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PubMed2003

Delayed K+ clearance associated with aquaporin-4 mislocalization: phenotypic defects in brains of alpha-syntrophin-null mice.

Recovery from neuronal activation requires rapid clearance of potassium ions (K+) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with alpha-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and alpha-syntrophin-null mice (alpha-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the alpha-Syn-/- mice, whereas labeling of the inwardly rectifying K+ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K+ clearance were studied in hippocampal slices to test whether AQP4 and K+ channels work in concert to achieve isosmotic clearance of K+ after neuronal activation. Microelectrode recordings of extracellular K+ ([K+]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K+ clearance was prolonged up to 2-fold in alpha-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the alpha-Syn-/-mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K+ after neuronal activation.

Animals↗
PubMed1999

Choline modulates cardiac membrane repolarization by activating an M3 muscarinic receptor and its coupled K+ channel.

Choline is a necessary substrate of the lipid membrane and for acetylcholine synthesis. Accumulating evidence indicates that besides being a structural component, choline is also a functional modulator of the membrane. It has been shown to be a muscarinic acetylcholine receptor (mAChR) agonist and can induce a novel K+ current in cardiac cells. However, the potential role of choline in modulating cardiac functions remained unstudied despite that mAChRs are known to be important in regulating heart functions. With microelectrode techniques, we found that choline produced concentration-dependent (0.1 approximately 10 mm) decreases in sinus rhythm and action potential duration in isolated guinea pig atria. The effects were reversed by 2 nm 4DAMP (an M3-selective antagonist). Whole-cell patch-clamp recordings in dispersed myocytes from guinea pig and canine atria revealed that choline is able to induce a K+ current with delayed rectifying properties. The choline-induced current was suppressed by low concentrations of 4DAMP (2 approximately 10 nm). Antagonists toward other subtypes (M1, M2 or M4) all failed to alter the current. The affinity of choline (Kd) at mAChRs derived from displacement binding of [3H]-NMS in the homogenates from dog atria was 0.9 mm, consistent with the concentration needed for the current induction and for the HR and APD modulation. Our data indicate that choline modulates the cellular electrical properties of the hearts, likely by activating a K+ current via stimulation of M3 receptors.

Animals↗
PubMed1996

Effects of cytochrome P450 inhibitors on potassium currents and mechanical activity in rat portal vein.

1. The effects of the cytochrome P450 inhibitors, proadifen, clotrimazole and 17-octadecynoic acid (17-ODYA) on K-currents in freshly-isolated single cells derived from rat portal vein and on mechanical activity in whole veins were studied. 2. When cells were stepped from -90 mV to a series of test potentials (from -80 to +50 mV), a delayed rectifier current (IK(V)) and an A-type current (IK(A)) could be identified. Proadifen (10 microM), clotrimazole (30 microM) and 17-ODYA (5 microM) each inhibited IK(V) but had little effect on IK(A). 3. When cells were held at -10 mV to inactivate the time-dependent K-currents, IK(V) and IK(A), levcromakalim (3 microM) induced a time-independent outward K-current (IK(ATP)) which was totally inhibited by clotrimazole (30 microM) and almost fully inhibited by proadifen (10 microM). 17-ODYA (5 microM) had no effect on IK(ATP) and exerted only a minor inhibitory action on this current at 20 microM. 4. 17-ODYA (5 microM) potentiated current flow through the large conductance, Ca-sensitive K-channel (BKCa). In contrast, proadifen (10 microM) had no effect on IBK(Ca) whereas clotrimazole (30 microM) exerted a small but significant inhibitory action. 5. Proadifen (10 microM) and clotrimazole (30 microM) each inhibited the magnitude but increased the frequency of spontaneous contractions in whole portal veins. 17-ODYA (5 microM) had no effect on spontaneous contractions but these were inhibited when the concentration of 17-ODYA was increased to 50 microM. 6. The spasmolytic effect of levcromakalim on spontaneous contractions was antagonized by proadifen (10-30 microM) in a concentration-dependent manner but 17-ODYA (up to 50 microM) was without effect. 7. These results in portal vein show that cytochrome P450 inhibitors exert profound effects on a variety of K-channel subtypes. This suggests that enzymes dependent on this cofactor may be important regulators of K-channel activity in smooth muscle. The relevance of these findings for the identification of the pathway involved in the synthesis of the endothelium-derived hyperpolarizing factor is discussed.

Animals↗
PubMedDate not supplied

The A-like potassium current of a leech neuron increases with age in cell culture.

Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane. However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection. To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration. Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed. Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component. The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca(2+) concentration, whereas the two other potassium currents remained unchanged. Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current.

Animals↗
PubMed2004

Autoimmunity against the second extracellular loop of beta(1)-adrenergic receptors induces early afterdepolarization and decreases in K-channel density in rabbits.

OBJECTIVES: We sought to define the electrophysiologic property of the rabbit heart associated with autoimmunity against the second extracellular loop of the beta(1)-adrenergic receptor. BACKGROUND: Sudden death of patients with cardiomyopathy, probably due to lethal ventricular arrhythmias, can be predicted by the presence of autoantibodies against the second extracellular loop of the beta(1)-adrenergic receptor. METHODS: Rabbits were immunized by repetitive subcutaneous administration of a synthetic peptide corresponding to the second extracellular loop of beta(1)-adrenergic receptors (beta group; n = 30) for a mean of 4.2 months. Control rabbits received only vehicle (control group; n = 30). RESULTS: One of the rabbits in the beta group died suddenly during the observation period, but none of the control animals died. The prevalence of sustained ventricular tachycardia was significantly higher in the beta group (beta: 4 of 27 vs. control: 0 of 30), and a standard microelectrode experiment revealed prolongation of the action potential duration (APD) in the right ventricular papillary muscle (beta: 156 +/- 5 ms vs. control: 131 +/- 4 ms; p < 0.05). Early afterdepolarization (EAD) was observed in one rabbit in the beta group (1 of 26), but not in any animals in the control group (0 of 17). A dose of 100 nmol/l of E-4031 induced EAD in the beta group (10 of 10), but not in the control group, except for one rabbit (1 of 10). The whole-cell, patch-clamp experiment on left ventricular M cells showed significant decreases in transient outward current (I(to1)) (-43%) and slowly activated delayed rectifier current (I(Ks)) densities (-33%), whereas the inward-rectifying K current (I(K1)) and rapidly activated delayed rectifier current (I(Kr)) densities remained unchanged. CONCLUSIONS: Long-term immunization against the second extracellular loop of the beta(1)-adrenergic receptor caused EAD and APD prolongation and decreased the K-channel density, suggesting that an arrhythmic substrate via autoimmune mechanisms is present in cardiomyopathic patients who have autoantibodies directed against the receptors.

Action Potentials↗
PubMed1983

Effects of physostigmine on the voltage dependent ionic conductances of skeletal muscle fibres.

The voltage dependent ionic conductances were studied by analysing the phase plane trajectories of action potentials evoked by electrical stimulation of the sartorius muscles of the frog (Rana esculenta). The delayed outward potassium current was measured also under voltage clamp conditions on muscle fibres of either the frog (Rana esculenta) or Xenopus laevis. On analysing the effect of physostigmine decreasing the peak amplitude, the rate of both the rising and falling phases of the action potentials, it was revealed that the alkaloid at a concentration of 1 mmol/l reduced significantly both the delayed potassium conductance and the outward ionic current values during the action potentials. The inhibition of sodium conductance and inward ionic current was less expressed. The maximum value of delayed potassium conductance measured under voltage clamp conditions was decreased by 1 mmol/l physostigmine. The time constant determined from the development of delayed potassium conductance was increased at a given membrane potential. The voltage vs. n relationship describing the membrane potential dependence of the delayed rectifier was not influenced by physostigmine. It has been concluded that physostigmine changes the time course of the action potentials by decreasing the value of both voltage dependent ionic conductances and by slowing down their kinetics. It is discussed that results obtained from the phase plane analysis of complex pharmacological effects can only be accepted with some restrictions.

Action Potentials↗
PubMed1999

Induction of seizures by the potent K+ channel-blocking scorpion venom peptide toxins tityustoxin-K(alpha) and pandinustoxin-K(alpha).

The scorpion venom peptide toxins tityustoxin-K(alpha) (TsTx-K(alpha)) and pandinustoxin-K(alpha) (PiTx-K(alpha)) are novel, highly potent and selective blockers of voltage-activated K+ channels. PiTx-K(alpha) preferentially blocks rapidly inactivating (A-type) K+ channels whereas TsTx-K(alpha) is selective for slowly inactivating (delayed rectifier-type) channels. K+ channel blockers are known to induce seizures, but the specific K channel types that can serve as convulsant targets are not well defined. To address this issue, we examined for convulsant activity the K+ channel type-specific scorpion toxins and the selective K+ channel antagonists 4-aminopyridine (4-AP), an inhibitor of A-type voltage-activated K+ channels, and paxilline, a selective blocker of large conductance (maxi K) Ca(2+)-activated K+ channels. Intracerebroventricular injection of recombinant TsTx-K(alpha) and PiTx-K(alpha) in mice produced limbic and clonic-tonic seizures. The severity of the seizures increased during the 60-min period following injection, culminating in continuous clonic seizure activity (status epilepticus), tonic hindlimb extension, and eventually in death. The estimated doses producing limbic and clonic seizures in 50% of animals (CD50) for TsTx-K(alpha) and PiTx-K(alpha) were 9 and 33 ng, respectively. 4-AP produced seizure activity similar to the toxins (CD50, 76 ng) whereas paxilline failed to induce seizures at doses up to 13.5 microg. Carbamazepine protected fully against the toxin- and 4-AP-induced seizures whereas phenytoin had variable activity against the clonic component although it was protective against tonic hindlimb extension. The AMPA receptor antagonist GYKI 52466 also conferred full protection against toxin-induced seizures, but the NMDA receptor antagonists (R)-CPP and dizocilpine failed to affect limbic and clonic seizures, although they protected against hindlimb extension. We conclude that selective blockade of delayed rectifier- or A-type voltage-activated K+ channels can produce limbic, clonic and tonic seizures, whereas blockade of maxi K-type Ca(2+)-activated K+ channels does not. The convulsant effects may be related to enhanced glutamate release and, in the case of the limbic and clonic convulsions, activation of AMPA receptors.

4-Aminopyridine↗
PubMed1990

Protein kinase C activation enhances the delayed rectifier potassium current in guinea-pig heart cells.

The possible involvement of protein kinase C in modulating membrane currents was investigated in isolated guinea-pig ventricular cells. In a Na(+)-and K(+)-free external solution, the delayed rectifier K+ current (IK) was increased by the activator of protein kinase C (PKC), 12-O-tetradecanoylphorbol-13-acetate (TPA). The amplitude of the IK tail elicited by a return from a depolarizing pulse for 3 s at + 50 mV to a holding potential of -30 mV was increased by 32 +/- 4% (mean +/- S.E., (n = 6) after the external application of 1 nM TPA, and by 60 +/- 17% (n = 5) after 10 nM. The increase in IK produced by 1 nM TPA was abolished by the inhibitor of PKC, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 10 microM). In addition, the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG, 125 microM) also increased IK (58 +/- 9%, n = 3). PKC purified from bovine brain remarkably increased IK (151 +/- 101%, n = 5) in the presence of 1 nM TPA when it was internally applied using the cell dialysis method. The concentration-response curve of IK for the intracellular concentration of Ca2+ was shifted to the left by 1 nM TPA, suggesting a Ca2(+)-dependent action of PKC and/or altered Ca2(+)-sensitivity of IK channels by phosphorylation. On the other hand, 1 nM TPA had no substantial influence on the Ca2+ current (decreased by 7 +/- 4%, n = 5) or the inward-rectifier K+ current (decreased by 5 +/- 5% in outward component, and 3 +/- 8% in inward component, n = 6). Therefore, the action of PKC was to specifically increase IK without affecting the other two currents.

Animals↗
PubMed1995

Investigation of the mechanism by which ketanserin prolongs the duration of the cardiac action potential.

Action potential duration (APD) lengthening is believed to underlie the cardiac arrhythmogenicity of ketanserin, a serotonin (5-HT)2A/2C receptor antagonist. We wished to determine (a) whether this activity involves blockade of 5-HT2A/2C receptors and (b) the precise mechanism of ketanserin-induced APD prolongation. APs were recorded in guinea pig isolated papillary muscles by conventional "floating" microelectrodes, and potassium currents in guinea pig isolated myocytes were recorded in the whole-cell configuration. Ketanserin (1-10 microM) increased APD (EC50 value for enhancing APD at 90% repolarization (APD90) 3.1 +/- 2.7 microM, n = 24), without affecting resting potential, maximum upstroke velocity (Vmax) or AP amplitude (APA). Pirenperone (10 microM), a ketanserin congener, similarly increased APD90 from 204 +/- 3 to 241 +/- 7 ms (p < 0.001, n = 6). No increase in APD was observed, however, with ritanserin or ICI 170809, even at high concentrations (10 microM, n = 6, respectively), two 5-HT2A/2C receptor antagonists chemically distinct from ketanserin, thereby excluding the involvement of 5-HT2A/2C receptors in mediating APD lengthening. That APD prolongation was mediated specifically by the benzolyl-piperidine moiety of ketanserin and pirenperone was confirmed by 1-propyl-4(4-fluorobenzoyl)piperidine (PFBP), which evoked APD lengthening effects remarkably similar to those produced by ketanserin and pirenperone (EC50 3.73 +/- 2.6 microM, n = 12). In isolated cardiomyocytes, ketanserin (1-32 microM) selectively and concentration-dependently reduced the IKr component of the delayed outward current (IK) without affecting the inward rectifier current, IK1. Thus, ketanserin (32 microM) significantly reduced IK at a potential value of -20 mV from 813 +/- 65 to 569 +/- 55 pA (p < 0.001, n = 6), whereas at a potential value of -110 mV, IK1 was not significantly affected (730 +/- 103 vs. 603 +/- 143 pA, respectively; n=6). The results demonstrate that APD is prolonged by ketanserin and congeners but not be chemically different 5-HT2A/2C receptor antagonists. The benzoyl-piperidine moiety appears to mediate the APD-prolonging effects of ketanserin and pirenperone specifically. Furthermore, ketanserin-induced APD lengthening does not appear to involve 5-HT2A/2C receptors but is consecutive to direct blockade of myocardial potassium channels.

Action Potentials↗
PubMed1996

Rate-dependent effects of sematilide on ventricular monophasic action potential duration and delayed rectifier K+ current in rabbits.

Our objective was to define the actions of sematilide in rabbits and to assess the contribution of the delayed rectifier (IK) to rate dependence of action potential duration (APD) in rabbit ventricular myocardium. In studies in vivo, New Zealand White rabbits were used to obtain dose/response curves of the effects of sematilide on APD from contact monophasic action potentials (MAP), ventricular effective refractory period (VERP), and ECG. Sematilide or placebo was administered as an i.v. bolus followed by a 45-min infusion in the following cumulative manner: infusion 1 (1 mg/kg bolus + 8 micrograms/kg/min); infusion 2 (2 mg/kg + 20 micrograms/kg/min); and infusion 3 (7 mg/kg + 68 micrograms/kg/min). At each infusion level, VERP and APD at 75% repolarization (APD75) were measured during cardiac pacing between 200- and 400-ms cycle length (CL). Serum sematilide levels were analyzed by high-performance liquid chromatography (HPLC). In studies in vitro, sematilide's effects on the delayed rectifier were assessed in isolated rabbit ventricular myocytes by using patch-clamp techniques. Sematilide infusion in vivo resulted in stable serum levels of 1.3 +/- 0.5, 3.7 +/- 1.4, and 13.4 +/- 1.8 micrograms/ml during infusions 1, 2, and 3, respectively. Maximal effects occurred at infusion 2, such that at 400 ms CL, sematilide widened predrug APD75 (145 +/- 5 ms) by 27 +/- 4% (p < 0.001 vs. placebo), and at 200-ms CL, sematilide prolonged predrug APD75 (115 +/- 10 ms) by only 18 +/- 4% (p < 0.001 vs. placebo; p < 0.05 vs. 400-ms CL). Similar effects were observed in VERP. Sematilide enhanced rate dependence of APD and produced the same degree of APD prolongation at a given CL, during accommodation to and recovery from rapid pacing. Rabbit ventricular myocytes appeared to have at least two types of delayed rectifier. Sematilide selectively blocked IKr, and block was not relieved by repetitive stimulation. In conclusion, the APD-widening effect of sematilide was independent of previous pacing history. Sematilide had little influence on background processes likely responsible for shortening APD because of rapid repetitive stimulation.

Action Potentials↗
PubMed1990

Characterization of an early afterhyperpolarization after a brief train of action potentials in rat hippocampal neurons in vitro.

1. In rat hippocampal pyramidal cells in vitro, a brief train of action potentials elicited by direct depolarizing current pulses injected through an intracellular recording electrode is followed by a medium-duration afterhyperpolarization (mAHP) and a longer, slow AHP. We studied the mAHP with the use of current-clamp techniques in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) to block the slow AHP and isolate the mAHP. 2. The mAHP evoked at hyperpolarized membrane potentials was complicated by a potential generated by the anomalous rectifier current, IQ. The mAHP is insensitive to chloride ions (Cl-), whereas it is sensitive to the extracellular potassium concentration ([K+]o). 3. At slightly depolarized levels, the mAHP is partially Ca2+ dependent, being enhanced by increased [Ca2+]o and BAY K 8644 and depressed by decreased [Ca2+]o, nifedipine, and Cd2+. The Ca2(+)-dependent component of the mAHP was also reduced by 100 microM tetraethylammonium (TEA) and charybdotoxin (CTX), suggesting it is mediated by the voltage- and Ca2(+)-dependent K+ current, IC. 4. Most of the Ca2(+)-independent mAHP was blocked by carbachol, implying that IM plays a major role. In a few cells, a small Ca2(+)- and carbachol-insensitive mAHP component was detectable, and this component was blocked by 10 mM TEA, suggesting it was mediated by the delayed rectifier current, IK. The K+ channel antagonist 4-aminopyridine (4-AP, 500 microM) did not reduce the mAHP. 5. We infer that the mAHP is a complex potential due either to IQ or to the combined effects of IM and IC. The contributions of each current depend on the recording conditions, with IC playing a role when the cells are activated from depolarized potentials and IM dominating at the usual resting potential. IQ is principally responsible for the mAHP recorded at hyperpolarized membrane potentials.

Action Potentials↗
PubMed2000

Effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes.

BDF 9198 (a congener of DPI 201 - 106 and BDF 9148) was found to be a positive inotrope on guinea-pig isolated ventricular muscle strips. The effects of BDF 9198 on action potentials and ionic currents from guinea-pig isolated ventricular myocytes were studied using the whole cell patch clamp method. In normal external solution, at 37 degrees C, action potential duration at 50% repolarization (APD(50)) was 167.4+/-8.36 ms (n=37). BDF 9198 produced a concentration-dependent increase in APD(50) (no significant increase at 1x10(-10) M; and APD(50) values of 273.03+/-35.8 ms at 1x10(-9) M; n=6, P<0.01 and 694.7+/-86.3 ms at 1x10(-7) M; P<0.001, n=7). At higher concentrations in the range tested, BDF 9198 also induced early and delayed and after-depolarizations. Qualitative measurements of I(Na) with physiological [Na](o) showed prolongation of the current by BDF 9198, and the appearance of transient oscillatory inward currents at high concentrations. Quantitative recording conditions for I(Na) were established using low external [Na] and by making measurements at room temperature. The current - voltage relation, activation parameters and time-course of I(Na) were similar before and after a partial blocking dose of Tetrodotoxin (TTX, 1 microM), despite a 2 fold difference in current amplitude. This suggests that voltage-clamp during flow of I(Na) was adequately maintained under our conditions. Selective measurements of I(Na) at room temperature showed that BDF 9198 induced a concentration-dependent, sustained component of I(Na) (I(Late)) and caused a slight left-ward shift in the current - voltage relation for peak current. The drug-induced I(Late) showed a similar voltage dependence to peak current in the presence of BDF 9198. Both peak current and I(Late) were abolished by 30 microM TTX and were sensitive to external [Na]. Inactivation of control I(Na) during a 200 ms test pulse to -30 mV followed a bi-exponential time-course. In addition to inducing a sustained current component, BDF 9198 left the magnitude of the fast inactivation time-constant unchanged, but increased the magnitude of the slow inactivation time-constant. Additional experiments with a longer pulse (1 s) raised the possibility that in the presence of BDF 9198, I(Na) inactivation may be comprised of more than two phases. No significant effects of 1x10(-6) M BDF 9198 were observed on the L-type calcium current, or delayed and inward rectifying potassium currents measured at 37 degrees C. It is concluded that the prolongation of APD(50) by BDF 9198 resulted from selective modulation of I(Na). Reduced current inactivation induced a persistent I(Na), increasing the net depolarizing current during the action potential. This action of the drug indicates a potential for 'QT prolongation' of the ECG. The observation of after-depolarizations suggests a potential for proarrhythmia at some drug concentrations.

Action Potentials↗
PubMed1994

External blockade of the major cardiac delayed-rectifier K+ channel (Kv1.5) by polyunsaturated fatty acids.

The present work shows that arachidonic acid and some other long chain polyunsaturated fatty acids such as docosahexaenoic acid, which is abundant in fish oil, produce a direct open channel block of the major voltage-dependent K+ channel (Kv1.5) cloned in cardiac cells. The inhibitory action of these selected fatty acids is seen when they are applied extracellularly but not when they are included in the patch pipette. Fatty acids then appear to bind to an external site on the Kv1.5 channel structure. Inhibition of Kv1.5 channel activity by polyunsaturated fatty acids (acceleration of the apparent inactivation and decrease of the peak current) is similar to that produced by the class III antiarrhythmic tedisamil. Docosahexaenoic acid and arachidonic acid also inhibit the delayed-rectifier K+ channel currents in cultured mouse and rat cardiomyocytes. These results are discussed in the light of the reported fatty acids effects on cardiac function in diseased states. Since Kv1.5 is also present in the brain, the results reported here could also have a significance in terms of processes such as long-term potentiation or depression.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1982

Effects of nystatin-mediated intracellular ion substitution on membrane currents in calf purkinje fibres.

1. Calf cardiac Purkinje fibres were exposed briefly to the ionophore nystatin to promote exchange of caesium for intracellular potassium. The effects of Cs loading were stable for at least 30 min, but they could be reversed by nystatin-mediated K loading.2. After Cs loading, the resting potential shifted to about -20 mV and the current-voltage relationship showed a strong inhibition of inwardly rectifying K channels.3. Anodal break stimulation evoked normal action potential upstrokes and twitch contractions. The early repolarization (phase 1) was markedly slowed.4. Cs loading simplified the pattern of current changes evoked by step depolarizations over the plateau range. Membrane current reached an inward peak and then declined monotonically.5. The current signal showed no hint of the transient outward current found in untreated or K-loaded preparations. Furthermore, Cs loading abolished the outward tails associated with deactivation of transient outward current, and occluded the blocking effect of the K-channel inhibitor 4-aminopyridine.6. Inhibition of transient outward current revealed a maximal inward current of about 5 muA/muF in 5.4 mM-Ca(o), which is considerably larger than the net inward current without Cs loading.7. The inward current was attributed to Ca channels on the basis of its sensitivity to membrane potential, extracellular Ca, D600, Mn and Cd.8. Cs loading also reduced slow current changes associated with delayed rectification and pace-maker depolarization.9. The results support the hypothesis that the transient outward current is carried by K(+) ions, while providing a method for unmasking inward Ca current.

Action Potentials↗
PubMed1993

Myosin light chain kinase occurs in bullfrog sympathetic neurons and may modulate voltage-dependent potassium currents.

A polyclonal antibody against myosin light chain kinase (MLCK) of chicken gizzard recognized a 130 kd peptide of bullfrog sympathetic ganglia as MLCK. MLCK immunoreactivity was confined to the neuronal cell body. A synthetic peptide corresponding to an inhibitory domain of MLCK (Ala783-Gly804) was applied intracellularly to isolated sympathetic neurons during whole-cell recordings of ionic currents. The peptide inhibitor reversibly decreased M-type potassium current (IM) while not affecting A-type of delayed rectifier-type potassium currents. Intracellular application of an active fragment of MLCK enhanced IM, whereas application of an inactive MLCK fragment did not. The results suggest that IM can be modulated by MLCK-catalyzed phosphorylation.

Amino Acid Sequence↗
PubMed1994

Injection of a K+ channel (Kv1.3) cRNA in fertilized eggs leads to functional expression in cultured myotomal muscle cells from Xenopus embryos.

The synthetic cRNA encoding for the major T lymphocyte K+ channel (Kv1.3) was injected into Xenopus fertilized eggs. Somites from embryos of stage 20-22 (about 40 h post-fertilization at 19 degrees C) were dissociated and myotomal muscle cells were cultured in vitro for 2 days. The whole cell configuration of the tight seal patch-clamp technique was used to record K+ channel activity in cultured myocytes. These myocytes have two endogenous delayed-rectifiers (sustained and transient) and an inward-rectifier K+ currents, all of which are insensitive to the scorpion toxin charybdotoxin. Cultured myocytes dissociated from embryos injected with the Kv1.3 cRNA expressed the exogenous Kv1.3 channel. The Kv1.3 channel was identified by its physiological (a very low recovery from inactivation) and its pharmacological properties (a high sensitivity to charybdotoxin). This work demonstrates that Xenopus cultured myotomal muscle cells represent a very efficient and practical assay system for the functional expression of cloned ion channels.

Animals↗
PubMed1993

Differential modulation of voltage-dependent currents in Hermissenda type B photoreceptors by serotonin.

1. In identified photoreceptors in the eyes of Hermissenda, serotonin (5-HT) enhances the peak and plateau phases of light-evoked generator potentials and modulates light-dependent and voltage-dependent currents. In addition, electrophysiological and morphological studies indicate that 5-HT may contribute to cellular plasticity detected in the visual system of Hermissenda produced by classical conditioning procedures. With the use of conventional two-electrode voltage-clamp techniques, we examined the effects of 5-HT on three distinct currents recorded across the soma membranes of identified lateral and medial type B photoreceptors in the isolated circumesophageal nervous system. 2. The inward rectifier (Iir), a putative K(+)-dependent conductance, activates in 0-Na artificial seawater at membrane potentials greater than -60 mV, is voltage dependent, noninactivating, and reaches steady-state within 800 ms to 3 s at -100 mV. Bath application of 10(-4) M 5-HT consistently enhanced the magnitude of Iir at all potentials tested (-60 to -100 mV) and, in some cases, allowed expression of Iir, which was not initially detectable before the application of 5-HT. 3. The major component of outward K+ current in type B photoreceptors with IA and IK(Ca) blocked is the delayed rectifier [IK(v)]. 5-HT (10(-4) M) produced both an increase as well as a decrease in the peak amplitude of IK(v) and consistently slowed its inactivation rate and reduced twin-pulse inactivation. 4. A previously identified outward K+ current in type B photoreceptors has properties that are similar to the A current (IA).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗