of voltage‐gated inward currents in osteoblasts from newborn rat calvaria. 2. In K+‐free solutions, three kinds of inward currents could be activated by depolarization: a voltage‐gated Na+ current and two different types of Ca2+ currents. 3. The Na+ current was activated by depolarization above ‐40 mV in all the cells. It was reduced by half by 10 nM‐TTX (tetrodotoxin). 4. In an isotonic Ba2+ external solution containing TTX, and with a Cs‐EGTA internal solution buffered at pCa 8, depolarizing jumps induced both a transient Ba2+ current and a sustained Ba2+ current. The relative proportions of these two currents varied greatly among cells. 5. The transient and sustained Ba2+ currents differ with respect to their time course and their voltage dependence. 6. The depolarization‐activated inward currents were also observed under more physiological conditions, in the presence of only 2 mM‐external Ca2+ and with a K+ internal solution buffered at pCa 7. 7. A few records obtained in current clamp showed that it is possible to induce action potentials in osteoblasts.

1. Aplysia neurones with an excitatory response to acetylcholine (ACh) were voltage‐clamped, and the ACh‐induced currents were studied using noise and relaxation techniques. The mean channel open time, tau, and the amplitude of the elementary current, iel, were determined from these experiments, and the variation of these parameters with the ionic content of the extracellular solution was analysed. The goal of this work was to test whether permeant ions may bind in a voltage‐dependent manner to channel sites and thereby hinder channel closing, as has been proposed before (Ascher, Marty & Neild, 1978a). 2. The relation between tau and the membrane potential V has a similar shape in normal sea water and after total replacement of Na ions with Li or Cs. In contrast, the shape of the tau(V) relation is modified if Na is replaced by Mg, Sr, or Ba. 3. Replacing the divalent cations (Mg and Ca) present in normal sea water with Na results in a decrease of tau and an increase of iel. Both effects are enhanced by cell hyperpolarization. 4. Similarly partial replacement of Na by Sr causes a voltage‐dependent decrease of iel. 5. Experiments were performed in solutions containing Na and sucrose, or Mg and mannitol. In both cases tau was smaller than in an isotonic Na or Mg solution. 6. None of the above observations can be accounted for on the sole basis of outer surface potential changes. 7. A quantitative model of the interaction between permeant ions and ACh‐sensitive channels is proposed. The possible relevance of this model for the interpretation of tau(V) curves in other systems is discussed.