Michael E. Bräu

Impact of TASK-1 in Human Pulmonary Artery Smooth Muscle Cells

The excitability of pulmonary artery smooth muscle cells (PASMC) is regulated by potassium (K+) conductances. Although studies suggest that background K+ currents carried by 2-pore domain K+ channels are important regulators of resting membrane potential in PASMC, their role in human PASMC is unknown. Our study tested the hypothesis that TASK-1 leak K+ channels contribute to the K+ current and resting membrane potential in human PASMC. We used the whole-cell patch-clamp technique and TASK-1 small interfering RNA (siRNA). Noninactivating K+ current performed by TASK-1 K+ channels were identified by current characteristics and inhibition by anandamide and acidosis (pH 6.3), each resulting in significant membrane depolarization. Moreover, we showed that TASK-1 is blocked by moderate hypoxia and activated by treprostinil at clinically relevant concentrations. This is mediated via protein kinase A (PKA)-dependent phosphorylation of TASK-1. To further confirm the role of TASK-1 channels in regulation of resting membrane potential, we knocked down TASK-1 expression using TASK-1 siRNA. The knockdown of TASK-1 was reflected by a significant depolarization of resting membrane potential. Treatment of human PASMC with TASK-1 siRNA resulted in loss of sensitivity to anandamide, acidosis, alkalosis, hypoxia, and treprostinil. These results suggest that (1) TASK-1 is expressed in human PASMC; (2) TASK-1 is hypoxia-sensitive and controls the resting membrane potential, thus implicating an important role for TASK-1 K+ channels in the regulation of pulmonary vascular tone; and (3) treprostinil activates TASK-1 at clinically relevant concentrations via PKA, which might represent an important mechanism underlying the vasorelaxing properties of prostanoids and their beneficial effect in vivo.

Effect of drugs used for neuropathic pain management on tetrodotoxin-resistant Na(+) currents in rat sensory neurons.

BackgroundTetrodotoxin-resistant Na+ channels play an important role in generation and conduction of nociceptive discharges in peripheral endings of small-diameter axons of the peripheral nervous system. Pathophysiologically, these channels may produce ectopic discharges in damaged nociceptive fibers, leading to neuropathic pain syndromes. Systemically applied Na+ channel–blocking drugs can alleviate pain, the mechanism of which is rather unresolved. The authors investigated the effects of some commonly used drugs, i.e., lidocaine, mexiletine, carbamazepine, amitriptyline, memantine, and gabapentin, on tetrodotoxin-resistant Na+ channels in rat dorsal root ganglia. MethodsTetrodotoxin-resistant Na+ currents were recorded in the whole-cell configuration of the patch-clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Half-maximal blocking concentrations were derived from concentration–inhibition curves at different holding potentials (−90, −70, and −60 mV). ResultsLidocaine, mexiletine, and amitriptyline reversibly blocked tetrodotoxin-resistant Na+ currents in a concentration- and use-dependent manner. Block by carbamazepine and memantine was not use-dependent at 2 Hz. Gabapentin had no effect at concentrations of up to 3 mm. Depolarizing the membrane potential from −90 mV to −60 mV reduced the available Na+ current only by 23% but increased the sensitivity of the channels to the use-dependent blockers approximately fivefold. The availability curve of the current was shifted by 5.3 mV to the left in 300 &mgr;m lidocaine. ConclusionsLess negative membrane potential and repetitive firing have little effect on tetrodotoxin-resistant Na+ current amplitude but increase their sensitivity to lidocaine, mexiletine, and amitriptyline so that concentrations after intravenous administration of these drugs can impair channel function. This may explain alleviation from pain by reducing firing frequency in ectopic sites without depressing central nervous or cardiac excitability.

Fundamental properties of local anesthetics: half-maximal blocking concentrations for tonic block of Na+ and K+ channels in peripheral nerve.

Local anesthetics suppress excitability by interfering with ion channel function.Ensheathment of peripheral nerve fibers, however, impedes diffusion of drugs to the ion channels and may influence the evaluation of local anesthetic potencies. Investigating ion channels in excised membrane patches avoids these diffusion barriers. We investigated the effect of local anesthetics with voltage-dependent Na+ and K+ channels in enzymatically dissociated sciatic nerve fibers of Xenopus laevis using the patch clamp method. The outside-out configuration was chosen to apply drugs to the external face of the membrane. Local anesthetics reversibly blocked the transient Na+ inward current, as well as the steady-state K+ outward current. Half-maximal tonic inhibiting concentrations (IC50), as obtained from concentration-effect curves for Na+ current block were: tetracaine 0.7 [micro sign]M, etidocaine 18 [micro sign]M, bupivacaine 27 [micro sign]M, procaine 60 [micro sign]M, mepivacaine 149 [micro sign]M, and lidocaine 204 [micro sign]M. The values for voltage-dependent K+ current block were: bupivacaine 92 [micro sign]M, etidocaine 176 [micro sign]M, tetracaine 946 [micro sign]M, lidocaine 1118 [micro sign]M, mepivacaine 2305 [micro sign]M, and procaine 6302 [micro sign]M. Correlation of potencies with octanol:buffer partition coefficients (logP0) revealed that ester-bound local anesthetics were more potent in blocking Na+ channels than amide drugs. Within these groups, lipophilicity governed local anesthetic potency. We conclude that local anesthetic action on peripheral nerve ion channels is mediated via lipophilic drug-channel interactions. Implications: Half-maximal blocking concentrations of commonly used local anesthetics for Na+ and K+ channel block were determined on small membrane patches of peripheral nerve fibers. Because drugs can directly diffuse to the ion channel in this model, these data result from direct interactions of the drugs with ion channels. (Anesth Analg 1998;87:885-9)

A K+ channel in Xenopus nerve fibres selectively blocked by bee and snake toxins: binding and voltage-clamp experiments.

1. The effects of mast cell degranulating peptide (MCDP), a toxin from the honey bee, and of dendrotoxin (DTX), a toxin from the green mamba snake, were studied in voltage‐clamp experiments with myelinated nerve fibres of Xenopus. 2. MCDP and DTX blocked part of the K+ current. About 20% of the K+ current, however, was resistant to the toxins even in high concentrations. In Ringer solution half‐maximal block was reached with concentrations of 33 nM‐MCDP and 11 nM‐DTX. In high‐K+ solution the potency of both toxins was lower. beta‐Bungarotoxin (beta‐BuTX), another snake toxin, also blocked part of the K+ current, but was less potent than MCDP and DTX. 3. Tail currents in high‐K+ solution were analysed and three K+ current components were separated according to Dubois (1981 b). Both MCDP and DTX selectively blocked a fast deactivating, slowly inactivating K+ current component which steeply activates between E = ‐60 mV and E = ‐40 mV (component f1). In concentrations around 100 nM, MCDP and DTX blocked neither the slow K+ current (component s) nor the fast deactivating, rapidly inactivating K+ current which activates between E = ‐40 mV and E = 20 mV (component f2). Similar results could be derived from K+ outward currents in Ringer solution. In high‐K+, IC50 of MCDP for component f1 was 99 nM, whereas it was 7.6 microM for f2. Corresponding values for DTX are 68 nM and 1.8 microM. 4. Binding studies with nerve fibre membranes of Xenopus reveal high‐affinity binding sites for 125I‐labelled DTX (KD = 22 pM in Ringer solution and 81 pM in high‐K+ solution). 125I‐labelled DTX can be displaced from its sites completely by unlabelled DTX, toxin I (black mamba toxin), MCDP, and partially by beta‐BuTX. 5. Immunocytochemical staining demonstrates that binding sites for DTX are present in nodal and paranodal regions of the axonal membrane. 6. The axonal membrane of motor and sensory nerve fibres is equipped with three types of well‐characterized K+ channels and constitutes so far the best preparation to study MCDP‐ and DTX‐sensitive K+ channels with electrophysiological and biochemical methods.

Blocking Mechanisms of Ketamine and Its Enantiomers in Enzymatically Demyelinated Peripheral Nerve as Revealed by Single-channel Experiments

Background Ketamine shows, besides its general anesthetic effect, a local anesthetic‐like action that is due to blocking of peripheral nerve sodium currents. In this study, the stereoselectivity of the blocking effects of the ketamine enantiomers S(+) and R(‐) was investigated in sodium and potassium channels in peripheral nerve membranes. Methods Ion channel blockade of ketamine was investigated in enzymatically dissociated Xenopus sciatic nerves in multiple‐channel and in single‐channel outside‐out patches. Results Concentration‐effect curves for the Na+ peak current revealed half‐maximal inhibiting concentrations (IC50) of 347 micro Meter and 291 micro Meter for S(+) and R(‐) ketamine, respectively. The potential‐dependent K+ current was less sensitive than the Na+ current with IC50 values of 982 micro Meter and 942 micro Meter. The most sensitive ion channel was the flickering background K+ channel, with IC50 values of 168 micro Meter and 146 micro Meter for S(+) and R(‐) ketamine. Competition experiments suggest one binding site at the flicker K+ channel, with specific binding affinities for each of the enantiomers. For the Na+ channel, the block was weaker in acidic (pH = 6.6) than in neutral (pH = 7.4) and basic (pH = 8.2) solutions; for the flicker K+ channel, the block was weaker in acidic and stronger in basic solutions. Conclusions Ketamine blockade of sodium and potassium channels in peripheral nerve membranes shows no stereoselectivity except for the flicker K+ channel, which showed a very weak stereoselectivity in favor of the R(‐) form. This potential‐insensitive flicker K+ channel may contribute to the resting potential. Block of this channel and subsequent depolarization of the resting membrane potential leads, besides to direct Na+ channel block, to inexcitability via Na+ channel inactivation.

Ketamine impairs excitability in superficial dorsal horn neurones by blocking sodium and voltage-gated potassium currents

Ketamine shows, besides its general anaesthetic effect, a potent analgesic effect after spinal administration. We investigated the local anaesthetic‐like action of ketamine and its enantiomers in Na+ and K+ channels and their functional consequences in dorsal horn neurones of laminae I–III, which are important neuronal structures for pain transmission receiving most of their primary sensory input from Aδ and C fibres. Combining the patch‐clamp recordings in slice preparation with the ‘entire soma isolation’ method, we studied action of ketamine on Na+ and voltage‐activated K+ currents. The changes in repetitive firing behaviour of tonically firing neurones were investigated in current‐clamp mode after application of ketamine. Concentration–effect curves for the Na+ peak current revealed for tonic block half‐maximal inhibiting concentrations (IC50) of 128 μM and 269 μM for S(+) and R(−)‐ketamine, respectively, showing a weak stereoselectivity. The block of Na+ current was use‐dependent. The voltage‐dependent K+ current (KDR) was also sensitive to ketamine with IC50 values of 266 μM and 196 μM for S(+) and R(−)‐ketamine, respectively. Rapidly inactivating K+ currents (KA) were less sensitive to ketamine. The block of KDR channels led to an increase in action potential duration and, as a consequence, to lowering of the discharge frequency in the neurones. We conclude that ketamine blocks Na+ and KDR channels in superficial dorsal horn neurones of the lumbar spinal cord at clinically relevant concentrations for local, intrathecal application. Ketamine reduces the excitability of the neurones, which may play an important role in the complex mechanism of its action during spinal anaesthesia.

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

SummaryA novel potassium-selective channel which is active at membrane potentials between — 100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mm-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mm-K and 17 pS with 2.5 mm-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (>0.2, but often >0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (τo= 0.09 msec), closed times with the sum of three exponentials (τc= 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mm). External Cs ions reduced the apparent unitary conductance for inward currents at Em= −90 mV (IC50 = 1.1 mm). Ba and, more potently, Zn ions lowered not only the apparent singlechannel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nm). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.

ATP-Dependent Potassium Channel in Rat Cardiomyocytes Is Blocked by Lidocaine Possible Impact on the Antiarrhythmic Action of Lidocaine

BACKGROUND During myocardial ischemia, lidocaine has favorable antiarrhythmic properties. Malignant arrhythmias result from heterogeneity between ischemic and nonischemic regions in extracellular potassium concentration and action potential duration. These effects have been attributed to the activation of ATP-dependent potassium (KATP) channels. In this study, we investigated the action of lidocaine on the KATP channels to test the possible link between the antiarrhythmic properties of lidocaine and its action on the KATP channel. METHODS AND RESULTS The patch-clamp technique was employed on enzymatic dissociated cardiomyocytes of adult rats. Lidocaine was applied to the outer side of excised membrane patches by means of a multibarrel perfusion system. Lidocaine reversibly blocked the mean current of the KATP channels in a concentration-dependent manner (IC50 = 43 +/- 4.7 mumol/L, E = 0 mV, n = 6), while the amplitude of the single-channel current remained unchanged. The half-maximum blocking concentration corresponds to the therapeutic range for the antiarrhythmic application of a lidocaine bolus in humans. CONCLUSIONS The open probability but not the conductance of the KATP channel in the membrane of rat cardiomyocytes is blocked by lidocaine. This action may explain, in part, the favorable antiarrhythmic properties of lidocaine during acute myocardial ischemia.

stereoselectivity of Bupivacaine in Local Anesthetic–sensitive Ion Channels of Peripheral Nerve

BACKGROUND The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)-and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel. METHODS In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k(-1)) were determined. RESULTS Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/- SEM) for tonic Na+ channel block were 29+/-3 microM and 44+/-3 microM, respectively. IC50 values for flicker K+ channel block were 0.15+/-0.02 microM and 11+/-1 microM, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k(-1) were 0.83+/-0.13x10(6) M(-1) x S(-1) and 0.13+/-0.03 s(-1), respectively, for R(+)-bupivacaine and 1.90+/-0.20x10(6) M(-1) x s(-1) and 8.3+/-1.0 s(-1), respectively, for S(-)-bupivacaine. CONCLUSIONS Bupivacaine block of Na+ channels shows no salient stereoselectivity. Block of flicker K+ channels has the highest stereoselectivity ratio of bupivacaine action known so far. This stereoselectivity derives predominantly from a difference in k(-1), suggesting a tight fit between R(+)-bupivacaine and the binding site. The flicker K+ channel may play an important role in yet unknown toxic mechanisms of R(+)-bupivacaine.

Block of Neuronal Tetrodotoxin-resistant Na+ Currents by Stereoisomers of Piperidine Local Anesthetics

Tetrodotoxin (TTX)-sensitive Na+ channels in the peripheral nervous system are the major targets for local anesthetics. In the peripheral nociceptive system, a Na+ channel subtype resistant to TTX and with distinct electrophysiological properties seems to be of importance for impulse generation and conduction. A current through TTX-resistant Na+ channels displays slower activation and inactivation kinetics and has an increased activation threshold compared with TTX-sensitive Na+ currents and may have different pharmacological properties. We studied the effects of stereoisomers of piperidine local anesthetics on neuronal TTX-resistant Na+ currents recorded with the whole-cell configuration of the patch clamp method in enzymatically dissociated dorsal root ganglion neurons of adult rats. Stereoisomers of mepivacaine, ropivacaine, and bupivacaine reversibly inhibited TTX-resistant Na+ currents in a concentration and use-dependent manner. All drugs accelerated time course of inactivation. Half-maximal blocking concentrations were determined from concentration-inhibition relationships. Potencies for tonic and for use-dependent block increased with rising lipid solubilities of the drugs. Stereoselective action was not observed. We conclude that block of TTX-resistant Na+ currents may lead to blockade of TTX-resistant action potentials in nociceptive fibers and consequently may be responsible for pain suppression during local anesthesia. Implications Tetrodotoxin-resistant Na+ channels are important in peripheral nociception. During local anesthesia, these channels are blocked by mepivacaine, ropivacaine, and bupivacaine in a concentration and use-dependent manner, but not stereoselectively.