Suction Pipette Research Articles

Voltage-dependent activation of potassium current in Helix neurones by endogenous cellular calcium.

1. The effect of endogenous Ca on potential-dependent K current IKD, was examined in identifiable neurones of Helix aspersa. The suction pipette method of internal perfusion was used along with a combined voltage-clamp method in which the membrane potential was measured by a separate glass micro-electrode and the current was passed by the suction pipette. Activation of the potential-dependent A current, IA, was prevented by using holding potentials of -40 mV where IA is inactivated and by the addition of the A-current blocker 4-aminopyridine. Activation of K currents by transmembrane Ca current, IKCa, was suppressed by Co substitution for Ca ion extracellularly. 2. Under these conditions, IKD rose to a peak value and then subsided to a steady level. The current-voltage (I-V) relationship for peak IKD had an upward bump at about +50 mV that gave it an S-shape. The I-V curve for steady IKD rose continuously. Peak and steady IKD were reduced by perfusing with EGTA or F ions intracellularly. The EGTA effect occurred at intracellular Ca activity levels below 10(-7) M. Increases in the concentration of EGTAi at constant Cai had no additional effect; however, recovery experiments do not allow us to rule out some direct action of EGTA on IKD. 3. Prolonged extracellular perfusion with Co-substituted solutions also reduced IKD and the effects occurred more quickly when the solutions were made hypertonic or caffeine was added to them. The peak transient was abolished, and the small remaining steady IKD (about 5-10% of normal peak IKD) was blocked by tetraethylammonium. IKD could be restored by the temporary reintroduction of Ca in the extracellular solution. 4. The S-shape of the peak I-V relationship for IKD may be due to Ca released from an endogenous site by membrane depolarization. The reduction of steady and peak IKD to very low values by Ca chelators or prolonged perfusion with Ca-free solutions indicates that Cai is important for activation of these K channels. 5. Three cellular structures were identified in electron micrographs of freeze-fractured neurones that could be involved in potential-dependent endogenous Ca release. These were a restricted extracellularly space, an intracellular membrane system of endoplasmic reticulum that may be fused to the internal face of the plasma membrane (the subsurface cisterns of Henkart & Nelson, 1979), and intracellular vesicles that also may be fused to the plasma membrane.

Accelerating actions of n-alcohols on the inactivation process of the Ca current in Helix neurones.

Effects of n-alcohols on the Ca current were investigated in snail neurones, perfused intracellularly and voltage-clamped by a suction pipette technique. All n-alcohols employed in this experiment (methanol, ethanol and buthanol), at low concentrations, decreased the peak amplitude of the Ca current and produced the acceleration of the current inactivation in a dose-dependent manner at all membrane potentials. These n-alcohols did not affect the rising phase of the current, capacitive, leakage and non-specific outward currents. Double-pulse experiments showed also the acceleration of the current inactivation in the presence of n-alcohols. Shortening the decay phase of the current appears to be due to acceleration of the normal inactivation mechanism rather than a time-dependent block of open Ca channels by alcohol molecules. The concentrations of n-alcohols for acceleration of the current inactivation decreased as the hydrocarbon chain of n-alcohols increased in length. The modification in the Ca current inactivation may result from conformational alterations in the lipid milieu surrounding the Ca channels by n-alcohols since the alcohols are known to perturb the structure of lipid bilayer membrane. We can not, however, rule out the possibility that n-alcohols affect the protein part of the Ca channel.

Interaction between GABA- and pentobarbital-induced responses

CI currents in GABA-sensitive neurons of the bullfrog dorsal root ganglion were separated from other Na, K and Ca currents using a suction pipette technique which allows internal perfusion and voltage clamp. GABA and pentobarbital (PB) induced CI current dose-dependently, and the number of drug molecules required to activate a receptor (n) was 1 for GABA and 2 for PB. In the presence of 10−4M PB, 10−6M GABA-induced CI currents were enhanced 6 to 10 times, and the dose-response curve gave n = 2. Moreover, the enhancement was potential dependent and strongly appeared at less membrane potentials than CI equilibrium potential. Power-spectrum analysis of GABA- and PB-induced CI current fluctuations showed two corner frequencies. In each agonist, the lower corner frequency was clearer than the higher one at low drug concentration,while the higher corner frequency became more clear at the high concentration. The coexistence of GABA and PB regardless of low concentration, however, markedly developed the higher corner frequency. Such a high corner frequency seems to reflect the bursting activities of single CI channel currents recorded by patch clamp technique. PB response was more sensitive to external or internal addition of Picrotoxin than in GABA response. The site of PB action on GABA response was discussed from all results.

Effects of pipecolic acid on the individual membrane currents of Helix neurones.

Recent pharmacological studies showed that pipecolic acid (PA) had potent actions on mammalian behaviors (Miyata et al., 1973) and potentiated the anticonvulsant activity of phénobarbital (Takahama et al. 1982). In addition, PA was reported to have an inhibitory action on unit discharge from cortical and hippocampal pyramidal cells (Kase’ et al., 1980). The present study described voltage clamp experiments designed to examine the effects of PA on individual membrane currents of the snail neurone by the suction pipette method. The results are summalized as follows: 1) PA at 0.01 to 0.03 mM started to reduce the maximal peak amplitude (MPA) of the Ca current, and increased concentrations of PA (0.1 to 1 mM) depressed the MPA by 13 to 23 %. 2) A high concentration (1 mM) of PA slightly reduced the peaks of delayed K current and fast K current. PA reduced the amplitude of the Ca current rather than that of the K current in a dose-dependent manner, suggesting that PA had an inhibitory effect on the excitability of the neurone. The concentrations of PA in mammalian tissues is, however, much lower than that of PA required for direct action on the membrane currents (Okano et al., 1983). The possible mechanism of actions of PA on the nervous system may be based on a functional connection of PA with GABAergic transmission as suggested previously (Kase’ et al., 1980; Takahama et al., 1982).

Differences in actions of organic and inorganic Ca-antagonists on the Ca current of Helix neurone

Differences in actions of organic and inorganic Ca-antagonists on the Ca current were investigated in snail (Helix aspersa) neurones perfused intracellularly and voltage-clamped by a suction pipette technique. Organic Ca-antagonists used were diltiazem, verapamil, nifedipine, nicardipine and KB-944; and inorganic Ca-antagonists used were cobalt, manganese and cadmiun. Results are summarized as follows: 1) The organic Ca-antagonists showed a time-dependent inhibition on the current amplitude during a depolarizing pulse lasting for 1 to 2 sec and a use-dependent depressant effect on the peak amplitude of the current. Double-pulse experiments showed that the organic Ca-antagonists also accelerated the current inactivation. Inorganic Ca-antagonists did not exhibit the accelerating actions on the current inactivation. 2) Diltiazem and verapamil showed almost equivalent inhibitory effects on the divalent cation currents carried by Ba, Ca and Mn ions which were permeant through the Ca channel. On the other hand, an inorganic Ca-antagonist such as cobalt ions greatly suppressed the peak amplitude of the Ba current, but slightly suppressed that of the Mn current. These results suggested that the site of action of organic Ca-antagonists partly differed from those of inorganic Ca-antagonists.

Effects of organic Ca-antagonists on the Na current in rabbit dipersed ventricular cells.

Effects of organic Ca-antagonists on the Ma current were investigated in rabbit dispersed ventricular cells by the suction pipette method. Drugs used were diltiazem (DL), verapamil (VR), nifedipine (NF) and nicardipine (NC). The Na current remained unaffected in the presence of MF and NC at concentrations below 0.1 μM, but VR and DL at a concentration of 0.1 μM started to reduce the maximal peak amplitude (MPA) of the Na current. Higher concentrations of the Ca-antagonists induced a decrease in the MPA of the Ma current, and the effects mere dnse-dependent. The current-voltage relationships showed that the current amplitude was reduced at all membrane potentials without any change in the potential at which the maximal inward current was induced. The effects of DL (1 μM), VR (1 μM), NF (1G μM) and NC (10 μM) on the steady state inactivation and the reactivation process of the Ma current were investigated. The Ca-antagonists shifted the midpoint of the steady-state inactivation curve in the hyperpoiarizing direction by 4 to 7 mV and made the curve slightly steeper. The half reactivation time in the control was 24 to 27 msec, but the time for half-reactivation in the presence of these agents was significantly prolonged to about 40 msec or more. The results allowed interpretation of mechanisms by which DL and VR exert antiarrhythmic actions at clinical doses.

Reversal of current through calcium channels in dialysed single heart cells.

Calcium channels contribute to important cellular functions such as impulse conduction, rhythmic activity, muscle contraction, and secretion1 and are a major target for modulation by neurohormones and drugs2,3. Despite their importance, Ca channels are much less well understood than Na and K channels because of several problems reviewed by Hagiwara and Byerly1. First, few preparations allow good control of electrical potential and ionic composition on both sides of the membrane, yet display strong Ca currents. Second, current carried by Ca channels is often obscured by ion movements through other membrane pathways. Third, outward current flow through Ca channels, carried by Ca2+ or any other ion, has been difficult to demonstrate. This is unfortunate because measurements of the potential at which current reverses (Erev) have been crucial in understanding ion permeation and selectivity in other ionic channels (see ref. 4). In molluscan neurones, attempts at recording outward current through the Ca channel have not succeeded, largely because of overlap by nonspecific outward current5–7. In multicellular cardiac muscle preparations, strong depolarizations produce a decaying outward current that Renter and Scholz attributed to K efflux through the Ca channel8. Their interpretation is controversial, however, since the evidence leaves open other explanations for the outward current—for example, Na efflux via Na–Ca exchange9, or K efflux through K-selective channels. To overcome these problems, we studied Ca channels in single isolated heart muscle cells10–12 using a suction pipette method13. We were able to record robust Ca currents with minimal interference from other time-dependent currents while controlling potential and ion composition on both sides of the membrane. Here we present experimental evidence for a genuine reversal of ionic current through Ca channels due to outward movement of K+ ions, in support of the hypothesis of Reuter and Scholz.

Further analysis of inhibitory effects of propranolol and local anaesthetics on the calcium current in Helix neurones.

1 The effects of propranolol and local anaesthetics on Ca2+ current (Ica), individually separated from other ionic currents, in Helix neurones were studied under voltage clamp, using a suction pipette technique. 2 Increases in external Ca2+ concentrations overcame the inhibitory action of propranolol on Ica. Double reciprocal plots for peak Ica versus external Ca2+ concentrations in the presence or absence of propranolol did not intersect at the ordinate. 3 Internal application of propranolol (10-4M) inhibited Ica to about 40-60% of the control in a time-dependent manner. 4 Lignocaine and procaine at concentrations of 10-3-10-2 M inhibited Ica without shifting the threshold in the I-V relationships. Internal application of lignocaine (10-3-10-2M) also inhibited Ica: the ratio of depression of the Ica was almost equivalent to that of the agent applied externally. 5 The results provide evidence that propranolol inhibits Ica in the noncompetitive manner with Ca2+ at the cell membrane, and suggest that the agents may occupy the receptor site in the Ca2+-channel somewhere between the outer surface and inner phase of the membrane.

Zinc current in Helix soma membrane

1. Characteristics of zinc inward current ( I Z n) were investigated in respect to its kinetics, voltage-dependence and pharmacological properties in the identified Heliix neurones using a suction pipette technique which allows internal perfusion and voltage clamp. 2. After complete blockage of Na + and K + currents, Zn 2+ evoked all-or-none actiona potentials with a prolonged plateau phase lasting for more than 30 sec. The action potentials were not affected in the presence of TTX but abolished by adding Ca 2+-blockers such as Co 2+ and verapamil. The overshoot was increased by 26 mV for a ten-fold increase in the external Zn 2+ concentration ([Zn 2+] 0). 3. Under voltage clamp conditions, the zinc inward current ( I Z n ) appeared around -20 mV and the maximum peak I Z n evoked at about +30mV. At more high voltage steps, I Z n became smaller and flattened, but did not reverse. 4. In spite of the presence of or absence of Mg 2+, increases of [Zn 2+] 0 induced a hyperbolic increase of I Z n and also shifted the threshold and peak potentials in the I- V curves along the voltage axis to the more positive direction. 5. I Z n was depressed dose- and time-dependently by externally and internally applied organic (verapamil) and inorganic (Co 2+) Ca 2+-blockers. Internal perfusion of Zn 2+ also depressed I Z n time-dependently without shifting the I- V curve and changing the leakage current. 6. Internal application of F − inhibited C n and I Z n time-depently and completely abolished both currents within 10 min. It is concluded that the voltage-dependent Ca channel in the identified Helix nerve cell bodies is permeable to Zn 2+. 7. Both activation ( τ m ) and inactivation ( τ h ) phases of I Z n were fitted by a single exponential function. Time constants for τ m and τ h for I Z n and I C a were voltage-dependent. The voltage dependency was greater for I Z n than for I C a , especially at high voltage steps. τ m and τ h values for I Z n were larger than those for I C a . 8. The steady-state inactivation value, h ∞, of I Z n showed dependence on either the voltage or duration of ‘conditioning’ prepulses. However, complete inactivation of I Z n at any given test pulses was not observed even in the prepulses duration of more than 12 sec. 9. Small amounts of Ca 2+ strongly reduced I Z n . The interaction of Ca 2+ on I Z n was 30 to 100 times stronger than that of Mg 2+. 10. Lower critical size which limits the ionic permeation through the Ca channel may exist just below of 0.74 Å which is the size of Zn 2+.

Calcium current-dependent and voltage-dependent inactivation of calcium channels in Helix aspersa.

1. Inactivation of the Ca channels has been examined in isolated nerve cell bodies of Helix aspersa using the suction pipette method for voltage clamp and internal perfusion.2. Satisfactory suppression of outward currents was essential. This was achieved over most of the voltage range by substitution of Cs ion for K ion and by the use of TEA intra- and extracellularly and 4-AP extracellularly. A small time- and voltage-dependent non-specific current remained at potentials above +60 mV.3. In these solutions, Ca current approaches E(Ca) but cannot be detected in the outward direction. The Ca channel appears to be impermeable to Cs and Tris ions.4. Inactivation of Ca currents occurs as a bi-exponential process. The faster rate is 10-20 times the slower rate and is about one twentieth the rate of activation. The development of inactivation during a single voltage-clamp step and the onset of inactivation produced by prepulses followed after brief intervals by a test pulse, have roughly similar time courses.5. The rates of inactivation increase monotonically at potentials more positive than about -25 mV. The amount of steady-state inactivation increases with membrane depolarizations to potentials of about +50 mV. At more positive potentials, steady-state inactivation is reduced.6. Intracellular EGTA slows the faster rate of inactivation of I(Ca) and reduces the amount of steady-state inactivation measured with a standard two pulse protocol. The effect is specifically related to Ca chelation and hydrogen ions are not involved. This component of inactivation is referred to as Ca current-dependent inactivation and is consistent with observations that increased Ca(i) inactivates the Ca channel. The process does not depend upon current flow alone since Ba currents of comparable or greater magnitude have smaller initial rates of inactivation. Furthermore, application of Ba ion intracellularly in large concentrations has no effect on steady-state inactivation.7. The bi-exponential inactivation process that persists in the presence of EGTA(i) is similar to that occurring when extracellular Ba ion carries current through the Ca channel. Steady-state inactivation also persists and is similar in the two cases. Therefore it is concluded that inactivation is voltage-dependent as well as Ca current-dependent.8. Diffusion models that included reasonable values for the effect of binding on diffusion, even when combined with declining influxes, did not account for this ;mixed' form of calcium- and voltage-dependent inactivation. A compartmental model in which the particular kinetic model of voltage-dependent inactivation was not critical described the Ca current-dependent inactivation.

Actions of verapamil, diltiazem and other divalent cations on the calcium-current of Helix neurones.

1 Effects of organic Ca(2+)-antagonists, verapamil and diltiazem, and cations, Ni(2+), Mn(2+), Co(2+) and La(3+) on Ca(2+) current (I(Ca)) separated from other ionic currents in a Helix neurone were studied. A suction pipette technique which allows internal perfusion of the cell body and voltage clamp was used.2 Verapamil and diltiazem (10(-6)-10(-4) M) increased the threshold, and decreased both the amplitude and rate of rise of the soma Ca(2+)-spike. Both agents inhibited I(Ca) over the entire range of the current-voltage (I-V) relationship dose-dependently, without shifting the threshold of the I-V relationship. Increases in external Ca(2+) overcame the inhibitory action of the agents.3 Divalent cations, Ni(2+), Mn(2+), Co(2+) and the trivalent cation, La(3+) inhibited I(Ca) dose-dependently, but induced shifts of the I-V relationship to more positive voltages. The order of potency of inhibition of I(Ca) among these cations was as follows; Ni(2+) > La(3+) > Mn(2+) > Co(2+).4 Double reciprocal plots for peak I(Ca) versus external Ca(2+) concentrations in the presence or absence of both organic and inorganic Ca(2+)-antagonists intersect at the ordinate. Results indicate that both organic and inorganic Ca(2+)-antagonists compete for Ca(2+) at the common binding site for Ca(2+).5 Internal application of the organic Ca(2+)-antagonists (10(-4) M) inhibited I(Ca) in a time-dependent manner to about 40-60% of the control. Ni(2+), when applied internally, also depressed I(Ca).6 The results provide evidence that organic Ca(2+)-antagonists occupy the binding site for Ca(2+) in a competitive manner at the surface of the soma membrane of the Helix neurone, while divalent and trivalent cations, in addition to inhibiting I(Ca) in a similar manner to the organic Ca(2+)-antagonists, change the surface charge of the soma membrane.

Inhibitory effects of propranolol on the calcium current of Helix neurones.

1 Calcium current (ICa) and potassium current (IK) in isolated nerve cell bodies of Helix aspersa were independently separated after suppression of Na+ and K+ currents, and Na+ and Ca2+ currents, respectively. The suction pipette method was used. Under voltage clamp conditions, the effects of propranolol on both the ICa and IK were examined. 2 Propranolol inhibits the ICa at relatively low concentrations (10(-6)-10(-5) M). The inhibitory action was dose-dependent. 3 The IK was moderately suppressed by the drug at high concentrations (10(-5)-5 X 10(-1) M). 4 Results provide evidence for a new aspect of the pharmacological action of propranolol on the excitable cell membrane.

Asymmetry of the acetylcholine channel revealed by quaternary anesthetics.

Tissue-cultured rat myoballs were examined electrophysiologically with a suction pipette, which was used for voltage clamping and internal perfusion. The lidocaine derivative QX-314 caused a time- and membrane potentia-dependent block of acetylcholine-induced current only when applied from the extracellular membrane surface. The same compounds caused a use-dependent block of the sodium channel only from the intracellular membrane surface. These experiments demonstrate a fundamental asymmetry of the acetylcholine receptor-channel complex.

Acetylcholine-induced current in perfused rat myoballs.

Spherical "myoballs" were grown under tissue culture conditions from striated muscle of neonatal rat thighs. The myoballs were examined electrophysiologically with a suction pipette which was used to pass current and perfuse internally. A microelectrode was used to record membrane potential. Experiments were performed with approximately symmetrical (intracellular and extracellular) sodium aspartate solutions. The resting potential, acetylcholine (ACh) reversal potential, and sodium channel reversal potential were all approximately 0 mV. ACh-induced currents were examined by use of both voltage jumps and voltage ramps in the presence of iontophoretically applied agonist. The voltage-jump relaxations had a single exponential time-course. The time constant, tau, was exponentially related to membrane potential, increasing e-fold for 81 mV hyperpolarization. The equilibrium current-voltage relationship was also approximately exponential, from -120 to +81 mV, increasing e-fold for 104 mV hyperpolarization. The data are consistent with a first-order gating process in which the channel opening rate constant is slightly voltage dependent. The instantaneous current-voltage relationship was sublinear in the hyperpolarizing direction. Several models are discussed which can account for the nonlinearity. Evidence is presented that the "selectivity filter" for the ACh channel is located near the intracellular membrane surface.

The suction pipette method for internal perfusion and voltage clamp of small excitable cells

A full description of a suction pipette method for voltage clamp and internal perfusion of isolated nerve cell bodies is presented. Particular emphasis is placed upon manufacture and assembly of the pipette and the limitations of the method.

Electrical Recording from the Contractile Ciliate Zoothamnium Geniculatum Ayrton

ABSTRACT The spherical body (200 μm in diameter) of this ciliate protozoan was easily immobilized on a suction pipette and penetrated with glass microelectrodes. No recordings could be made from the contractile stalk, but in the body a negative potential of 30· 3 ± 12· 5 mV was observed. Approximately 9 ms after the onset of a mechanical stimulus, a rapid depolarization was observed. This was apparently simultaneous throughout the body surface. The amplitude was 38· 9 ±13· 7 mV. Recordings with a photomultiplier showed that contraction of the stalk began 10 ms later. A slower depolarization of complex form was observed during systole of the contractile vacuole.

Pipette suction for atraumatic extraction of intraventricular cysticercosis cysts

✓ The authors use a slender Pasteur pipette for the atraumatic extraction of Cysticercus cysts from the deep ventricular cavities. Their technique is described and illustrated.

Development of electrical coupling and action potential synchrony between paired aggregates of embryonic heart cells.

Pairs of spheroidal aggregates of embryonic chick heart cells, held in suction pipettes were brought into contact and allowed to synchronize their spontaneous action potentials. Contractions were suppressed with cytochalasin B. Both intracellular and extracellular electrodes were used to analyze the development of synchrony. Electric coupling occurred in three phases. During phase I electrical interactions were absent despite close physical contact. Phase II was characterized by partial synchrony. Action potentials in the faster aggregate (F) induced small depolarizations in the other member of the pair (S). These depolarizations sometimes triggered action potentials in S depending on when during the diastolic depolarization in S they occurred. In these cases both the latency between the action potentials (L) and the fluctuations in latency (VL) were large. At the end of phase II the aggregates often passed through a brief period when functuation in interbeat interval in both increased noticeably. In phrase III, beginning about 8 min after initial contact, action potentials were completely entrained at a certain L. During the subsequent 20--40 min L fell along an approximately exponential time course from about 130 to less than 1 msec, while VL declined in parallel. When well-coupled aggregates were pulled apart and immediately pressed back together, they re-established synchronization according to the usual three-phase time course. Synchronized aggregates could be partially decoupled by separating them just far enough to reduce the area of mutual contact. Pairs joined only by cellular strands maintained entrained action potentials with long latencies for many minutes. These results indicate that electronic junctions form between the paired heart cell aggregates causing the gradual development of action potential synchrony.

Sodium current in single heart muscle cells

A COMPLETE description of the sodium current, INa, in heart muscle has not been obtained despite the practical importance of such information as a rational basis for the therapy of arrhythmias. INa has been examined under voltage clamp using micropipette1 or sucrose gap methods2, but the adequacy of spatial control of voltage has been questioned and temporal resolution of the early kinetics has been unsatifactory and results therefore inconclusive3. These problems reflect the fact that heart muscle is a syncytium with restricted extracellular compartments. One of the more favourable preparations is rabbit Purkinje fibres4 and Colatsky and Tsien5 have recently performed voltage clamp studies on them with the two micro-pipettes technique. They have demonstrated the feasibility of this approach when proper precautions are taken5. Quite a different approach has been taken by other investigators such as Mehdi and Sachs6 and Powell et al.7–9, who have dispersed individual heart muscle cells and examined them with micropipette techniques. In this way the complex geometry is greatly simplified although the enzymes used in dispersion may pose another set of problems. We have followed this second approach and have isolated individual heart muscle cells from adult rats with a modified version of the dispersion method reported by Powell and Twist9. To perform electrophysiological studies we have sucked single cells into a suction pipette and used a technique which allows voltage clamp and internal perfusion10. INa has been isolated by suppression of other cardiac currents and its kinetics separated from the capacitative current transient. The success of the present approach indicates its potential usefulness in cardiac electrophysiology.

The units of calcium conduction in Helix neurones.

VOLTAGE-DEPENDENT membrane calcium currents, ICa, occur in muscle1,2, axons3,4 and nerve cell bodies5–9 but are not well characterised because of difficulties in separating them from other ionic currents, and inadequate spatial and temporal control of the clamp voltages used to examine them. As a result, information on the conduction process underlying ICa is limited. Recently two new techniques have been reported6,9,10 which overcome most of the problems associated with experimental investigations of ICa. We have used the suction pipette technique9,10, which combines methods of internal perfusion and voltage clamp, to study ICa in isolated nerve cell bodies of the snail, Helix aspersa. The time course of ICa in response to step changes in membrane potential was readily examined and, in addition, very small fluctuations (noise) in current were also observed. Assuming that the fluctuations represent current contributions from a population of independent two-state (conducting/nonconducting) unit conductances, 〈ICa〉, the average value of the fluctuations in the steady state, and σ2, the variance of the fluctuations can be used to estimate a single unit conductance, γca. Analysis of spontaneous current noise has been used previously to obtain γ values for the Na and K systems in several axon membranes11–14, and γ values for acetylcholine-sensitive conductances at the neuromuscular junction15,16 and in molluscan neurones17, but γCa is unknown. Unit ion conductances of a few picosiemens or greater have been reported and where comparisons can be made, these values are similar to those estimated using quite different techniques such as tetrodotoxin binding18–21. We found γCa to be less than 1 pS however, and we suggest that the low value results from the effect of a Ca2+ binding site in the conductance.