ABSTRACT
Whole-cell recordings were made from dissociated bullfrog sympathetic neurons to examine the actions of quinine (1-100 microM) on the steady-state activation and inactivation curves of a delayed rectifier-type potassium current (I(K)). Quinine (EC50 approximately 8 microM) caused a hyperpolarizing shift (approximately 31 mV with 30 microM) in the inactivation curve of I(K) without significantly affecting its activation curve. Quinine (20 microM) was without effects on the voltage-dependence of a rapidly-inactivating A-type potassium current (I(A)). It is concluded that quinine can selectively modulate the voltage-dependence of I(K) in amphibian autonomic neurons.
Subject(s)
Cell Membrane/drug effects , Ganglia, Sympathetic/drug effects , Membrane Potentials/drug effects , Muscle Relaxants, Central/pharmacology , Neurons/drug effects , Potassium Channels/drug effects , Quinine/pharmacology , Animals , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Ganglia, Sympathetic/cytology , Ganglia, Sympathetic/metabolism , Kinetics , Membrane Potentials/physiology , Neurons/metabolism , Potassium Channels/metabolism , Rana catesbeianaABSTRACT
The effects of lanthanides (La(3+), Gd(3+), Lu(3+) and Sm(3+)) on voltage-dependent potassium currents were studied in dissociated bullfrog sympathetic neurons. A-type current (I(A)) and M-type current (I(M)) were blocked by lanthanides (0.1-30 microM) with I(M) being much less sensitive to these ions than I(A). The order of potency was Gd(3+)>/=Lu(3+) approximately La(3+) approximately Sm(3+) for I(A) and Gd(3+)&z.Gt;Lu(3+) approximately La(3+)>Sm(3+) for I(M). The I(M) block occurred independently of its activation kinetics while the I(A) block was associated with a positive shift of the activation and inactivation curves. Gd(3+) (100 microM) blocked the delayed rectifier-type current (I(K)) by less than 20%; Lu(3+), La(3+) and Sm(3+) (100 microM for each) were without effect on I(K). It is concluded that I(A) was the most sensitive to lanthanides, and Gd(3+) was the most potent for all the currents in amphibian autonomic neurons.
Subject(s)
Ganglia, Sympathetic/drug effects , Metals, Rare Earth/pharmacology , Neurons/drug effects , Potassium Channels/drug effects , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Electric Stimulation , Electrophysiology , Ganglia, Sympathetic/cytology , Ganglia, Sympathetic/metabolism , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurons/cytology , Neurons/metabolism , Potassium Channels/metabolism , Rana catesbeianaABSTRACT
Whole-cell/voltage-clamp recordings were made from dissociated bullfrog sympathetic neurons to examine the channel blocking actions of barium (3-2000 microM) on an M-type potassium current (I(M)). Barium (IC(50) approximately 105 microM) blocked I(M) without affecting the 50%-activation voltage ( approximately -35 mV) and the slope factor ( approximately 11 mV) of the activation curve. The results indicate that the barium block is independent of the kinetics of I(M).
Subject(s)
Barium/pharmacology , Neurons/physiology , Potassium Channel Blockers , Sympathetic Nervous System/physiology , Action Potentials/drug effects , Animals , Autonomic Nerve Block/methods , Neurons/drug effects , Patch-Clamp Techniques , Potassium Channels/drug effects , Potassium Channels/metabolism , Rana catesbeiana , Sympathetic Nervous System/drug effects , Sympatholytics/pharmacologyABSTRACT
Whole-cell/voltage-clamp recordings were made from dissociated bullfrog sympathetic neurons to examine the sensitivity of potassium currents to a potassium channel blocker quinine (1-500 microM). Among three currents tested, a rapidly inactivating A-type current (I(A)) was the most sensitive to the block by quinine (IC50 approximately 22 microM). A non-inactivating M-type current (I(M)) was the least sensitive (IC50 approximately 445 microM), and the sensitivity of a slowly inactivating delayed rectifier-type current (I(K)) was in between (IC50 approximately 115 microM). Results suggest that the ability of quinine to block different types of potassium currents such as I(A) and I(M) with significantly different IC50 values would be of help for the potassium channel pharmacology in amphibian autonomic ganglion cells.
Subject(s)
Ganglia, Sympathetic/physiology , Neurons/drug effects , Potassium Channels/drug effects , Quinine/pharmacology , Rana catesbeiana , Animals , Ganglia, Sympathetic/cytology , Neurons/physiology , Patch-Clamp TechniquesABSTRACT
Theoretical background has been reviewed for inward rectification due to a potassium current termed IRK. The Eyring rate theory in which the thermodynamic rate coefficient for chemical reactants (channels and ions in this case) can be described in terms of energy barriers for potassium ions can mimic not only the polarity and degree of rectification but also the voltage-dependence of the barium-induced IRK block. The model predicts that the blocking site locates 30-70% depth from the outer margin of the IRK channel.
Subject(s)
Models, Biological , Potassium Channels, Inwardly Rectifying , Potassium Channels/physiology , Animals , Energy Transfer , HumansABSTRACT
The actions of zinc on A-type potassium current (I(A)) were studied in dissociated bullfrog sympathetic neurons. Zinc (1-300 microM) caused a parallel shift in the activation and inactivation curves to a depolarizing direction, thereby enhancing I(A) around physiological resting potential. An EC50 value was 70-100 microM for these actions. The zinc actions were non-selective in a sense that zinc inhibited M-type potassium current (I(M)) with an IC50 value of 300 microM. Zinc was without effect on the maximum conductance for I(A) and the kinetic behavior for I(M). The ability of low concentrations of zinc to modulate separate set of potassium currents such as I(A) and I(M) in conceptually distinct manner may therefore assume pathophysiological importance for autonomic neurons.
Subject(s)
Ganglia, Sympathetic/drug effects , Ganglia, Sympathetic/physiology , Neurons/drug effects , Neurons/physiology , Potassium/physiology , Zinc/pharmacology , Animals , Dose-Response Relationship, Drug , Electric Conductivity , Ganglia, Sympathetic/cytology , Kinetics , Osmolar Concentration , Potassium/antagonists & inhibitors , Time FactorsABSTRACT
The effect of barium (1 mM) on a delayed rectifier-type potassium current was examined in bullfrog sympathetic neurons. An M-type potassium current was eliminated by pretreatment of the cells with a microbial product, wortmannin (10 microM). An A-type potassium current was continuously inactivated by setting a holding potential at -65 mV. In treated cells (n = 10), the delayed rectifier at 0 mV averaged 2200 +/- 107 pA in the presence of barium (1 mM) as compared to 2308 +/- 110 pA in the controls, and 2085 +/- 103 pA after washing out the barium. It is concluded that the delayed rectifier is insensitive to barium blockage in amphibian autonomic neurons.
Subject(s)
Androstadienes/pharmacology , Barium/pharmacology , Ganglia, Sympathetic/drug effects , Potassium Channel Blockers , Potassium Channels, Voltage-Gated , Potassium Channels/drug effects , Animals , Cells, Cultured , Delayed Rectifier Potassium Channels , Ganglia, Sympathetic/physiology , Potassium Channels/physiology , Rana catesbeiana , WortmanninABSTRACT
Whole-cell/voltage-clamp recordings were made from dissociated bullfrog sympathetic neurons to examine the inhibitory actions of barium (0.01-3 mM) on a rapidly inactivating A-type potassium current (IA). The IC50 value was about 0.9 mM. Barium (1 mM) approximately halved the maximum amplitude of IA (approximately 1.7 nA near 0 mV) without significantly affecting a voltage for the 50%-activation (approximately -40 mV) and that for the 50%-inactivation (approximately -90 mV), nor did it affected the time course of IA. The results suggest that the barium block is independent of the kinetics of the A-channels in bullfrog sympathetic neurons.
Subject(s)
Barium/pharmacology , Ganglia, Sympathetic/drug effects , Neurons/drug effects , Potassium Channels/drug effects , Animals , Ganglia, Sympathetic/cytology , Kinetics , Patch-Clamp Techniques , Rana catesbeiana , Time FactorsABSTRACT
Intracellular Ca2+ concentration ([Ca]i) was measured following the activation of an inward Ca2+ current and subsequent potentiation of an M-type K+ current (IM) in bullfrog sympathetic neurons. Fura-2 was used as an indicator for [Ca]i. The fluorescence ratio at 340 and 380 nm (F340/F380) was elevated from 0.36 to 1.22 when IM was potentiated by 68% following the Ca2+ current. Based on the in vivo calibration curve obtained from cells permeabilized with digitonin (20 microM), the F340/F380 value of 1.22 was equivalent to a [Ca]i of 0.97 microM. We therefore propose that a rise in [Ca]i into the micromolar range can lead to the potentiation of IM in amphibian autonomic neurons.
Subject(s)
Calcium/metabolism , Neurons/physiology , Potassium Channels/physiology , Sympathetic Nervous System/cytology , Action Potentials/physiology , Animals , Electric Conductivity , Electrophysiology , Fura-2 , Neurons/chemistry , Rana catesbeianaABSTRACT
Whole-cell/voltage-clamp recordings were made from dissociated sympathetic neurons of Xenopus laevis. Step depolarization (10-70 mV, 0.5 s) from the holding potential of -65 mV activated a non-inactivating potassium current which was selectively blocked by barium (1 mM). The current did not require an inward calcium current for its activation. The steady-state activation curve of the current was centered at -35 mV with a slope factor of 8.6 mV. The time constant for activation of the current was about 115 ms when measured at -35 mV and the effective valence of the voltage-sensing particle was 2.9. This potassium current was identified as a particular set of potassium currents usually referred to as M-current.
Subject(s)
Ganglia, Sympathetic/physiology , Potassium Channels/physiology , Animals , In Vitro Techniques , Patch-Clamp Techniques , Xenopus laevisABSTRACT
Active and passive properties of dissociated bullfrog sympathetic ganglion cells were examined in the whole-cell configuration. Electrical constants were 107 k omega cm2 and 1 microFcm2 for the unitary cell membrane area. Under conditions in which the equilibrium potential was 55 mV for sodium ions and -101 mV for potassium ions, the action potential and its after-hyperpolarization peaked at 50 and -83 mV, respectively, when elicited from the resting potential of -63 mV. These observations are consistent with those reported previously for other autonomic neurons. The reversal potential of the after-hyperpolarization was approximately 10 mV less negative than the potassium equilibrium potential. The basis of this difference has been discussed using a hypothetical equivalent circuit.
Subject(s)
Ganglia, Sympathetic/physiology , Neurons/physiology , Rana catesbeiana/physiology , Action Potentials/physiology , Animals , Cell Membrane/physiology , Electrophysiology , Ganglia, Sympathetic/cytology , Membrane Potentials/physiology , Neurons/cytologyABSTRACT
1. The effects of multivalent cations on membrane currents recorded from Xenopus oocytes were studied. 2. The hyperpolarization-activated chloride current was reversibly blocked by lanthanum; half-maximal block occurred at a concentration of 8 microM. Zinc, cadmium, cobalt and nickel were less potent than lanthanum, and gadolinium, manganese, barium and strontium had no effect at a concentration of 100 microM. 3. The calcium-activated chloride current was blocked by gadolinium (50 microM), and lanthanum, cadmium, cobalt, nickel and manganese were equally effective. The actions of gadolinium and lanthanum were almost irreversible, while partial (30-80%) recovery was observed with the other cations. Zinc (100 microM) had no effect. 4. In lanthanum (100 microM), membrane depolarizations from -70 mV activated an outward potassium current that was partially blocked by barium (0.1-2 mM). The barium-sensitive current was confined to potentials less negative than -70 mV. The current consisted of a time-independent as well as a time-dependent component, the latter of which had voltage dependence similar to the M-current. 5. It is proposed that lanthanum, gadolinium and barium can usefully separate these endogenous membrane currents in Xenopus oocytes.
Subject(s)
Barium/pharmacology , Chloride Channels/physiology , Gadolinium/pharmacology , Lanthanum/pharmacology , Oocytes/physiology , Potassium Channels/physiology , Animals , Cations/pharmacology , Chloride Channels/drug effects , Female , In Vitro Techniques , Kinetics , Membrane Potentials/drug effects , Oocytes/drug effects , Potassium Channels/drug effects , Time Factors , Xenopus laevisABSTRACT
Whole-cell recordings were made from dissociated bullfrog sympathetic neurons. Tetraethylammonium (30 mM) and apamin (100 nM) were added to the superfusate to eliminate the known calcium-activated potassium currents termed Ic and IAHP. Under these conditions, the action potential carried by calcium ions was followed by a prolonged (10-60 s) after-hyperpolarization. A current component (IAC) underlying the after-hyperpolarization was eliminated by barium (2 mM) and showed voltage-dependence identical to that of a M-type potassium current. I concluded that the after-hyperpolarization is caused not only by IAHP but also by the calcium-dependent potentiation of M-current.
Subject(s)
Calcium/pharmacology , Membrane Potentials/physiology , Sympathetic Nervous System/physiology , Animals , Patch-Clamp Techniques , Rana catesbeianaABSTRACT
Whole-cell voltage-clamp recordings were made from cultured bullfrog sympathetic neurons to measure the steady-state activation curve of M-type potassium current. When measured with a calcium-deficient (10 nM) pipette solution M-conductance was 4.8 nS at -35 mV having the 50%-activation voltage at-20 mV. Respective values were 17.2 nS at -35 mV with the 50%-activation voltage at -42 mV when measured with a calcium-rich (1 microM) solution, indicating the hyperpolarizing displacement of the activation curve with high internal calcium. It is suggested that intracellular calcium ions can modulate kinetics of M-current which thereby regulate the number of M-channels being open at given membrane potentials.
Subject(s)
Calcium/physiology , Neurons/physiology , Potassium Channels/physiology , Sympathetic Nervous System/physiology , Animals , Electrophysiology , In Vitro Techniques , Kinetics , Membrane Potentials/physiology , Patch-Clamp Techniques , Rana catesbeiana , Sympathetic Nervous System/cytologyABSTRACT
Membrane current was recorded from neurons in rabbit vesical parasympathetic ganglia, utilizing single electrode voltage clamp techniques. ATP (0.1-1 mM) caused an inward current (IATP) associated with an increased conductance at a holding potential of -50 mV. ADP (0.1-1 mM) and 5'-O-3-thiotriphosphate (0.1-0.6 mM) but not AMP (0.3-2 mM) and adenosine (0.1-2 mM) mimicked the actions of ATP. The IATP reversed its polarity at -12.1 +/- 1.4 mV. The amplitude of the IATP was depressed in low sodium solutions and in nominally calcium-free solutions but not in low chloride solutions. Suramin (10-100 microM) and reactive blue 2 (10-100 microM), P2-antagonists, reversibly depressed the IATP. In contrast, hexamethonium (100 microM) did not affect the IATP. These data suggest that ATP activates cation channels through P2X receptor subtypes in parasympathetic neurons.
Subject(s)
Ion Channels/physiology , Membrane Potentials/physiology , Parasympathetic Nervous System/physiology , Receptors, Purinergic/physiology , Animals , Atropine/pharmacology , Male , Rabbits , Suramin/pharmacologyABSTRACT
The mechanism underlying the over-recovery of an M-type potassium current following the washout of muscarine (20 microM) has been examined. Whole-cell recordings were made from single neurons dissociated from bullfrog sympathetic ganglia. During over-recovery, the maximum M-conductance decreased by about 2.8 nS while the steady-state M-current activation curve was displaced in the hyperpolarizing direction by about 13 mV. These data suggest that a hyperpolarizing shift in the kinetics of M-current causes over-recovery in amphibian autonomic neurons.
Subject(s)
Ganglia, Sympathetic/drug effects , Membrane Potentials/physiology , Muscarine/pharmacology , Potassium Channels/drug effects , Animals , Patch-Clamp Techniques , Rana catesbeiana , Time FactorsABSTRACT
Whole-cell voltage-clamp recordings were made from cultured bullfrog dorsal root ganglion cells to examine the channel blocking activity of 20 microM of 1, 9-dideoxy forskolin (d-FSK) on the delayed rectifier potassium current. The drug reduced the maximum amplitude of the current (10-20 nA at +65 mV) by 40-80%. However, the voltages for 50%-activation (approximately -12 mV) and for 50%-inactivation (approximately -37 mV) remained unaffected by d-FSK. These results indicate that the channel block by d-FSK occurs in a voltage-independent manner.
Subject(s)
Colforsin/analogs & derivatives , Ganglia, Spinal/drug effects , Potassium Channel Blockers , Potassium Channels, Voltage-Gated , Animals , Cells, Cultured , Colforsin/pharmacology , Delayed Rectifier Potassium Channels , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Potassium Channels/physiology , Rana catesbeianaABSTRACT
Actions of myosin light chain kinase inhibitors were tested on delayed rectifier potassium current (IK) in dissociated bullfrog sympathetic neurons. A microbial product, wortmannin (10 microM, extracellularly) and a synthetic peptide, SM-1 (20 microM, intracellularly) caused approximately 35 mV hyperpolarizing shift of the inactivation curve. Substitution of ATP (1.15 mM) in the pipette solution with 5'-adenylylimidodiphosphate mimicked the actions of wortmannin and SM-1. Results suggest that phosphorylation of myosin may modulate kinetics for the inactivation of IK.
Subject(s)
Myosin-Light-Chain Kinase/antagonists & inhibitors , Neurons/metabolism , Potassium Channels/metabolism , Sympathetic Nervous System/metabolism , Adenylyl Imidodiphosphate/pharmacology , Androstadienes/pharmacology , Animals , Electrophysiology , Enzyme Inhibitors/pharmacology , Ganglia, Sympathetic/cytology , Ganglia, Sympathetic/drug effects , In Vitro Techniques , Myosin-Light-Chain Kinase/pharmacology , Neurons/drug effects , Peptide Fragments/pharmacology , Potassium Channels/drug effects , Rana catesbeiana , Sympathetic Nervous System/cytology , Sympathetic Nervous System/drug effects , WortmanninABSTRACT
1. The actions of wortmannin, an inhibitor of myosin light chain kinase (MLCK), on M-type potassium current of dissociated bullfrog sympathetic neurones have been examined. 2. The amplitude of M-current was measured by whole cell recordings from cells pretreated with wortmannin (0.01-10 microM) or the wortmannin vehicle, dimethylsulphoxide (0.0001-0.1 vol%), for 30 min. Internal (recording pipette) solutions having three different pCa values (6, 7 and 8) were used for the measurements. 3. Irrespective of the pCa, M-current was not detectable when the cells were pretreated with 10 microM wortmannin. Wortmannin, 3 microM, produced 85-95% inhibition of the M-current. Pretreatment with 10-30 nM wortmannin was without effect on M-current. 4. The M-current inhibition by wortmannin at concentrations of 0.1-1 microM depended on the pCa of the internal solution. Inhibition occurred only when the calcium-rich (pCa = 6) internal solution was used. 5. Pre-treatment of the cells with wortmannin (10 microM) did not affect rapidly-inactivating A-type or delayed rectifier-type potassium currents not did it alter inwardly rectifying sodium-potassium current (IH). 6. These observations show that M-current inhibition by wortmannin has two pharmacological profiles. One is calcium-dependent and occurs at lower concentrations (0.1-1 microM), and is attributed to inhibition of MLCK by wortmannin. At higher concentrations (3-10 microM), wortmannin has an additional, calcium-independent action, inhibiting the M-current by an unknown mechanism.
Subject(s)
Androstadienes/pharmacology , Enzyme Inhibitors/pharmacology , Neurons/metabolism , Potassium Channels/metabolism , Sympathetic Nervous System/metabolism , Animals , Calcium/pharmacology , Calcium/physiology , Calmodulin/pharmacology , Culture Techniques , Cyclic AMP/physiology , Membrane Potentials/drug effects , Myosin-Light-Chain Kinase/metabolism , Neurons/drug effects , Patch-Clamp Techniques , Potassium Channels/drug effects , Rana catesbeiana , Sympathetic Nervous System/cytology , Sympathetic Nervous System/drug effects , WortmanninABSTRACT
Although Ca(2+)-induced Ca2+ release (CICR) via ryanodine receptors has been found to occur in intact neurons, little is known about the physiological processes that regulate it. We studied the effects of cyclic ADP-ribose (cADPR) on CICR in cultured bullfrog sympathetic neurons by fura-2 fluorescence recording and patch-clamp techniques. cADPR applied through a patch pipette augmented action potential- or depolarizing pulse-induced rises in intracellular Ca2+ without a change in Ca2+ entry initiating the responses, but not in the presence of ryanodine. Likewise, cADPR enhanced a single or oscillatory rise(s) in intracellular Ca2+ induced by caffeine. These results strongly suggest that cADPR can be an endogenous modulator of ryanodine receptors in neurons.
