Rat hippocampal neurons in culture: potassium conductances
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Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)Keywords:
Tetrodotoxin
Tetraethylammonium
Hyperpolarization
Reversal potential
4-Aminopyridine
Current clamp
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A two-microelectrode current-voltage clamp and Cl(-)-selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (aiCl) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500 microM) produced a mono- or biphasic depolarization (amplitude < or = 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500 microM) was associated with an increase in aiCl which indicates that the depolarizing action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500 microM-GABA (EGABA) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (ECl). ECl was 2-7 mV more negative than the resting membrane potential. 5. EGABA (measured using pulses of 500 microM-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500 microM-muscimol, a GABA agonist that is a poor substrate of the Na(+)-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500 microM-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200 microM), the depolarizing effect of 500 microM-GABA was enhanced and the transient hyperpolarizing shifts were abolished. 8. In the presence of picrotoxin, GABA (> or = 2 microM) produced a concentration-dependent monophasic inward current which had a reversal potential of +30 to +60 mV. This current was inhibited in the absence of Na+ and by the GABA uptake blocker, nipecotic acid. Unlike the channel-mediated current, the picrotoxin-insensitive current was activated without delay also at low (2-10 microM) concentrations of GABA.(ABSTRACT TRUNCATED AT 400 WORDS)
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1. Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.
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1. Inwardly rectifying ionic currents were studied using patch‐clamp recording methods in oscillatory‐type and spike‐type hair cells and supporting cells dissociated from the goldfish sacculus. These cells had different types of inwardly rectifying currents. The biophysical properties of these currents were investigated. 2. A unique potassium current (Isc) was the sole ionic current recognized in supporting cells. Isc was active throughout the membrane potential range between +30 and ‐170 mV, but showed weak inward rectification and no inactivation. 3. In spike‐type hair cells, inwardly rectifying current (Ik1) was selectively permeable to K+ (K+:Na+ permeability ratio, 1:0.0021). Ik1 could underlie the high negative resting potential of these hair cells because it is partially active at this potential. The strong inward rectification of Ik1 contributed to the low negative plateau potential seen in spike‐type hair cells. 4. In oscillatory‐type hair cells, hyperpolarization‐activated potassium‐sodium current (Ih), which had properties similar to that in photoreceptor and other neurons, was present instead of inwardly rectifying K+ current. 5. In the cell‐attached and inside‐out modes with 125 microM external K+ ([K+]o), IK1 channel had a unitary conductance of 27 pS and showed inactivation with increasing hyperpolarization. Putative Ih and Iso single channels had unitary conductances of 7 and 61 pS, respectively, in the cell‐attached mode with 125 microM Ko+.
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The effects of tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP) on membrane currents and on single channel K currents in smooth muscle cells isolated from canine trachea were examined by use of tight seal whole cell‐ and patch‐clamp techniques. Depolarizing current applied through a recording pipette did not elicit an action potential under current clamp. A strong outward rectification was observed. In most cells under voltage‐clamp, only an outward current was observed upon depolarization from −60 mV when a pipette solution contained mainly KCl. The outward current consisted of three components; a large initial transient, a following sustained component and an additional component of irregular small transients on the sustained one. The two transient components were almost abolished when extracellular and pipette solutions contained 2.2 m m Cd 2+ (0 m m Ca 2+ ) and 10 m m EGTA, respectively. The sustained component was well maintained under these conditions. TEA at low concentrations (<1 m m ) effectively decreased the transient components and made the outward current smooth; it also suppressed the sustained component at higher concentrations. In outside‐out patches, external 1 m m TEA reduced the single channel conductance of Ca‐activated K channels by about 87% whereas 3 m m 4‐AP did not. 4‐AP at low concentrations (<3 m m ) selectively reduced the sustained component of the outward current. A Ca current recorded after the suppression of outward current by internal Cs + had a peak of approximately 200 pA at + 10 mV (holding potential: −60 mV). The half inactivation voltage in the steady‐state was approximately −30 mV. Simultaneous application of 1 m m TEA and 4‐AP reduced the outward current and unmasked a Ca current. Under these conditions, an action potential with overshoot was easily elicited under current clamp. It is concluded that the low excitability of canine tracheal smooth muscle cell upon depolarization is due to a large outward K current which consists of Ca‐dependent and Ca‐independent components. The peak amplitude of the Ca current is similar to that in highly excitable smooth muscle cells such as those of the ureter.
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Purkinje fibers
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1. The outward rectifying K+ conductance and underlying single channel behaviour in mouse small intestine (MSI) smooth muscle cells was studied using microelectrode impalement and the patch clamp technique. 2. At 37 degrees C, smooth muscle cells in MSI explants had a resting membrane potential around ‐65 mV and showed spontaneous electrical and mechanical activity. 3. Under whole‐cell voltage clamp, depolarization of smooth muscle cells in the explants evoked a methoxyverapamil (D600)‐sensitive, partially inactivating inward current and a non‐inactivating outward current. The outward current was also observed in enzymatically dispersed cells from neonatal mouse small intestine. 4. The reversal potential of the outward current as established in tail current experiments was ‐70.2 mV. Tail currents could be fitted with a single exponential, suggesting the participation of only one population of channels. 5. The outward current was sensitive to 4‐aminopyridine (10(‐4) M), Ba2+ (1 mM) and to the presence of Cs+ in the pipette, but not to D600 (10(‐6) M), or the presence of ATP (1 mM) in the pipette. 6. In the cell‐attached patch configuration, a unitary outward current was observed that showed increased activity upon depolarization of the patch. The current‐voltage relationship was close to linear with a slope conductance of 186 pS. 7. With normal K+ (6 mM) in the pipette, the extrapolated reversal potential for the unitary current was around ‐75 mV, while with high K+ (120 mM) the reversal potential was close to 0 mV. 8. Averaging single channel traces recorded under a depolarizing pulse protocol resulted in a trace with similar time characteristics as the outward current observed in the whole‐cell configuration. 9. The burst behaviour of the channel was described by a simple model consisting of two closed states, Cf (intraburst closed state) and Cs (interburst closed state) and an open state (O). The rate constants in the model showed differential sensitivity to potential changes, channel blockade by Ba2+ and equimolar K+ conditions. 10. It was concluded that the outward rectifying potassium current in MSI smooth muscle cells is mediated by a 186 pS bursting channel. Voltage dependency and Ba2+ blockade are mainly reflected by changes in the transition rate from the open channel state to the interburst closed state.
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1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)
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1. The membrane characteristics of the slowly adapting stretch receptor from the crayfish, Astacus fluviatilis, were examined with electrophysiological techniques consisting of membrane potential recording, voltage clamp and ion‐sensitive microelectrodes. 2. The passive membrane current (Ip) following step changes of the membrane potential to levels above 0 mV required more than a minute to decay to a steady‐state level. 3. The stretch‐induced current (SIC, where SIC = Itotal‐‐Ipassive) was not fully developed until the Ip had decayed to a steady state. 4. With Ip at the steady state and the stretch‐induced current at the O‐current potential, a slow stretch‐induced inward current was isolated. The latter reaches a maximum after 1 sec of stretch and declines even more slowly after stretch. The I‐V relation of the slow current had a negative slope and reversed sign near the resting potential. It is suggested that this current is due to a Cl‐ conductance change. 5. The stretch‐induced current, consisting of a rapid transient phase and a steady component can be isolated from the slow stretch‐induced current at a holding potential corresponding to the resting potential. 6. The SIC‐Em relation is non‐linear and reverses sign at about +15 mV. 7. In a given cell, the reversal potential of the stretch‐induced potential change obtained with current clamp coincided with the 0‐current potential of the stretch‐induced current obtained by voltage clamp. The average value from twenty‐six cells was +13 +/‐ 6.5 mV; cell to cell variability seemed to be correlated with dendrite length. 8. Tris (mol. wt. 121) or arginine (mol. wt. 174) susbstituted for Na+ reduces but does not abolish the stretch‐induced current. 9. The permeability ratios of Tris:Na and arginine:Na were estimated from changes in the 0‐current potential as these cations replaced Na+ in the external medium. The PTris:PNa was somewhat higher (0.31) than the Parginine:PNa ratio (0.25). 10. Changes in the external Ca2+ concentration had no effect on the 0‐current potential in Na or Tris saline. However, reducing Ca2+ did augment the stretch‐induced current in either saline. A tenfold reduction of Ca2+ increased the conductance (at the 0‐current level) about twofold. 11. Intracellular K+ and Cl‐ activities were obtained with ion sensitive electrodes. The average values from six cells were aiK = 133 +/‐ 34 mM and aiCl = 15.2 +/‐ 1.8 mM S.D.). EK was about 20 mV more negative than Em and ECl was about 10 mV more positive than Em. 12. aik and resting Em undergo large changes in K+‐free solutions. After 60 min, ak was reduced eightfold and Em was reduced from ‐67 to ‐40 mV. Reduced Ca2+ in K+‐free augments the rate of these changes. Receptor potential amplitude was also reduced in K+‐free solution but could be restored upon polarizing the membrane to the pre‐existing resting level.
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