The effects of 4-aminopyridine (4-AP) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free artificial seawater (ASW) containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External (and internal) 4-AP blocks the delayed K+ current in a dose-dependent manner but does not block the leakage current. Our results show that one 4-AP molecule combines with a single receptor site and that the block is voltage dependent with an apparent dissociation constant (K4-AP) of approximately 0.8 mM at 0 mV. K4-AP increases e-fold for a 32-mV change in potential, which is consistent with the block occurring approximately 0.8 of the distance through the membrane electrical field. The 4-AP block appears to depend upon stimulus frequency as well as upon voltage. The greater speed of onset of the block produced by internal 4-AP relative to when it is used externally suggests that 4-AP acts from inside the cell. The Ca2+-activated K+ current was measured in Ca2+-free ASW containing TTX, using internal Ca2+-ion injection to directly activate the K+ conductance. Low external 4-AP concentrations (less than 2 mM) have no effect on the Ca2+-activated K+ current, but concentrations of 5 mM or greater increase the K+ current. Internal 4-AP has the same effect. The opposing effects of 4-AP on the two components of the K+ current can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW and during the repolarizing phase of the action potential.

The effects of tetraethylammonium (TEA) on the delayed K+ current and on the Ca2+-activated K+ current of the Aplysia pacemaker neurons R-15 and L-6 were studied. The delayed outward K+ current was measured in Ca2+-free ASW containing tetrodotoxin (TTX), using brief depolarizing clamp pulses. External TEA blocks the delayed K+ current reversibly in a dose-dependent manner. The experimental results are well fitted with a Michaelis-Menten expression, assuming a one-to-one reaction between TEA and a receptor site, with an apparent dissociation constant of 6.0 mM. The block depends on membrane voltage and is reduced at positive membrane potentials. The Ca2+-activated K+ current was measured in Ca2+-free artificial seawater (ASW) containing TTX, using internal Ca2+ ion injection to directly activate the K+ conductance. External TEA and a number of other quaternary ammonium ions block the Ca2+-activated K+ current reversibly in a dose-dependent manner. TEA is the most effective blocker, with an apparent dissociation constant, for a one-to-one reaction with a receptor site, of 0.4 mM. The block decreases with depolarization. The Ca2+-activated K+ current was also measured after intracellular iontophoretic TEA injection. Internal TEA blocks the Ca2+-activated K+ current (but the block is only apparent at positive membrane potentials), is increased by depolarization, and is irreversible. The effects of external and internal TEA can be seen in measurements of the total outward K+ current at different membrane potentials in normal ASW.

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

1. Membrane currents from the bursting pace‐maker neurone R‐15 of Aplysia were measured under conditions designed to simulate membrane oscillations. Changes in the absorbance of the Ca²⁺‐sensitive dye arsenazo III were used to monitor changes in the free intracellular Ca²⁺ concentration, [Ca]_i, under these conditions. In addition, changes in the extracellular K⁺, concentration [K]_o were measured with K⁺‐sensitive electrodes.2. In normal external ionic conditions the depolarizing phase of pace‐maker activity was associated with a slow inward current and the hyperpolarizing phase with a slow outward current.3. In cells where the early inward Na⁺ current was blocked by tetrodotoxin and outward K⁺ currents were suppressed by intracellular EGTA and extracellular tetraethylammonium and 4‐aminopyridine, the slow inward current was significantly larger in amplitude and was suppressed by removal of external Ca²⁺ or the addition of external La³⁺, but not by the removal of external Na⁺.4. The slow inward current was increased when [Ca]_o was raised and decreased when it was reduced in the manner expected for current flow through a Ca²⁺ channel. The selectivity of the slow inward current for divalent cations was [ ].5. The slow inward current was only slightly reduced by a 10 °C reduction in temperature.6. In normal external and internal ionic conditions changes in dye absorbance occurred when the membrane was depolarized with slow triangular voltage ramps or long depolarizing steps within the pace‐maker oscillation range. The obsorbance change, and thus the increase in Ca²⁺, [Ca]_i, was well correlated with the appearance of the slow inward current. Moreover, the magnitude of the slow outward current was dependent upon the change in [Ca]_i.7. The slow inward current and a substantial fraction of the outward current, as well as the change in [Ca]_i, were reduced appreciably by the addition of La³⁺ ions (3 mM) to the external medium.8. The increase in [Ca]_i during prolonged depolarization was not affected by external tetrodotoxin or by the removal of external Na⁺, but was abolished by a Ca²⁺‐free external medium containing EGTA. Nevertheless, significant changes occurred in [Ca]_i during depolarization in 0·1 mM‐external Ca²⁺.9. In normal external and internal ionic conditions extracellular K⁺, [K]_o, increased during the depolarizing phase of the pace‐maker cycle and decayed during the hyperpolarizing phase.10. There was a measurable increase in [K]_o during small prolonged depolarizing steps which produced a net inward current, indicating that inward and outward currents overlap under normal conditions.11. In the absence of action potential discharge, [Ca]_i increased during the depolarizing phase and decreased during the hyperpolarizing phase of the membrane oscillation.12. It is proposed that pace‐maker oscillations depend upon three separate but linked systems which include a voltage‐dependent Ca²⁺ current, the free intracellular Ca²⁺ concentration and the Ca²⁺‐activated K⁺ current.

Homocysteine is a non-proteinogenic sulfhydryl-containing amino acid derived from methionine and is a homologue of cysteine