A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

Whole-cell and single channel currents were studied in cells from frog (R. pipiens and R. catesbiana) skin epithelium, isolated by collagenase and trypsin treatment, and kept in primary cultures up to three days. Whole-cell currents did not exhibit any significant time-dependent kinetics under any ionic conditions used. With an external K gluconate Ringer solution the currents showed slight inward rectification with a reversal potential near zero and an average conductance of 5 nS at reversal. Ionic substitution of the external medium showed that most of the cell conductance was due to K and that very little, if any, Na conductance was present. This confirmed that most cells originate from inner epithelial layers and contain membranes with basolateral properties. At voltages more positive than 20 mV outward currents were larger with K in the medium than with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion transport mechanism. Whole-cell K current was inhibited by external Ba and quinidine. Blockade by Ba was strongly voltage dependent, while that by quinidine was not. In the presence of high external Cl, a component of outward current that was inhibited by the anion channel blocker diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This component was strongly outwardly rectifying and reversed at a potential expected for a Cl current. At the single channel level the event most frequently observed in the cell-attached configuration was a K channel with the following characteristics: inward-rectifying I-V relation with a conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal potential, one open and at least two closed states, and open probability that increased with depolarization. Quinidine blocked by binding in the open state and decreasing mean open time. Several observations suggest that this channel is responsible for most of the whole-cell current observed in high external K, and for the K conductance of the basolateral membrane of the intact epithelium. On a few occasions a Cl channel was observed that activated upon excision and brief strong depolarization. The I-V relation exhibited strong outward rectification with a single channel conductance of 48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed the presence of two open and at least two closed states. Open time constants and open probability increased markedly with depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.     

Calcium- and voltage-activated potassium channels in human macrophages.

Single calcium-activated potassium channel currents were recorded in intact and excised membrane patches from cultured human macrophages. Channel conductance was 240 pS in symmetrical 145 mM K+ and 130 pS in 5 mM external K+. Lower conductance current fluctuations (40% of the larger channels) with the same reversal potential as the higher conductance channels were noted in some patches. Ion substitution experiments indicated that the channel is permeable to potassium and relatively impermeable to sodium. The frequency of channel opening increased with depolarization and intracellular calcium concentration. At 10(-7) M (Ca++)i, channel activity was evident only at potentials of +40 mV or more depolarized, while at 10(-5) M, channels were open at all voltages tested (-40 to +60 mV). In intact patches, channels were seen at depolarized patch potentials of +50 mV or greater, indicating that the ionized calcium concentration in the macrophage is probably less than 10(-7) M.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

K channel kinetics during the spontaneous heart beat in embryonic chick ventricle cells.

By averaging the current that passes through cell-attached patches on beating heart cells, while measuring action potentials with a whole-cell electrode, we were able to study K channels during beating. In 7-d chick ventricle in 1.3 mM K physiological solutions at room temperature, delayed-rectifier channels have three linear conductance states: 60, 30, and 15 pS. The 60 and 15 pS conductances can exist alone, but all three states may appear in the same patch as interconverting conductance levels. The delayed-rectifier conductance states have low densities (less than 10 channels per 10-microns diam cell), and all have a reversal potential near -75 mV and the same average kinetics. Outward K current through delayed-rectifier channels follows the upstroke without appreciable delay and lasts throughout the action potential. No inward current flows through delayed-rectifier channels during beating. The early outward channel has a nonlinear conductance of 18-9 pS depending on the potential. It also turns on immediately after the upstroke of the action potential and lasts on average only 50 ms. The early outward channel has an extrapolated reversal potential near -30 mV; no inward current flows during beating. The inward-rectifier has an extrapolated conductance and reversal potential of 2-3 pS and -80 mV in 1.3 mM K. Channel kinetics are independent of external K between 10 and 120 mM, and the channel conducts current only during the late repolarization and diastolic phases of the action potential. No outward current flows through inward-rectifier channels during beating. This work parallels a previous study of Na channels using similar techniques (Mazzanti, M., and L. J. DeFelice. 1987, Biophys. J. 52:95-100).     

Calcium currents in squid giant axon.

Voltage-clamp experiments were carried out on intracellularly perfused squid giant axons in a Na-free solution of 100 mM CaCl2+sucrose. The internal solution was 25 mM CsF+sucrose or 100 mM RbF+50mM tetraethylammonium chloride+sucrose. Depolarizing voltage clamp steps produced small inward currents; at large depolarizations the inward current reversed into an outward current. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward current was seen with 100 mM MgCl2+sucrose as internal solution. It is concluded that the inward current is carried by Ca ions moving through the sodium channel. The reversal potential of the tetrodotoxin-sensitive current was +54mV with 25 mM CsF+sucrose inside and +10 mV with 100 mM RbF+50 mM tetraethylammonium chloride+sucrose inside. From the reversal potentials measured with varying external and internal solutions the relative permeabilities of the sodium channel for Ca, Cs and Na were calculated by means of the constant field equations. The results of the voltage-clamp experiments are compared with measurements of the Ca entry in intact axons.     

Effects of external cesium and rubidium on outward potassium currents in squid axons

We have studied the effects of external cesium and rubidium on potassium conductance of voltage clamped squid axons over a broad range of concentrations of these ions relative to the external potassium concentration. Our primary novel finding concerning cesium is that relatively large concentrations of this ion are able to block a small, but statistically significant fraction of outward potassium current for potentials less than approximately 50 mV positive to reversal potential. This effect is relieved at more positive potentials. We have also found that external rubidium blocks outward current with a qualitatively similar voltage dependence. This effect is more readily apparent than the cesium blockade, occurring even for concentrations less than that of external potassium. Rubidium also has a blocking effect on inward current, which is relieved for potentials more than 20-40 mV negative to reversal, thereby allowing both potassium and rubidium ions to cross the membrane. We have described these results with a single-file diffusion model of ion permeation through potassium channels. The model analysis suggests that both rubidium and cesium ions exert their blocking effects at the innermost site of a two-site channel, and that rubidium competes with potassium ions for entry into the channel more effectively than does cesium under comparable conditions.     

Whole-Cell K+ Currents across the Plasma Membrane of Tobacco Protoplasts from Cell-Suspension Cultures

The whole-cell configuration of the patch clamp technique was used to study both outward and inward ion currents across the plasma membrane of tobacco (Nicotiana tabacum) protoplasts from cell-suspension cultures. The ion currents across the plasma membrane were analyzed by the application of stepwise potential changes from a holding potential or voltage ramps. In all protoplasts, a voltage- and time-dependent outward rectifying current was present. The conductance increased upon depolarization of the membrane potential (to >0 mV) with a sigmoidal time course. The reversal potential of the outward current shifted in the direction of the K+ equilibrium potential upon changing the external K+ concentration. The outward current did not show inactivation. In addition to the outward rectifying current, in about 30% of the protoplasts, a time- and voltage-dependent inward rectifying current was present as well. The inward rectifying current activated upon hyperpolarization of the membrane potential (<-100 mV) with an exponential time course. The reversal potential of the inward conductance under different ionic conditions was close to the K+ equilibrium potential.     

Regulation of a voltage-dependent,calcium-activated K conductance by cyclic GMP in dissociated flounder enterocytes

Enterocytes from the winter flounder (Pseudopleuronectes americanus) were isolated by collagenase digestion and maintained in flounder Ringer's solution. Whole cell currents were studied using the amphotericin-perforated whole-cell patch clamp technique. The mean resting membrane potential and capacitance values or dissociated cells were-45±7 mV and 5±0.4 pF, respectively. Enterocytes held at-20 mV and treated with 1 mol·l^-1 ionomycin exhibited outward currents when cells were stepped through a series of voltages from-60 to +110 mV. The reversal potential of this current in flounder Ringer's solution was-55 mV and the voltage at which half-maximal activation occurred was +20 mV. Voltage-dependent inhibition of outward current was observed at +60 mV and above. When cells were bathed in symmetric K Ringer's solution the reversal potential shifted to zero mV and no inhibition of current was observed at voltages between-60 and 140 mV. When the holding potential of the cell was changed from-20 to-80 mV and stepped from-60 to +110 mV, a second [previously characterized, O'Grady et al. (1991)] K current with delayed-rectifier properties was identified. This observation demonstrated that the delayed rectifier K channel and the Ca²⁺-activated K channel described in this study exist in the same cell. Extracellular addition of 2 mmol·l^-1 Ba²⁺ to cells bathed in symmetric K Ringer's solution resulted in nearly complete inhibition of outward current. Charybdotoxin produced only minor effects on this current. Addition of 8-Br cGMP to the bathing solution also inhibited outward current and this effect could be partially reversed following washout of 8-Br cGMP from the bathing solution. The results of this study indicated that a Ca²⁺-activated K conductance in winter flounder enterocytes is potentially inhibited by agents that increase intracellular cGMP. A similar effect of cGMP on a delayed rectifier K channel in flounder enterocytes was previously demonstrated.Abbreviations ANP atrial natriuretic peptide - CTX charybdotoxin - EPPS N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid     

On the block of outward potassium current in rabbit Schwann cells by internal sodium ions.

Currents through delayed rectifier-type K+ channels in Schwann cells cultured from rabbit sciatic nerve were studied with patch-clamp techniques. When the internal and external solutions contained physiological concentrations of sodium, the amplitude of these outward currents declined as the cell was depolarized to potentials above about +40 mV, despite the increased driving force. This reduction in the amplitude of outward K+ currents was observed in many cells before the subtraction of leakage currents; it was also observed for ensemble currents recorded in outside-out patches. It was therefore not the result of a leak-subtraction artefact nor of inadequate voltage-clamp control. Several lines of evidence also suggested that it was not the result of the extracellular accumulation of K+. By contrast, when the Na+ ion concentration of the internal solution was nominally zero, the reduction in the amplitude of outward K+ currents at positive membrane potentials was not observed. The apparent amplitude of single-channel currents through two types of K+ channel was reduced by 30 mM internal Na+, apparently as the result of a rapid 'flickery' block. The results suggest that channel block by internal Na+ is largely responsible for the negative slope conductance seen in current-voltage plots of whole-cell K+ currents at positive membrane potentials. In addition, our analysis of single-channel currents suggests that the current-voltage curve for a delayed rectifier channel in rabbit Schwann cells (in the absence of internal Na+) is roughly linear with internal and external K+ concentrations of 140 mM and 5.6 mM, respectively.     

Ionic currents underlying the action potential of Rana pipiens oocytes

Ionic currents in immature, ovulated Rana pipiens oocytes (metaphase I) were studied using the voltage-clamp technique. At this stage of maturity the oocyte can produce action potentials in response to depolarizing current or as an "off response" to hyperpolarizing current. Reducing external Na+ to 1/10 normal (choline substituted) eliminated the action potentials and both the negative-slope region and zero-crossing of the I-V relation. Reducing external Cl- to 1/10 or 1/100 normal (methanesulfonate substituted) lengthened the action potential. The outward current was reduced and a net inward current was revealed. By changing external Na+, Cl-, and K+ concentrations and using blocking agents (SITS, TEA), three voltage- and time-dependent currents were identified, INa, IK and ICl. The Na+ current activated at about 0 mV and reversed at very positive values which decreased during maturation. Inward Na+ current produced the upstroke of the action potential. During each voltage-clamp step the Na+ current activated slowly (seconds) and did not inactivate within many minutes. The Na+ current was not blocked by TTX at micromolar concentrations. The K+ current was present only in the youngest oocytes. Because IK was superimposed on a large leakage current, it appeared to reverse at the resting potential. When leakage currents were subtracted, the reversal potential for IK was more negative than -110 mV in Ringer's solution. IK was outwardly rectifying and strongly activated above -50 mV. The outward K+ current produced an after hyperpolarization at the end of each action potential. IK was blocked completely and reversibly by 20 mM external TEA. The Cl- current activated at about +10 mV and was outwardly rectifying. ICl was blocked completely and reversibly by 400 microM SITS added to the bathing medium. This current helped repolarize the membrane following an action potential in the youngest oocytes and was the only repolarizing current in more mature oocytes that had lost IK. The total leakage current had an apparently linear I-V relation and was separated into two components: a Na+ current (IN) and a smaller component carried by as yet unidentified ions.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

An odorant-suppressed Cl– conductance in lobster olfactory receptor cells

Odorants evoke an outward current in cultured lobster olfactory receptor neurons voltage clamped at -60 mV. The reversal potential of the outward current is independent of the reversal potential of potassium, but shifts with imposed changes in the reversal potential of chloride. The slope of the current-voltage relationship is negative, suggesting that the current is mediated by the odorant suppressing a steady-state conductance. Anthracene-9-carboxylic acid, a specific chloride channel blocker, reversibly inhibits the steady-state conductance. Local application of odorants to the outer dendrites evokes a hyperpolarizing receptor potential in lobster olfactory receptor neurons current-clamped at -70 mV in situ. Consistent with the current characterized in the cultured cells, hyperpolarizing receptor potentials in some cells are voltage sensitive, blocked by anthracene-9-carboxylic acid and associated with a decrease in membrane conductance. These results support the hypothesis that odorants suppress a steady-state chloride conductance in lobster olfactory receptor neurons. Evidence that the chloride conductance can coexist with a 4-aminopyridine-blockable potassium conductance reported earlier in these cells suggests that two distinct mechanisms can mediate odorant-evoked inhibition in lobster olfactory receptor neurons.     

Characterization of the inward-rectifying potassium current in cat ventricular myocytes

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.     

Ionic currents in the soma of an identified cockroach motoneurone recorded under voltage-clamp

1. Membrane currents have been recorded from the soma of a bifunctional basalar/coxal depressor motoneurone in the metathoracic ganglion of the cockroach (Periplaneta americana) using a two-electrode voltage-clamp technique. 2. This motoneurone cell body is normally inexcitable when studied under current-clamp. Appropriate depolarizing command steps evoke rapid transient outward currents and late outward currents. 3. Late outward currents are dominated by a Ca-dependent component that confers an N-shaped I-V relationship on the neurone. 4. The Ca-dependent outward current is suppressed by Cd2+ (1 mM), Mn2+ (5 mM) or verapamil (50 microM). 5. Externally applied tetraethylammonium ions (TEA+) (25 mM) block the Ca-dependent current, but also appear to suppress a component of the late outward current that is independent of Ca2+. 6. Aminopyridines cause only minor suppression of late outward currents, but shift the peak in the N-shaped I-V relationship to more negative potentials. 7. The reversal potential of tail currents recorded following pre-pulses to +50 mV were dependent upon the pre-pulse duration; increasing the duration from 10 to 50 msec caused a +17 mV shift in tail current reversal potential. 8. A five-fold increase in the K+ concentration of the solution bathing the preparation only produced small and inconsistent changes in the reversal potential of tail currents. 9. Five-fold reduction in external Cl- caused no change. 10. The dependence of tail current reversal potential upon pre-pulse duration and the limited effect of alterations in the composition of the bathing solution are discussed in the context of restricted ion movements near the external surface of the cell membrane.     

Fluid Recirculation in Necturus Intestine and the Effect of Alanine

Previous studies in our laboratory have shown that Na absorption across the porcine endometrium is stimulated by PGF_2α and cAMP-dependent activation of a barium-sensitive K channel located in the basolateral membrane of surface epithelial cells. In this study, we identify and characterize this basolateral, barium-sensitive K conductance. Porcine uterine tissues were mounted in Ussing chambers and bathed with KMeSO₄ Ringer solution. Amphotericin B (70 μm) was added to the luminal solution to permeabilize the apical membrane and determine the current-voltage relationship of the basolateral K conductance after activation by 100 μm CPT-cAMP. An inwardly rectifying current was identified which possessed a reversal potential of −53 mV when standard Ringer solution was used to bathe the serosal surface. The K:Na selectivity ratio was calculated to be 12:1. Administration of 5 mm barium to the serosal solution completely inhibited the current activated by cAMP under these conditions. In addition to these experiments, amphotericin-perforated whole cell patch clamp recordings were obtained from primary cultures of porcine surface endometrial cells. The isolated cells displayed an inwardly rectifying current under basal conditions. This current was significantly stimulated by CPT-cAMP and blocked by barium. These results together with our previous studies demonstrate that cAMP increases Na absorption in porcine endometrial epithelial cells by activating an inwardly rectifying K channel present in the basolateral membrane. Similar patch clamp experiments were conducted using cells from a human endometrial epithelial cell line, RL95-2. An inwardly rectifying current was also identified in these cells which possessed a reversal potential of −56 mV when the cells were bathed in standard Ringer solution. This current was blocked by barium as well as cesium. However, the current from the human cells did not appear to be activated by cAMP, indicating that distinct subtypes of inwardly rectifying K channels are present in endometrial epithelial cells from different species. Received: 6 February 1997/Revised: 10 July 1997     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

T-type Ca2+ channel lowers the threshold of spike generation in the newt olfactory receptor cell

Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of -70 +/- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na+ concentration. After a complete removal of Na+, however, cells still showed action potentials which was abolished either by Ca2+ removal or by an application of Ca2+ channel blocker (Co2+ or Ni2+), indicating an involvement of Ca2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to -40 mV from the holding voltage of -100 mV induced a fast transient inward current, which consisted of Na+ (INa) and T-type Ca2+ (ICa.T) currents. The amplitude of ICa,T was about one fourth of that of INa. Depolarization to more positive voltages also induced L-type Ca2+ current (ICa,L). ICa,L was as small as a few pA in normal Ringer solution. The activating voltage of ICa,T was approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Ni2+ (a specific T-type Ca2+ channel blocker) even in the presence of Na+. These results suggest that ICa,T contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation.     

Single potassium channels of human glioma cells.

Single potassium channels in the membrane of human malignant glioma cells U-118MG were studied using the technique of patch clamp in cell-attached and inside-out configurations. Three types of potassium channels were found which differed from each other under conditions close to physiological in their conductance and gating characteristics. The lowest-conductance channel (20 pS near the reversal potential) showed a mild outward rectification up to 45 pS at positive voltages and spontaneous modes of high and low activity. At extreme values of potentials its activity was generally low. The intermediate conductance channel had an S-shaped I-V curve, giving a conductance of 63 pS at reversal, and a low and voltage independent opening probability. The high-conductance (215 pS) channel was found to be activated by both membrane potential and Ca2+ ions and blocked by internal sodium at high voltages. The current-voltage curves of all three channel types displayed saturation.