Effects of flufenamic acid on smooth muscle of the carotid artery isolated from spontaneously hypertensive rats.

Endothelium-removed carotid artery strips from stroke-prone spontaneously hypertensive rats spontaneously developed a tonic myogenic contraction. Flufenamic acid reduced the resting tone observed during superfusion with Tyrode's solution, in a concentration-dependent manner. Flufenamic acid also inhibited contractions produced by high-K solutions in a concentration-dependent manner. The resting membrane potential of smooth muscle cells in the artery was around -32 mV, with occasional oscillatory potentials. Flufenamic acid hyperpolarized the membrane in a concentration-dependent manner. The voltage-dependent outward currents recorded in isolated cells with micropipettes filled with high-K+ solution (holding potential, -60 mV) were enhanced by flufenamic acid and inhibited by tetraethylammonium. When the recording micropipette was filled with high Cs to inhibit the K+-current, depolarizing step pulses evoked nifedipine-sensitive inward currents. Flufenamic acid inhibited the inward currents. These results indicate that flufenamic acid inhibits the spontaneous active tone of the carotid artery by inhibiting L-type Ca2+-channels and possibly by membrane hyperpolarization through activation of the voltage-dependent K+-channels.     

Potassium currents in octopus cells of the mammalian cochlear nucleus.

Octopus cells in the posteroventral cochlear nucleus (PVCN) of mammals are biophysically specialized to detect coincident firing in the population of auditory nerve fibers that provide their synaptic input and to convey its occurrence with temporal precision. The precision in the timing of action potentials depends on the low input resistance (approximately 6 MOmega) of octopus cells at the resting potential that makes voltage changes rapid (tau approximately 200 micros). It is the activation of voltage-dependent conductances that endows octopus cells with low input resistances and prevents repetitive firing in response to depolarization. These conductances have been examined under whole cell voltage clamp. The present study reveals the properties of two conductances that mediate currents whose reversal at or near the equilibrium potential for K(+) over a wide range of extracellular K(+) concentrations identifies them as K(+) currents. One rapidly inactivating conductance, g(KL), had a threshold of activation at -70 mV, rose steeply as a function of depolarization with half-maximal activation at -45 +/- 6 mV (mean +/- SD), and was fully activated at 0 mV. The low-threshold K(+) current (I(KL)) was largely blocked by alpha-dendrotoxin (alpha-DTX) and partially blocked by DTX-K and tityustoxin, indicating that this current was mediated through potassium channels of the Kv1 (also known as shaker or KCNA) family. The maximum low-threshold K(+) conductance (g(KL)) was large, 514 +/- 135 nS. Blocking I(KL) with alpha-DTX revealed a second K(+) current with a higher threshold (I(KH)) that was largely blocked by 20 mM tetraethylammonium (TEA). The more slowly inactivating conductance, g(KH), had a threshold for activation at -40 mV, reached half-maximal activation at -16 +/- 5 mV, and was fully activated at +30 mV. The maximum high-threshold conductance, g(KH), was on average 116 +/- 27 nS. The present experiments show that it is not the biophysical and pharmacological properties but the magnitude of the K(+) conductances that make octopus cells unusual. At the resting potential, -62 mV, g(KL) contributes approximately 42 nS to the resting conductance and mediates a resting K(+) current of 1 nA. The resting outward K(+) current is balanced by an inward current through the hyperpolarization-activated conductance, g(h), that has been described previously.     

Voltage-dependent currents in isolated vestibular afferent calyx terminals

Na(+) currents were studied by whole cell patch clamp of chalice-shaped afferent terminals attached to type I hair cells isolated from the gerbil semicircular canal and utricle. Outward K(+) currents were blocked with intracellular Cs(+) or with extracellularly applied 20 microM linopirdine and 2.5 mM 4-aminopyridine (4-AP). With K(+) currents blocked, inward currents activated and inactivated rapidly, had a maximum mean peak amplitude of 0.92 +/- 0.60 (SD) nA (n = 24), and activated positive to -60 mV from holding potentials of -70 mV and more negative. The transient inward currents were blocked almost completely by 100 nM TTX, confirming their identity as Na(+) currents. Half-inactivation of Na(+) currents occurred at -82.6 +/- 0.9 mV, with a slope factor of 9.2 +/- 0.8 (n = 7) at room temperature. In current clamp, large overshooting action potential-like events were observed only after prior hyperpolarizing current injections. However, spontaneous currents consistent with quantal release from the hair cell were observed at holding potentials close to the zero-current potential. This is the first report of ionic conductances in calyx terminals postsynaptic to type I hair cells in the mammalian vestibular system.     

A voltage-dependent transient K(+) current in rat dental pulp cells

We characterized a voltage-dependent transient K(+) current in dental pulp fibroblasts on dental pulp slice preparations by using a nystatin perforated-patch recording configuration. The mean resting membrane potential of dental pulp fibroblasts was -53 mV. Depolarizing voltage steps to +60 mV from a holding potential of -80 mV evoked transient outward currents that are activated rapidly and subsequently inactivated during pulses. The activation threshold of the transient outward current was -40 mV. The reversal potential of the current closely followed the K(+) equilibrium potential, indicating that the current was selective for K(+). The steady-state inactivation of the peak outward K(+) currents described by a Boltzmann function with half-inactivation occurred at -47 mV. The K(+) current exhibited rapid activation, and the time to peak amplitude of the current was dependent on the membrane potentials. The inactivation process of the current was well fitted with a single exponential function, and the current exhibited slow inactivating kinetics (the time constants of decay ranged from 353 ms at -20 mV to 217 ms at +60 mV). The K(+) current was sensitive to intracellular Cs(+) and to extracellular 4-aminopyridine in a concentration-dependent manner, but it was not sensitive to tetraethylammonium, mast cell degranulating peptide, and dendrotoxin-I. The blood depressing substance-I failed to block the K(+) current. These results indicated that dental pulp fibroblasts expressed a slow-inactivating transient K(+) current.     

Two types of voltage-gated K(+) currents in dissociated heart ventricular muscle cells of the snail Lymnaea stagnalis

We have used a combination of current-clamp and voltage-clamp techniques to characterize the electrophysiological properties of enzymatically dissociated Lymnaea heart ventricle cells. Dissociated ventricular muscle cells had average resting membrane potentials of -55 +/- 5 mV. When hyperpolarized to potentials between -70 and -63 mV, ventricle cells were capable of firing repetitive action potentials (8.5 +/- 1.2 spikes/min) that failed to overshoot 0 mV. The action potentials were either simple spikes or more complex spike/plateau events. The latter were always accompanied by strong contractions of the muscle cell. The waveform of the action potentials were shown to be dependent on the presence of extracellular Ca(2+) and K(+) ions. With the use of the single-electrode voltage-clamp technique, two types of voltage-gated K(+) currents were identified that could be separated by differences in their voltage sensitivity and time-dependent kinetics. The first current activated between -50 and -40 mV. It was relatively fast to activate (time-to-peak; 13.7 +/- 0.7 ms at +40 mV) and inactivated by 53.3 +/- 4.9% during a maintained 200-ms depolarization. It was fully available for activation below -80 mV and was completely inactivated by holding potentials more positive than -40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and by concentrations of tetraethylammonium chloride (TEA) >10 mM. These properties characterize this current as a member of the A-type family of voltage-dependent K(+) currents. The second voltage-gated K(+) current activated at more depolarized potentials (-30 to -20 mV). It activated slower than the A-type current (time-to-peak; 74.1 +/- 3.9 ms at +40 mV) and showed little inactivation (6.2 +/- 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below -80 mV with a proportion of the current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1-3 mM TEA. These properties characterize this current as a member of the delayed rectifier family of voltage-dependent K(+) currents. The slow activation rates and relatively depolarized activation thresholds of the two K(+) currents are suggestive that their main role is to contribute to the repolarization phase of the action potential.     

Calcium and potassium systems of a giant barnacle muscle fibre under membrane potential control

1. Single barnacle muscle fibres from Megabalanus psittacus (Darwin) were internally perfused and the effects of various internal and external solutions on voltage clamp currents were examined.2. The usual internal solution was 180 mM-K(+) aspartate (osmotic pressure adjusted to 1000 m-osmole by adding sucrose). Fibres perfused with this solution gave an average resting potential of -55 +/- 5 mV (all potentials are referred to the external solutions as ground). Further increase in internal K concentration depolarized the fibres.3. With membrane current control the total capacitance, referred to apparent membrane surface area, was 21.2 +/- 2.4 muF/cm(2).4. Under normal conditions, with demonstrably good longitudinal space clamp control, voltage clamp currents associated with certain depolarizing pulses showed oscillations. These oscillations were reduced in frequency and magnitude by lowering the temperature from 20 to 10 degrees C, by eliminating the inward currents with external Ca-free saline or by reducing the outward currents with internal tetraethylammonium (TEA) or replacement of internal K by Cs.5. With a Na- and Ca-free, 60 mM-MgCl(2) solution outside depolarizing voltage clamp pulses produced only outward currents. On repolarization the current tail reversed direction at about -70 mV for pulses of less than 50 msec duration. For longer pulses this reversal potential was less negative, suggesting an accumulation of external, or depletion of internal K.6. Both the size of the outward currents and the rate at which they reached their maximum value increased with temperature. The activation energy for the rate constant was about 63 kJ/mole.7. Fibres bathed in Na- and Mg-free, 60 mM-CaCl(2) saline were excitable. After replacement of the internal K(+) with Cs(+) or adding 60 mM-TEA to the internal solution only sustained inward currents were recorded with depolarization.8. Sustained inward currents could be reduced by external application of 5 mM-LaCl(3). Tetrodotoxin was not effective even at a concentration of 1000 nM.9. The rate at which these inward currents reached a maximum value increased with increase in temperature of the bathing solution with an activation energy of the order of 42 kJ/mole.10. The reversal potential of the inward currents changed with the level of internal Ca ions. For a fibre perfused without ethyleneglycol-bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) this reversal potential was 175 mV (internal free Ca 5 x 10(-7)M), and was 196 mV for a fibre perfused with 20 mM-Tris EGTA (internal free Ca 0.26 x 10(-8)M).11. We propose an electrical equivalent circuit to account for most of the observed electrical properties of barnacle muscle fibres. In this model the Ca and the K system are located at different anatomical places and they interact through a series resistance.     

Na(+) entry through AMPA receptors results in voltage-gated k(+) channel blockade in cultured rat spinal cord motoneurons

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor currents, evoked with the agonist kainate, were studied with the gramicidin perforated-patch-clamp technique in cultured rat spinal cord motoneurons. Kainate-induced currents could be blocked by the AMPA receptor antagonist LY 300164 and displayed an apparent strong inward rectification. This inward rectification was not a genuine property of AMPA receptor currents but was a result of a concomitant decrease in outward current at potentials positive to -40.5 +/- 1.3 mV. The AMPA receptor current itself was nearly linear (rectification index 0.91). The kainate-inhibited outward current had a reversal potential close to the estimated K(+) equilibrium potential and was blocked by 30 mM tetraethylammonium. When voltage steps were applied, it was found that kainate inhibited both the delayed rectifier K(+) current K(V) and the transient outward K(+) current, K(A). The kainate-induced inhibition of K(+) currents was dependent on ion flux through the AMPA receptor, because no change in the membrane conductance was noticed in the presence of LY 300164. Removing extracellular Ca(2+) had no effect, whereas replacing extracellular Na(+) or clamping the membrane close to the estimated Na(+) equilibrium potential during kainate application attenuated the inhibition of the K(+) current. Sustained Na(+) influx induced by application of the Na(+) ionophore monensin could mimic the effect of kainate on K(+) conductance. These findings demonstrate that Na(+) influx through AMPA receptors results in blockade of voltage-gated K(+) channels.     

Single channel analysis of the inward rectifier K current in the rabbit ventricular cells

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.     

Ion channels in spinal cord astrocytes in vitro. I. Transient expression of high levels of Na+ and K+ channels.

1. Na+ and K+ channel expression was studied in cultured astrocytes derived from P--0 rat spinal cord using whole cell patch-clamp recording techniques. Two subtypes of astrocytes, pancake and stellate, were differentiated morphologically. Both astrocyte types showed Na+ channels and up to three forms of K+ channels at certain stages of in vitro development. 2. Both astrocyte types showed pronounced K+ currents immediately after plating. Stellate but not pancake astrocytes additionally showed tetrodotoxin (TTX)-sensitive inward Na+ currents, which displayed properties similar to neuronal Na+ currents. 3. Within 4-5 days in vitro (DIV), pancake astrocytes lost K(+)-current expression almost completely, but acquired Na+ currents in high densities (estimated channel density approximately 2-8 channels/microns2). Na+ channel expression in these astrocytes is approximately 10- to 100-fold higher than previously reported for glial cells. Concomitant with the loss of K+ channels, pancake astrocytes showed significantly depolarized membrane potentials (-28.1 +/- 15.4 mV, mean +/- SD), compared with stellate astrocytes (-62.5 +/- 11.9 mV, mean +/- SD). 4. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp, when clamp potential was more negative than resting potential. Both depolarizing and hyperpolarizing current injections elicited overshooting responses, provided that cells were current clamped to membrane potentials more negative than -70 mV. Anode-break spikes were evoked by large hyperpolarizations (less than -150 mV). AP-like responses in these hyperpolarized astrocytes showed a time course similar to neuronal APs under conditions of low K+ conductance. 5. In stellate astrocytes, AP-like responses were not observed, because the K+ conductance always exceeded Na+ conductance by at least a factor of 3. Thus stellate spinal cord astrocyte membranes are stabilized close to EK as previously reported for hippocampal astrocytes. 6. It is concluded that spinal cord pancake astrocytes are capable of synthesizing Na+ channels at densities that can, under some conditions, support electrogenesis. In vivo, however, AP-like responses are unlikely to occur because the cells' resting potential is too depolarized to allow current activation. Thus the absence of electrogenesis in astrocytes may be explained by two mechanisms: 1) a low Na-to-K conductance ratio, as in stellate spinal cord astrocytes and in other previously studied astrocyte preparations; or, 2) as described in detail in the companion paper, a mismatch between the h infinity curve and resting potential, which results in Na+ current inactivation in spinal cord pancake astrocytes.     

CO-induced K(+) currents in rat glomus cells are insensitive to light unlike carotid body neural discharge and Vo(O(2))

The hypothesis that the light sensitive properties of CO-induced chemosensory nerve (CSN) discharge and oxygen consumption of the carotid body (CB) were shared by the pre-synaptic glomus cells was tested. The light effect on K(+) currents were measured before and during perfusion of the isolated rat glomus cells with high P(CO) of 550 Torr during nomoxia (P(O(2)approximately equal 100 Torr) at extra-cellular pH 7.0 and intracellular pH 6.8 with HEPES buffer. CO increased the K(+) currents with a left ward shift of the reversal potential, which showed no light effect. Thus the K(+) permeability of the glomus cell membrane were not shared by the light-sensitive CSN discharge of the CB and oxygen consumption in the presence of high P(CO.)     

Physiological significance of hyperpolarization-activated inward currents (I h) in smooth muscle cells from the circular layers of pregnant rat myometrium

The properties of hyperpolarization-activated current in pregnant rat uterus (17-19 days gestation) were investigated using microelectrode and patch-clamp techniques, and isometric tension recording. The resting membrane potentials were -58.4 mV and -48.5 mV in longitudinal and circular muscle cells, respectively. Application of hyperpolarizing current pulses produced a time-dependent anomalous inward rectification of membrane potential only in circular muscle cells. Under voltage-clamp conditions, inward currents (Ih) were activated by long hyperpolarizing pulses below -60 mV in circular but not in longitudinal muscle cells. Application of extracellular but not intracellular Cs+ reduced the amplitude of I(h) in a concentration-dependent manner (an IC50( of 0.15 mM). The reversal potential for Ih was -26.2 mV and the slope conductance was 5 nS/pF. Changes in [K+]o and [Na+]o shifted the reversal potential, and Ih amplitude increased with excess [K+]o and decreased with low [Na+]o. The steady-state activation of Ih was well fitted by a Boltzmann equation with a half-activation potential of -84.3 mV and a slope factor of 9.6 mV. Time courses of activation and deactivation of the current strongly depended on membrane potential, and were well fitted by a single exponential function. The activation time constant of Ih was dependent on temperature. In isometric tension recording, application of extracellular Cs+ to the circular muscles reduced the frequency, but not the amplitude, of spontaneous contractions in a concentration-dependent manner. It is concluded that in pregnant rat uterus Ih channels are predominantly distributed in smooth muscle cells from the circular layer. Since Ih is activated at the resting membrane potential, it is likely that this current contributes to the maintenance of resting membrane potential and spontaneous activity in circular smooth muscle cells of late pregnant rats.     

Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-dependent chloride current induced by axotomy in rat sympathetic neurons.

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after-depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single-electrode voltage-clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between -10 and -20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage-clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately -15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non-axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)-dependent chloride current responsible for the ADP observed following spike firing.     

Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular cells

We studied the effects of lysophosphatidylcholine (LPC), a toxic metabolite of ischemia, on the inward rectifier potassium channel current in isolated guinea pig ventricular cells. LPC (10-50 microM) added to the external solution decreased the resting membrane potential and occasionally induced repetitive action potential discharges, with or without loss of repolarization. In voltage clamp studies, LPC (20 microM) decreased the conductance at the levels of resting potentials (approximately equal to -80 mV) from 26 +/- 8 nS to 16 +/- 3 nS (mean and SD, n = 4) within 10 min. Prolonged application of LPC (greater than 12 min) produced transient inward currents after depolarizing clamp pulses, thereby suggesting that the LPC elevated intracellular Ca2+ concentrations. The effect of LPC on the single inward rectifier K channel current was examined using the patch clamp technique in a cell-attached mode. LPC decreased the single channel conductance, depending on the concentration (5-100 microM). The slope conductance in the presence of 150 mM K+ in the pipette decreased from 45 +/- 7 pS (control) to 32 +/- 17, 20 +/- 19, and 14 +/- 10 pS for 5, 20 and 100 microM LPC, respectively. LPC induced little change with regard to probability of the channel opening. These results suggest that LPC depolarizes membrane by decreasing single channel conductance of the inward rectifier K channel. This reduction partially contributes to the alleged LPC-induced abnormal automaticities and conduction disturbances in the heart.     

Inward rectification in rat nucleus accumbens neurons

1. Intracellular recordings were made from neurons in slices cut from the rat nucleus accumbens septi. Membrane currents were measured with a single-electrode voltage-clamp amplifier in the potential range -50 to -140 mV. 2. In control conditions (2.5 mM potassium), the resting membrane potential of the neurons was -83.4 +/- 1.1 (SE) mV (n = 157). Steady state membrane conductance was voltage dependent, being 34.8 +/- 1.7 nS (n = 25) at -100 mV and 8.0 +/- 0.7 nS (n = 25) at -60 mV. 3. Barium (1 microM) markedly reduced the inward rectification and caused a small inward current (40.6 +/- 8.7 pA, n = 8) at the resting potential. These effects became larger with higher barium concentrations, and, in 100 microM barium, the current-voltage relation was straight. 4. The block of the inward current by barium (at -130 mV) occurred with an exponential time course; the time constant was approximately 1 s at 1 microM barium and less than 90 ms with 100 microM. Strontium had effects similar to those of barium, but 1000-fold higher concentrations were required. Cesium chloride (2 mM) and rubidium chloride (2 mM) also blocked the inward rectification; their action reached steady state within 50 ms. 5. It is concluded that the nucleus accumbens neurons have a potassium conductance with many features of a typical inward rectifier and that this contributes to the potassium conductance at the resting potential.     

A Ba(2+)-sensitive K(+) current contributes to the resting membrane potential of neurons in rat suprachiasmatic nucleus

A Ba(2+)-sensitive K(+) current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba(2+)-sensitive K(+) current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba(2+) (500 microM) reversibly depolarized the membrane potential by 6.7 +/- 1.3 mV (n = 22) and concomitantly Ba(2+) induced an increase in the spontaneous firing rate of 0.8 +/- 0.2 Hz (n = 12). The Ba(2+)-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba(2+)-sensitive current was observed. Voltage-clamp experiments showed that Ba(2+) (500 microM) reduced a fast-activating, voltage-dependent K(+) current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba(2+)-sensitive K(+) current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 microM). A component of Ba(2+)-sensitive K(+) current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba(2+)- and TEA-sensitive K(+) current make it unlikely that this current is a delayed rectifier, Ca(2+)-activated K(+) current, ATP-sensitive K(+) current, M-current or K(+) inward rectifier. Our data are consistent with the Ba(2+)- and TEA-sensitive K(+) current in SCN neurons being an outward rectifying K(+) current of a novel identity or belonging to a known family of K(+) channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.     

Differential inhibition of glial K(+) currents by 4-AP

Spinal cord astrocytes express four biophysically and pharmacologically distinct voltage-activated potassium (K(+)) channel types. The K(+) channel blocker 4-aminopyridine (4-AP) exhibited differential and concentration-dependent block of all of these currents. Specifically, 100 microM 4-AP selectively inhibited a slowly inactivating outward current (K(SI)) that was insensitive to dendrototoxin (< or = 10 microM) and that activated at -50 mV. At 2 mM, 4-AP inhibited fast-inactivating, low-threshold (-70 mV) A-type currents (K(A)) and sustained, TEA-sensitive noninactivating delayed-rectifier-type currents (K(DR)). At an even higher concentration (8 mM), 4-AP additionally blocked inwardly rectifying, Cs(+)- and Ba(2+)-sensitive K(+) currents (K(IR)). Current injection into current-clamped astrocytes in culture or in acute spinal cord slices induced an overshooting voltage response reminiscent of slow neuronal action potentials. Increasing concentrations of 4-AP selectively modulated different phases in the repolarization of these glial spikes, suggesting that all four K(+) currents serve different roles in stabilization and repolarization of the astrocytic membrane potential. Our data suggest that 4-AP is an useful, dose-dependent inhibitor of all four astrocytic K(+) channels. We show that the slowly inactivating astrocytic K(+) currents, which had not been described as separate current entities in astrocytes, contribute to the resting K(+) conductance and may thus be involved in K(+) homeostatic functions of astrocytes. The high sensitivity of these currents to micromolar 4-AP suggests that application of 4-AP to inhibit neuronal A-currents or to induce epileptiform discharges in brain slices also may influence astrocytic K(+) buffering.     

Membrane properties of a barnacle photoreceptor examined by the voltage clamp technique

1. Electrical properties of the membrane of photoreceptor cells in the lateral ocelli of barnacles, Balanus amphitrite and B. eburneus were investigated by intracellular recording, polarization and voltage-clamp techniques.2. The resting potential of a dark adapted cell was 36.3 +/- 6.6 mV (S.D.) and depended mainly on the external K(+) concentration.3. Current-voltage relations obtained from voltage-clamp experiments in the absence of light were non-linear and varied with time after the onset of a step change in membrane potential; the steady state was reached after about 0.5 sec.4. Illumination resulted in a membrane potential change under current clamp and in a change of membrane current (light-initiated membrane current (L.I.C.): total membrane current with illumination minus current without illumination) under voltage-clamp conditions. Amplitudes and time course of L.I.C. depended on the light intensity as well as membrane potential.5. The L.I.C.-voltage relation was non-linear and corresponded with a slope conductance increase with increasing positive membrane potential.6. The reversal potential of L.I.C. was independent of the light intensity and the time after onset of illumination; the average value obtained in normal saline was +26.9 +/- 5.0 mV.7. The membrane conductance estimated from instantaneous L.I.C.-voltage relations agreed with the chord conductance of the non-linear L.I.C.-voltage relation.8. Decreasing external Na(+) concentration decreased the inward component of L.I.C. but not the outward component.9. Decreasing external Ca(2+) concentration increased the inward as well as the outward component of L.I.C.10. The reversal potential shifted in the negative direction with decreasing external Na(+) concentration (the rate was 10-15 mV for a tenfold change in concentration) and the rate was augmented in the absence of Ca(2+) but did not exceed 21 mV.11. The change of reversal potential with changes of external Ca(2+) concentration was negligible in normal Na(+) media but was significant in the absence of Na(+) (rate as high as 20 mV).12. Alteration of the external K(+) or Cl(-) concentrations did not affect the amplitude or reversal potential of L.I.C.13. The results indicate that illumination increases the membrane permeability mainly to Na(+) ions and that the primary effect of Ca(2+) ions is suppression of the permeability increase; Ca(2+) permeability may increase slightly during illumination.     

Inward rectification in the inner segment of single retinal cone photoreceptors.

1. Single cone photoreceptors were dissociated from the retina of a lizard with the aid of papain. The majority of the cells lost their outer segments but had well-preserved, large synaptic pedicles. Electrical properties of the cells were studied with tight-seal electrodes in the whole cell configuration. On the average, cone inner segments had a resting potential of -55 mV, and at this potential their input resistance was 2.6 G omega and their capacitance was 8 pF. 2. Under current clamp the cones exhibited a pronounced anomalous voltage rectification in response to hyperpolarizing currents. The voltage rectification was eliminated by external Cs+. 3. The Cs(+)-sensitive current underlying voltage rectification was isolated by blocking other currents present in the cone. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and tetraethylammonium (TEA)+ blocked a delayed-rectifier K+ current. 4. The Cs(+)-sensitive current was activated by hyperpolarization to potentials more negative than -50 mV, and its current-voltage (I-V) relationship exhibited inward rectification. 5. The inward-rectifying current was selective for K+, but not exclusively. Increasing external K+ concentration 10-fold shifted the reversal potential by 13 mV. If Na ions also permeate through the inward-rectifying channels, the ratio of permeabilities (PK+/PNa+) in normal solution is approximately 3.9. 6. The kinetics of the inward-rectifying current were described by the sum of two exponentials, the amplitudes and time constants of which were voltage dependent. 7. The voltage dependence of the inward-rectifying current was described by Boltzmann's function, with half-maximum activation at -79 mV and a steepness parameter of 7.5 mV. 8. The voltage dependence and kinetics of the inward-rectifying current suggest that it is inactive in a cone photoreceptor in the dark. However, it becomes activated in the course of large hyperpolarizations generated by bright-light illumination. This activity will modify the waveform of the photovoltage--the current will generate a depolarizing component that opposes the light-generated hyperpolarization.     

Muscarine induces two distinct current responses in adrenal chromaffin cells of the guinea-pig

Muscarine-induced membrane responses were studied in dissociated chromaffin cells of the guinea-pig adrenal medulla, using the whole-cell version of the patch-clamp technique. Bath application of muscarine (1-10 microM) produced two distinct current responses at a holding potential of -40 mV. One is an inward current associated with an increase in current noise. This current response was sustained during stimulation and had a reversal potential of 4.5 +/- 3.4 mV (n = 6) with a negative slope conductance below about -30 mV in 12.5 mM K(+)-containing perfusate. The other is a transient outward current. This was evoked at membrane potentials more positive than -60 mV and completely suppressed by addition of 2 mM TEA to the bath solution, suggesting a possible involvement of the Ca2(+)-dependent K+ channel. Generation of the outward current response was suppressed for at least 60-90 s following 25 s muscarinic stimulation and was facilitated by activation of the nicotinic receptor. The maximum inward current seemed to be produced by 3 microM, whereas the threshold concentration required for generation of the outward current was somewhere between 3 and 10 microM. The outward current was evoked less often in cells treated with 2% collagenase for 1 h than in those treated with 0.2% for 30 min. The results suggest that guinea-pig chromaffin cells have two muscarinic receptors: one is coupled with a cation nonselective channel and the other may be related to a Ca2(+)-dependent K+ channel.