Andreas Scholz

Properties and functions of calcium-activated K+ channels in small neurones of rat dorsal root ganglion studied in a thin slice preparation

1 Properties, kinetics and functions of large conductance calcium‐activated K+ channels (BKCa) were investigated by the patch‐clamp technique in small neurones (Aδ‐ and C‐type) of a dorsal root ganglion (DRG) thin slice preparation without enzymatic treatment. 2 Unitary conductance of BKCa channels measured in symmetrical high K+ solutions (155 mm) was 200 pS for inward currents, and chord conductance in control solution was 72 pS. Potentials of half‐maximum activation (V1/2) of the channels were linearly shifted by 43 mV per log10[Ca2+]i unit (pCa) in the range of −28 mV (pCa 4) to +100 mV (pCa 7). Open probabilities increased e‐times per 15–32 mV depolarization of potential. 3 In mean open probability, fast changes with time were mainly observed at pCa > 6 and at potentials > +20 mV, without obvious changes in the experimental conditions. 4 BKCa channels were half‐maximally blocked by 0.4 mm TEA, measured by apparent amplitude reductions. They were completely blocked by 100 nm charybdotoxin and 50 nm iberiotoxin by reduction of open probability. 5 Two subtypes of small DRG neurones could be distinguished by the presence (type I) or absence (type II) of BKCa channels. In addition, less than 10 % of small neurones showed fast (∼135 V s−1) and short (∼0.8 ms) action potentials (AP). 6 The main functions of BKCa channels were found to be shortening of AP duration, increasing of the speed of repolarization and contribution to the fast after‐hyperpolarization. As a consequence, BKCa channels may reduce the amount of calcium entering a neurone during an AP. 7 BKCa channel currents suppressed a subsequent AP and prolonged the refractory period, which might lead to a reduced repetitive activity. We suggest that the BKCa current is a possible mechanism of the reported conduction failure during repetitive stimulation in DRG neurones.

Human axons contain at least five types of voltage-dependent potassium channel

1 We investigated voltage‐gated potassium channels in human peripheral myelinated axons; apart from the I, S and F channels already described in amphibian and rat axons, we identified at least two other channel types. 2 The I channel activated between ‐70 and ‐40 mV, and inactivated very slowly (time constant 13.1 s at ‐40 mV). It had two gating modes: the dominant (‘noisy’) mode had a conductance of 30 pS (inward current, symmetrical 155 mM K+) and a deactivation time constant (τ) of 25 ms (‐80 mV); it accounted for most (≈50‐75 %) of the macroscopic K+ current in large patches. The secondary (‘flickery’) gating mode had a conductance of 22 pS, and showed bi‐exponential deactivation (τ= 16 and 102 ms; ‐80 mV); it contributed part of the slow macroscopic K+ current. 3 The I channel current was blocked by 1 μM α‐dendrotoxin (DTX); we also observed two other DTX‐sensitive K+ channel types (40 pS and 25 pS). The S and F channels were not blocked by 1 μM DTX. 4 The conductance of the S channel was 7‐10 pS, and it activated at slightly more negative potentials than the I channel; its deactivation was slow (τ= 41.7 ms at ‐100 mV). It contributed a second component of the slow macroscopic K+ current. 5 The F channel had a conductance of 50 pS; it activated at potentials between ‐40 and +40 mV, deactivated very rapidly (τ= 1.4 ms at ‐100 mV), and inactivated rapidly (τ= 62 ms at +80 mV). It accounted for the fast‐deactivating macroscopic K+ current and partly for fast K+ current inactivation. 6 We conclude that human and rat axonal K+ channels are closely similar, but that the correspondence between K+ channel types and the macroscopic currents usually attributed to them is only partial. At least five channel types exist, and their characteristics overlap to a considerable extent.

Two types of TTX‐resistant and one TTX‐sensitive Na+ channel in rat dorsal root ganglion neurons and their blockade by halothane

The clinically employed general anaesthetic halothane was shown to exert action on the peripheral nervous system by suppressing spinal reflexes, but it is still unclear which mechanisms underlie this action. The present study addressed the question whether blockade of tetrodotoxin‐sensitive (TTXs) and ‐resistant (TTXr) Na+‐channels in rat dorsal root ganglia (DRG) neurons by halothane could explain its peripheral effects. Two types of TTXr Na+‐currents, fast and slow, with distinct activation and inactivation kinetics were found in small (< 25 μm) and medium sized (25–40 μm) DRG neurons. These currents were blocked by halothane with IC50 values of 5.4 and 7.4 mmol/L, respectively. Additionally, in a concentration‐dependent manner halothane accelerated the inactivation kinetics of both currents and shifted the inactivation curves to more hyperpolarized potentials. Neither the activation curves of both TTXr Na+‐currents were influenced by halothane nor a voltage‐dependent block at test potentials of the currents was seen. In contrast to that of fast current, the time‐to‐peak for slow current was changed in the presence of halothane. The TTXs Na+‐current which prevailed in large neurons (> 40 μm) was blocked by halothane with an IC50 of 12.1 mmol/L. Its inactivation curve was also shifted to more hyperpolarized potentials and the inactivation kinetics accelerated with increasing halothane concentration. Similarly to TTXr Na+‐currents, the activation curve of TTXs Na+‐current and its time‐to‐peak were not influenced by halothane. It is suggested that two types of TTXr Na+‐currents can explain the heterogeneity in kinetic data for TTXr Na+‐currents. Furthermore, the incomplete blockade of Na+‐currents might underlie the incomplete reduction of spinal reflexes at clinically used concentrations of halothane.

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.

Tetrodotoxin-resistant action potentials in dorsal root ganglion neurons are blocked by local anesthetics.

&NA; Evidence from animal models and studies of human sensory nerves demonstrate that tetrodotoxin (TTX)‐resistant Na+ channels are present in sensory neurons and might play an important role in pain conduction and chronic pain. Recent investigations suggest that TTX‐resistant Na+ channels in the peripheral nervous system are less sensitive to local anesthetics than TTX‐sensitive Na+ channels. To test the effects of the clinically used local anesthetics lidocaine and bupivacaine on TTX‐resistant action potentials (APs) in sensory neurons, we performed electrophysiological experiments on small dorsal root ganglion (DRG) neurons from young rats. Amplitudes, time to peak and duration of TTX‐resistant APs were measured in A&dgr;‐ and C‐type neurons using the patch‐clamp technique in a thin slice preparation (150 &mgr;m), thus avoiding enzymatic treatment. With increasing concentrations of the local anesthetics, the AP amplitude was gradually reduced but the AP did not disappear abruptly. The concentrations needed to reduce the amplitudes of TTX‐resistant APs by half were 760 &mgr;M for lidocaine and 110 &mgr;M for bupivacaine. Time to peak and duration of TTX‐resistant APs were prolonged by local anesthetics. Trains of APs could be elicited in some neurons by long‐lasting current injections, and the half‐maximal concentrations needed to suppress these trains were 30 &mgr;M lidocaine or 10 &mgr;M bupivacaine. We suggest that the reduction in firing frequency at low concentrations of local anesthetic may explain the phenomenon of paresthesia when sensory information is gradually suppressed during spinal anesthesia.

Ethanol reduces excitability in a subgroup of primary sensory neurons by activation of BKCa channels

Ethanol effects on the central nervous system have been well investigated and described in recent years; modulations, by ethanol, of several ligand‐gated and voltage‐gated ion channels have been found. In this paper, we describe a shortening of action potential duration (APD) by ethanol in ≈ 40% of small diameter neurons in rat dorsal root ganglia (DRG). In these neurons, designated as group A neurons, we observed an ethanol‐induced increase in whole‐cell outward‐current. As iberiotoxin, a specific blocker of large‐conductance calcium‐activated K+ channels (BKCa channels), blocks the effects of ethanol, we investigated the interaction between these channels and ethanol in outside‐out patches. Open probability of BKCa channels was increased 2–6 × depending on the concentration (40–80 mm≈ 2–4‰ v/v) of ethanol. Functional consequences were a prolongation of the refractory period, which was reversible after addition of iberiotoxin, and reduced firing frequency during ethanol application. In contrast, another type of neuron (group B) showed a prolonged APD during application of ethanol which was irreversible in most cases. In 90% of cases, neurons of group A showed a positive staining for isolectin B4 (I‐B4), a marker for nociceptive neurons. We suggest that the activation of BKCa channels induced by clinically relevant concentrations of ethanol, the resulting modulations of APD and refractory period of DRG neurons, might contribute to clinically well‐known ethanol‐induced analgesia and paresthesia.

Cooling inhibits capsaicin-induced currents in cultured rat dorsal root ganglion neurones.

Whole-cell and single-channel recordings from rat dorsal root ganglion neurones were used to investigate the temperature dependence of currents through the capsaicin receptor (vanilloid receptor 1, VR1). Reducing the temperature from 31 to 14 degrees C inhibited the current induced by 0.5 microM capsaicin by 80%. The Q(10) (temperature coefficient over a 10 degrees C range) of the whole-cell capsaicin-induced current was 2.3 between 10 and 30 degrees C. Single-channel recordings showed that this inhibition by cooling was due to a marked reduction in the open probability (Q(10)=8.2 between 10 and 30 degrees C). This effect can explain the pain relief and reduction in inflammation caused by strong cooling of the skin.

Moderate hypoxia influences excitability and blocks dendrotoxin sensitive K+ currents in rat primary sensory neurones

Hypoxia alters neuronal function and can lead to neuronal injury or death especially in the central nervous system. But little is known about the effects of hypoxia in neurones of the peripheral nervous system (PNS), which survive longer hypoxic periods. Additionally, people have experienced unpleasant sensations during ischemia which are dedicated to changes in conduction properties or changes in excitability in the PNS. However, the underlying ionic conductances in dorsal root ganglion (DRG) neurones have not been investigated in detail.Therefore we investigated the influence of moderate hypoxia (27.0 ± 1.5 mmHg) on action potentials, excitability and ionic conductances of small neurones in a slice preparation of DRGs of young rats. The neurones responded within a few minutes non-uniformly to moderate hypoxia: changes of excitability could be assigned to decreased outward currents in most of the neurones (77%) whereas a smaller group (23%) displayed increased outward currents in Ringer solution. We were able to attribute most of the reduction in outward-current to a voltage-gated K+ current which activated at potentials positive to -50 mV and was sensitive to 50 nM α-dendrotoxin (DTX). Other toxins that inhibit subtypes of voltage gated K+ channels, such as margatoxin (MgTX), dendrotoxin-K (DTX-K), r-tityustoxin Kα (TsTX-K) and r-agitoxin (AgTX-2) failed to prevent the hypoxia induced reduction. Therefore we could not assign the hypoxia sensitive K+ current to one homomeric KV channel type in sensory neurones. Functionally this K+ current blockade might underlie the increased action potential (AP) duration in these neurones. Altogether these results, might explain the functional impairment of peripheral neurones under moderate hypoxia.

Hypoxic increase in nitric oxide generation of rat sensory neurons requires activation of mitochondrial complex II and voltage-gated calcium channels

Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide (NO) synthase (eNOS) resulting in enhanced NO production associated with mitochondria which contributes to resistance against hypoxia. Extracellular calcium is essential to this effect. In the present study on rat DRG slices, we set out to determine what types of calcium channels operate under hypoxia, and which upstream events contribute to their activation, thereby focusing upon mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit voltage-gated calcium channels and T-type channels, respectively, and verapamil and nifedipine, typical blocker of L-type calcium channels completely prevented the hypoxic neuronal NO generation. Inhibition of complex II by thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic acid at the substrate binding site largely diminished hypoxic-induced NO production while having an opposite effect under normoxia. An additional blockade of voltage-gated calcium channels entirely abolished the hypoxic response. The complex II inhibitor malonate inhibited both normoxic and hypoxic NO generation. These data show that complex II activity is required for increased hypoxic NO production. Since succinate dehydrogenase activity of complex II decreased at hypoxia, as measured by histochemistry and densitometry, we propose a hypoxia-induced functional switch of complex II from succinate dehydrogenase to fumarate reductase, which subsequently leads to activation of voltage-gated calcium channels resulting in increased NO production by eNOS.

Lidocaine and octanol have different modes of action at tetrodotoxin-resistant Na(+) channels of peripheral nerves.

Local anesthetics and alcohols block impulse conduction in peripheral nerves by inhibiting Na+ currents. In small peripheral nerve fibers, tetrodotoxin-resistant (TTX-r) Na+ channels play an important role in impulse generation. We investigated the effects of lidocaine and the alcohol octanol on TTX-r Na+ channels. Currents were recorded with the whole-cell patch-clamp method from enzymatically isolated rat dorsal root ganglion cells (data evaluation: nonlinear least-squares fitting). Lidocaine and octanol blocked the TTX-r Na+ current in a reversible and concentration-dependent manner (50% inhibitory concentration values: 177 ± 25 and 455 ± 25 &mgr;M, respectively). Lidocaine additionally produced a strong use-dependent block. Both drugs showed a strong dynamic block (i.e., block developed during the time course of current activation and inactivation). Double-pulse protocols showed a slow dissociation of lidocaine from the channel during repolarization (time constant: 1763 ± 63 ms; 300 &mgr;M). The dissociation of octanol was too quick to be distinguished from normal current repriming kinetics of 2.2 ms. Lidocaine and octanol acted noncompetitively in the Na+ channel. Lidocaine and octanol have different blocking properties on the TTX-r Na+ current and bind to different channel sites.