Suction Pipette Research Articles

Synaptic transmission in the guinea-pig vas deferens: The role of nerve action potentials

To determine how transmitter release is related to presynaptic nerve activities, pre- and postsynaptic electrical events of the vas deferens in the guinea-pig were recorded with a suction electrode. Stimulation of the hypogastric nerve elicited excitatory junction currents and nerve action potentials. Intermittence of excitatory junction currents was observed. In some instances, this was related to the absence of nerve action potentials, suggesting failure of impulse propagation into the nerve terminals. Facilitation of both the nerve action potentials and the excitatory junction currents was also observed. Internal perfusion of the recording electrode with tetrodotoxin blocked the nerve impulse, and the polarity of the excitatory junction current became positive. Similar effects on the polarity of the excitatory junction current were observed with α,β -methylene ATP. Perfusion of the suction pipette with 4-aminopyridine or tetraethylammonium increased the amplitude of the excitatory junction currents and prolonged the nerve action potential duration. These experiments show that: (1) transmission failure in some cases can be related to conduction block into the terminal region; (2) facilitation of excitatory junction currents may be related to facilitation of the nerve action potentials; (3) enhancement of transmitter release by potassium channel blockers may be related to prolongation of the duration of the nerve action potential. It is concluded that transmitter release is intimately related to presynaptic nerve activities.

Two types of sodium channel block by class-I antiarrhythmic drugs studied by using [formula omitted] of action potential in single ventricular myocytes

The state-dependent sodium channel block by mexiletine, tocainide, lidocaine, OPC-88117, aprindine, quinidine, disopyramide and AN-132 was investigated in single ventricular myocytes isolated from guinea-pig hearts. A single conditioning clamp pulse with a duration from 10 ms to 1000 ms was applied from the resting potential (−82 mV) to 0 mV level using a suction pipette whole-cell voltage clamp technique, and the maximum upstroke velocity ( V ̇ max ) of a test action potential elicited 100 ms after termination of the clamp pulse was measured as an index of sodium channel availability. In myocytes treated with the eight drugs, such clamp pulse caused a significant decrease in V ̇ max . With mexiletine, tocainide, lidocaine, OPC-88117 and aprindine, the V ̇ max reduction was enhanced progressively as the clamp pulse duration was prolonged. With quinidine, disopyramide and AN-132, an appreciable V ̇ max reduction at the shortest clamp pulse was followed by an additional small enhancement of the V ̇ max decay. These findings suggest that the former group of drugs may block the sodium channel mainly during the inactivated state (inactivation blockers), whereas the latter one may do so mainly during the activated state (activation blockers). Multiple short clamp pulses caused a greater V ̇ max reduction than a single prolonged clamp pulse for the activation blockers, and vice-versa for the inactivation blockers. Molecular dimensions of the eight drugs, which were estimated by X-ray diffraction of crystals, did not satisfy a simple size criterion as proposed by Courtney (1988) to explain such different types of sodium channel block by Class-I drugs.

Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium.

1. Light adaptation has been studied in isolated red-sensitive cone photoreceptors of the salamander, using suction pipette recordings of circulating current. 2. In the presence of background illumination, the response to incremental dim flashes became desensitized according to the Weber-Fechner law. The recovery phase of the flash response was accelerated significantly, although the time-to-peak was reduced only slightly, and for dim backgrounds the rising phase was unaltered. 3. The role of cytoplasmic calcium concentration, Cai2+, in mediating cone adaptation was investigated by minimizing light-induced changes in Cai2+, either by incorporating calcium buffer into the cytoplasm or by exposing the outer segment to low-Ca2+, 0-Na+ solution. Both treatments appeared to slow dramatically or even to eliminate the onset of light adaptation in the cone. 4. When the low-Ca2+, 0-Na+ solution was presented in darkness, responses to subsequent illumination were affected in a characteristic manner: (i) the response-intensity relation was steepened and shifted to lower intensities, (ii) the response to a step of light could be predicted by integration and compression of the flash response, and (iii) the flash sensitivity declined steeply as a function of background intensity. 5. After extended exposure of the cone to bright backgrounds, the sensitivity in darkness failed to return to its original level. The flash response kinetics were faster and more biphasic than for dark-adapted responses or for responses desensitized to a comparable degree by exposure to steady background illumination. 6. The results indicate that, in cones isolated from the pigment epithelium, the primary factor influencing the adaptational state of the cell is the cytoplasmic concentration of free calcium, but that at high intensities the effects of pigment bleaching are likely to be significant.

Influences of external Ca 2+ on the GABA-induced chloride current and the efficacy of diazepam in internally perfused frog sensory neurons

The effects of extracellular Ca 2+ on the γ-aminobutyric acid (GABA)-induced Cl - current and the efficacy of diazepam in the facilitation of GABA response were studied in frog isolated sensory neurons, using a ‘concentration clamp’ technique which combines a suction pipette (internal perfusion and voltage clamp) and a rapid drug application system. When nominal Ca 2+-free external solution was changed to the solution containing 2 mM Ca 2+, the response elicited by 1 × 10 −5 M GABA was reduced by about 40% of the control obtained in nominal Ca 2+-free solution. The dose-response curve for GABA was shifted to the right without affecting the maximum response. It can be suggested that the application of external Ca 2+ modulates the affinity of the GABA receptor to its agonist, GABA. Diazepam at the concentration of 3 × 10 −6 M shifted the dose-response curve for GABA to the left without changing the maximum reponse with or without external Ca 2+. However, the augmentatory action of diazepam on the GABA response was reduced in the presence of external Ca 2+. Possible mechanisms for inhibitory action of external Ca 2+ on the GABA-gated response and the reduced efficacy of diazepam are discussed.

Outward potassium currents in freshly isolated smooth muscle cell of dog coronary arteries.

Outward membrane currents were characterized in single coronary smooth muscle cells of adult beagle dogs. The cells averaged 96.4 x 7.1 microns and had a resting potential of -50.7 mV, an input resistance of 307.9 M omega, a capacitance of 32.3 pF, and a calculated membrane surface area of 4,037 microns2. The cells contracted in response to external application of acetylcholine or high K+. In voltage clamp by use of the suction pipette method, outward current began to appear at -50 mV and reached 15.2 nA at 50 mV with a current density of 376.5 microA/cm2. The current was reduced by external tetraethylammonium, Ba2+, and internal Cs+, and its reversal potential had a Nernst relation to external K+ concentration. Elevation of external Ca2+ (Ca2+o) from 0 to 0.3 mM increased total K+ current by up to 300%; elevation of internal Ca2+ (Ca2+i) to 5 x 10(-7) M by internal perfusion increased total outward current to a similar extent, suggesting a large difference in Ca2+ transmembrane sensitivity. Total whole-cell K+ current consisted of two components: an initial time-independent current (Ii) followed by a time-dependent current (It). Ii and It were through separate K+ channels based on differences in a) sensitivity to Ca2+09b) modulation by an inward Ca2+ current, c) current amplitudes and activation kinetics, and d) responses to pharmacological agents. It was the largest component, measuring 4.5 nA in 0 mM Ca2+o but increasing to 11.9 nA in 0.3 mM Ca2+o with a steep 2.5 power function. It activated with a biexponential time course; in Ca2+o-free solution, its time course was relatively insensitive to voltage changes but became voltage sensitive in the presence of Ca2+o. Further, such sensitivity was abolished or enhanced by Co2+ or Bay K 8644, respectively. We concluded that there are two types of Ca2+-sensitive K+ currents, Ii and It, in coronary smooth muscle cells. Via an inward Ca2+ channel Ca2+o strongly modulates It, both in amplitude and kinetics.

New observations on the mechanisms of antiarrhythmic actions of disopyramide on cardiac membranes

Electrophysiologic effects of disopyramide on cardiac membranes were studied using the microelectrode technique applied to papillary muscles and the suction pipette whole-cel damp method applied to isolated ventricular myocytes from guinea-pig hearts. In contrast to previous reports, the development and recovery from the V max Mocks of action potentials by disopyramide and lidocaine were best expressed by 2 exponential functions, not 1, suggesting that there might be 2 different processes for the sodium current block. A near-therapeutic concentration (11 μM) of disopyramide depressed V max at frequencies of 0.1 to 2.0 Hz decreased action potential amplitude and shortened plateau phase in ventricular myocytes. Action potential duration was prolonged by the drug in most of the preparations but shortened in some. Disopyramide prolonged the refractory period and increased threshold current for excitation. The drug was also shown to depress both the calcium current and the delayed outward potassium current. These multiple actions of disopyramide may explain its variety of antiarrhythmic properties.

Ionic diffusion in voltage-clamped isolated cardiac myocytes. Implications for Na,K-pump studies

The whole-cell voltage-clamp technique employing electrolyte-filled micro-pipette suction electrodes is widely used to investigate questions requiring an electrophysiological approach. With this technique, the ionic composition of the cytosol is assumed to be strongly influenced (as result of diffusion) by the ionic composition of the solution contained in the electrode. If this assumption is valid for isolated cardiac myocytes, the technique would be particularly powerful for studying the dependence of their Na,K-pump on the intracellular [Na+]. However, the relationship between the concentrations of ions in the solution filling the electrode and those in the cytosol has not been established. The relationship was investigated to determine in particular whether the [Na+] at the intracellular cation ligand binding sites for the Na-pump ([ Na+]ps) can be set and clamped by [Na+] in the pipette electrode ([ Na+]pip). If [Na+]pip can set and clamp [Na+]ps, this would provide a means for defining the dependence of the Na,K-pump on intracellular [Na+]. The relationship between [Na+]pip and [Na+]ps was analyzed using two approaches. First, a mathematical model of three-dimensional ionic diffusion within a whole-cell patch-clamped myocyte was developed and the effects of experimental parameters on mean [Na+]ps were investigated. When typical experimental values were simulated, the time course to achieve steady state mean [Na+]ps was found to be most sensitive to variations in electrode pore size, cell length and the Na+ pumping rate, but at steady state, mean [Na+]ps varies from [Na+]pip by 5% or less depending on pump rate. Second, to provide experimental support for the validity of the simulations, isolated ventricular myocytes were voltage-clamped and the reversal potential for the Na current was determined in order to estimate steady state intracellular [Na+]. The results of the mathematical and experimental analyses suggest that steady state [Na+]ps can be regulated by the [Na+] in suction pipette electrodes. These findings, while also having a broader significance, indicate for isolated cardiac myocytes that whole-cell suction micro-electrodes can provide a means to assess the dependence of the Na,K-pump on [Na+]ps.

Is there a second external lidocaine binding site on mammalian cardiac cells?

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

Comparative study on barbiturates using isolated single neurons: GABA-mimetic action and augmentatory action on GABA response

The comparison among barbiturate derivatives in both GABA-mimetic action and augmentatory effect on GABA response were made. Experiments were performed on single neurones isolated from frog dorsal root ganglia, using a concentration clamp technique which combines a suction pipette technique (internal perfusion and voltage-clamp) with a rapid external solution exchange within 2 ms. Barbiturates employed here were secobarbital, pentobarbital, hexobarbital and phenobarbital. All barbiturates at the concentrations over1 × 10 −4 M evoked the chloride currents ( I CI) dose-dependently. In the GABA-mimetic action of barbiturates the potency was in the order of secobarbital > pentobarbital > hexobarbital > phenobarbital. All barbiturates at the concentrations over1 × 10 −6 M (but up to1 × 10 −3 M) augmented the GABA-induced I CI. Potency order of barbiturates in augmenting GABA response was the same as that in their GABA-mimetic actions when the concentration was1 × 10 −4 M or less. However at the concentration of1 × 10 −3 M the efficacies of secobarbital and pentobarbital in augmenting GABA response were greatly reduced while the other two barbiturates produced further augmentation.

Two components of calcium channel current in embryonic chick skeletal muscle cells developing in culture

The properties of the Ca channel currents in chick skeletal muscle cells (myoballs) in culture were studied using a suction pipette technique which allows internal perfusion and voltage clamp. The Ca channel currents as carried by Ba ions were recorded, after suppression of currents through ordinary Na, K and Cl channels by absence of Na, K and Cl ions, by external TEA, by internal EGTA and by observing the Ba currents instead of the Ca currents. Two components of Ba current could be distinguished. One was present only if the myoballs were held at relatively negative holding potentials below −50 mV. This component first became detectable at clamp potentials of about −50 mV and reached a maximum between −10 and −20 mV. During long clamp steps, it became inactivated completely. The inactivation process of this component at a clamp potential of−30 mV was well fitted to a single exponential with a time constant of about −20 ms. Half-maximal steady-state inactivation was observed at −63 mV. The other component persisted even at relatively positive holding potentials above −40 mV, was observed during clamp pulses to −20 mV and above, and reached a maximum between +10 and +20 mV. This component inactivated very little; a substantial fraction of this component remained at the end of clamp pulses lasting 1 s. The inactivation process of this component at a clamp potential of −10 mV apparently followed a single exponential with a time constant of about 1 s. Half-maximal steady-state inactivation was attained at −33 mV. Both components of Ba current were blocked by Co ions, but organic Ca channel blocker D600 preferentially blocked the high-threshold, slowly inactivating component. The relationship between the current amplitude and the concentration of the external Ba ions was different between the two components. Furthermore, the two components of Ba current also differed in their developmental profile. These findings demonstrate the existence of two distinct types of Ca channels in the early stages of chick muscle cell development.

Measurement of the effective thickness of the mucosal unstirred layer in Necturus gallbladder epithelium.

The effective thickness of the unstirred fluid layer (USL) adjacent to an epithelial barrier can be estimated from the time course for the accumulation or depletion of a solute at the membrane surface. In 1985 we reported an unstirred layer thickness of approximately 70 microns for Necturus gallbladder epithelium. In our earlier studies the delay caused by noninstantaneous bulk solution mixing was not taken into account and thus the USL thickness was systematically overestimated. In the present studies we describe an analysis of the time course of solute arrival at the membrane surface that takes into account noninstantaneous bulk solution mixing. We also describe a simple technique to monitor the accumulation or depletion of a solute at the membrane surface. The time course for the change in the concentration of either tetramethylammonium (TMA+) or tetrabutylammonium (TBA+) upon elevation of bulk solution concentration is sensed at the membrane surface with an ion-sensitive microelectrode. Because of the high selectivity of the ion-sensitive resin for TMA+ or TBA+ over other monovalent cations in the solution (Na+ and K+), a low concentration (1-2 mM) of the probe can be used. By measuring the time course of the arrival of first one probe and then the other, under identical superfusion conditions, sufficient information is obtained to eliminate multiple fits to the data, obtained when only one probe is used. Neglecting bulk solution mixing caused an error greater than 50% in estimated apparent USL thickness. The effective thickness of the USL depends critically upon chamber geometry, flow rate, and the position of superfusion and suction pipettes. Under our experimental conditions the effective USL at the mucosal surface of Necturus gallbladder epithelium was approximately 40 microns.

Kinetics of the membrane current mediated by serotonin 5‐HT3 receptors in cultured mouse neuroblastoma cells.

1. Ionic currents mediated by serotonin 5-HT3 receptors were studied in the mouse neuroblastoma cell line N1E-115, using suction pipettes for intracellular perfusion and voltage clamp recording. The dependence of the kinetics of the membrane current on serotonin concentration was investigated. 2. At a holding potential of -70 mV application of 5-HT (5-hydroxytryptamine creatinine sulphate) causes a transient inward current. The i-V curve of the peak amplitude is linear between -80 and 60 mV. The reversal potential is 20 +/- 4 mV (mean +/- S.D.). The kinetics of the transient ionic current are independent of the holding potential. 3. In the presence of 5-HT the membrane current decays to a small steady-state level with a single-exponential time course. The time constant of decay decreases with increasing concentration of the agonist, to a minimum value of 6.5 +/- 1.5 s for concentrations of 5-HT greater than or equal to 3 microM. 4. When the agonist is rapidly removed, single-exponential decay of the ionic current is observed. The time constant of this decay in the absence of 5-HT amounts to 6.9 +/- 1.5 s and is independent of the membrane potential and of the concentration of 5-HT used. 5. In the presence of low concentrations of 5-HT the peak amplitude of the inward current evoked with a high concentration of agonist is gradually reduced. The onset of this desensitization follows the same time course as the decay of the membrane current. In the range from 0.7 to 1.5 microM-5-HT both kinetic processes show the same steep concentration dependence. 6. Recovery from desensitization, measured at variable intervals after removal of the agonist, can be fitted by a single-exponential function with a time constant of 18 +/- 4 s. 7. The results show that the kinetic properties of the 5-HT3 receptor-mediated ionic current can only be described by a complex, co-operative model.

A stretch-activated K+ channel sensitive to cell volume.

The role of K+ channels in cell osmoregulation was investigated by using the patch-clamp technique. In cell-attached patches from Necturus proximal tubule, the short-open-time K+ channel at the basolateral membrane could be stretch-activated by pipette suction, where a negative pressure of 6 cm H2O (588.6 Pa) was sufficient to increase the open probability of the channel by a factor of 4.0 +/- 0.8 (n = 7 tubules). A 50% reduction in bath osmolarity increased cell volume by 66 +/- 10% and increased the K+-channel open probability by a factor of 5.8 +/- 1.4 (n = 7) in the same cell-attached patches that were activated by pipette suction. A kinetic analysis indicates one open state and at least two closed states for this epithelial K+ channel. Both suction and swelling shorten the longest time constant of the closed-time distribution by a factor of 3, without significant effect on either the mean open time or the shorter closed-state time constant. The similar effect of suction and swelling is consistent with the hypothesis that stretch-activated K+ channels mediate the increase in macroscopic K+ conductance that occurs during osmoregulation of amphibian proximal tubules. Calculations based on a simple model indicate that small increments in cell volume could produce statistically significant increases in K+-channel activity.

Calcium‐sensitive and insensitive transient outward current in rabbit ventricular myocytes.

1. A suction pipette whole-cell voltage-clamp technique was used to record membrane currents and potentials of isolated ventricular myocytes from rabbit hearts. 2. Transient outward current (Ito) was activated by voltage steps positive to -20 mV, increasing in amplitude with further depolarization to reach a maximum around +70 mV. The current attained its peak within 10 ms and then it inactivated for 100-200 ms. 3. A large portion of Ito still remained after the calcium current (ICa) was blocked when depolarizing pulses were applied at a frequency of 0.1 Hz or less. Therefore, this current component is referred to as calcium-insensitive Ito or It. 4. It showed voltage- and time-dependent inactivation similar to that observed in Purkinje fibres and other cardiac preparations. 5. The reversal potential of It depended on external K+ concentration, [K+]o, with a slope of 32 mV per 10-fold change in the presence of a normal [Na+]o (143 mM), while the slope was 48 mV per 10-fold change in low [Na+]o (1.0 mM). 6. It was completely inhibited by 2-4 mM-4-aminopyridine. Ito in the presence of ICa was also partially blocked by 4-aminopyridine and the remainder was abolished by 5 mM-caffeine. 7. The calcium-insensitive and caffeine-sensitive Ito differed in their decay rates as well as in their recovery time courses. The former was predominantly available at a slow pulsing rate, while the latter increased its amplitude with high-frequency depolarization. 8. The caffeine-sensitive Ito was inhibited by a blockade of ICa, by replacing Ca2+ with Sr2+, by external application of ryanodine and by internal application of EGTA. This indicates that the current is calcium-sensitive and is dependent on increased myoplasmic Ca2+ through Ca2+ influx via the sarcolemma and Ca2+ release from the sarcoplasmic reticulum. The current is therefore designated as IK, Ca. 9. The physiological functions of IK, Ca and It are indicated by their contribution to ventricular repolarization at fast and slow heart rates, respectively.

Effects of sulfhydryl binding reagents on the photoresponses of amphibian retinal rods

1. 1. We have studied the effects of four sulfhydryl binding reagents (NEM, PHMB, PCMP and IAA) and the disulfide reducing agent, DTT, on the photocurrent of vertebrate rods by recording the ERG across the aspartate-treated retina of the frog, and by suction pipette recording from isolated rods of the tiger salamander. 2. 2. SH-reagents brought about three types of effects on rods: (1) a fast transitory increase in light sensitivity and photocurrent; (2) a “leakage” current that could not be turned off by light; and (3) a slower irreversible loss of sensitivity. 3. 3. The fast effects, including the leakage current, are attributable in part to direct action on the sodium channels in the plasma membrane. 4. 4. NEM, PHMB and PCMP were able to affect the transduction machinery inside the rod, which first contributed to the growth of photoresponses, but gradually depressed light sensitivity irreversibly. 5. 5. Typically, the reagents also induced large ERG transients of non-receptor (glial) origin. 6. 6. The fast effects of DTT on isolated rods were similar to those of the SH-reagents. This drug) however, had no clear effect on ERG photoresponses, suggesting that the oxidation of sodium-channel SH-groups is modest in the intact retina as compared with isolated rods.

Incorporation of analogues of GTP and GDP into rod photoreceptors isolated from the tiger salamander.

1. Analogues of GTP and GDP were introduced into isolated rod photoreceptors using the whole-cell patch clamp technique, while simultaneously recording the photocurrent with a suction pipette. After several minutes of whole-cell recording the patch pipette was disengaged, thus trapping the analogue inside the cell. 2. During the introduction of the hydrolysis-resistant GTP analogues guanosine-5'-O-(3-thio-triphosphate) (GTP-gamma-S) and guanylyl-imidodiphosphate (GMP-PNP) the dark current progressively declined, and the duration of responses to flashes of light which had previously been just-saturating increased slightly. The form of the rising phases of the responses to dim or bright flashes was little affected. 3. Following the incorporation of these GTP analogues the response to an intense flash was prolonged by a factor of up to 300, and the circulating current remained suppressed for up to 1 h. Ultimately the circulating current recovered and the duration of the flash response returned to near its control value. 4. Superfusion of the outer segment with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) during the extended period of saturation resulted in a rapid increase in the circulating current, suggesting that the analogues had their major effect on the duration of phosphodiesterase activation by light. 5. Introduction of the phosphorylation-resistant GDP analogue guanosine-5'-O-(2-thio-diphosphate) (GDP-beta-S) resulted in a decrease in light sensitivity and a reduction in the slope of the rising phase of the flash response. 6. The response to an intense flash was also prolonged in cells containing GDP-beta-S, recovery becoming progressively slower on successive presentations of the flash following the withdrawal of the patch pipette. This observation suggests that GDP-beta-S may be slowly converted within the cell to form a hydrolysis-resistant product. 7. These results indicate that the presence of a hydrolysis-resistant analogue of GTP within the cell causes light activation of the transduction mechanism for an extended period. Our interpretation of this finding is that hydrolysis of the bound guanosine nucleotide is necessary for the quenching of activated GTP-binding protein.

External and internal actions in the response of salamander retinal rods to altered external calcium concentration.

1. The membrane current was recorded from retinal rods isolated from Ambystoma tigrinum using the suction pipette and whole-cell patch pipette techniques, while the concentration of calcium bathing the outer segment was rapidly reduced. 2. The increase in outer segment current induced by lowered external calcium in darkness could be resolved into two components, one as rapid as the time course of the solution change (as judged by the junction current) and the other somewhat slower. 3. Introduction of the calcium buffer BAPTA (1,2-bis(o-aminophenoxy)ethane-N ,N ,N' ,N'-tetraacetic acid) into the cell from a patch pipette led to a progressive slowing of the second component of current increase. 4. When several minutes had elapsed following rupture of the patch, to allow a substantial amount of BAPTA into the cell (ca. 10 mM in the patch pipette), the second component was slowed by a factor of about 20-fold, while the first component continued to have the same rapid time course as the solution change. 5. The rapid component is attributed to a direct effect of external calcium, Ca2+o, and the delayed component to an indirect effect mediated by a reduction in internal calcium, Ca2+i. 6. These results confirm that, in previous experiments in which BAPTA was introduced into photoreceptors, the internal calcium concentration was very significantly buffered. 7. When Ca2+o drops from 1 mM to less than 10(-8) M, the rapid external component corresponds to an increase in circulating current of 3- to 4-fold, and the internal component corresponds to an increase of at least 4- to 5-fold. However, the total current at late times is limited by electrical factors, so that the size of the internal effect is bound to be considerably greater.

Calcium current restitution in mammalian ventricular myocytes is modulated by intracellular calcium.

Restitution of the conventional L-type calcium current (ICa) was studied in dog or guinea pig ventricular myocytes to understand its time course and regulation. Whole-cell ICa free of other overlapping currents was recorded with a suction pipette. The intracellular environment was varied by intracellular dialysis. The properties of ICa were similar in dog and guinea pig ventricular myocytes, except that the amplitude of ICa was larger in the latter (2.2 +/- 0.5 nA in guinea pig cells and 0.9 +/- 0.2 nA in dog cells, n = 8 for both). In both types of cells during restitution a holding voltage (Vh) negative to -50 mV induced a transient increase in ICa above the control level (ICa overshoot). This overshoot was inhibited by substituting barium for calcium, lowering [Ca]0, increasing intracellular calcium buffering capacity, ryanodine (1-2 microM), or caffeine (10 mM). The overshoot peaked 30-100 msec after repolarization from the conditioning depolarization and gradually declined over the following 2-3 seconds. During the overshoot, although the amplitude of ICa was larger its half-time of decay was longer than the control. The maximum overshoot occurred following a conditioning step to plateau voltages and it was decreased by prolonging the conditioning step from 50 to 100 or 500 msec. It is concluded that intracellular calcium regulates restitution of the L-type calcium channels in mammalian ventricular myocytes and that the sarcoplasmic reticulum is involved in this process.

Permeability to various cations of the voltage-dependent sodium channel of isolated rat hippocampal pyramidal neurons

The permeability to various cations of the voltage-dependent sodium channel of isolated rat hippocampal pyramidal neurons was investigated under the voltage-clamp condition. The neurons were dispersed enzymatically and mechanically and perfused internally using a suction pipette technique. The permeability sequence, estimated from the reveral potential of the inward current was Li + > Na + > hydrazine + > formamidine + > guanidine + > methylguanidine + > monomethylamine +. Thus the ionic selectivity of the voltage-dependent sodium channel of rat central nervous system neurons is similar to that in the squid axon, myelinated frog nerve fiber and rat ventricle muscle.

Electrical properties of the gap junctional membrane studied in rat liver cell pairs

Cell pairs isolated from adult rat liver were used to study the electrical properties of gap junctions. Each cell of a cell pair was connected to a suction pipette so as to enable whole cell tight-seal recording. A double voltage-clamp approach was adopted to control the voltage gradient across the gap junction and measure the transjunctional current. The current-voltage relationship of the gap junctional membrane was linear over the voltage range tested (+/- 50 m V). Under control conditions, the resistance of the gap junction, rj, was 15 M omega (n = 27; range, 4.6 to 45.8 M omega), corresponding to a conductance gj of 67 nS. rj was insensitive to the nonjunctional membrane potential, Vm (voltage range,-90 m V to + 40 m V). There was no indication of a time-dependent gating of rj (time range, 20 ms to 10 s). Dialysis with 1 mM CaCl2 produced irreversible electrical uncoupling without affecting the linearity of the relationship Vj/Ij.