Background potassium channel block and TRPV1 activation contribute to proton depolarization of sensory neurons from humans with neuropathic pain

Protons cause a sustained depolarization of human dorsal root ganglion (DRG) neurons [Baumann et al. (1996) Pain, 65, 31-38]. In the present study we sought to determine which ion channels are expressed in human DRG neurons that could mediate the sustained responses observed in the patch-clamp recordings. RT-PCR of material from the DRG tissue revealed the presence of mRNAs for a nonselective cation channel that is activated by protons (TRPV1) and background potassium channels that are blocked by protons (TASK-1, TASK-3 and Kir2.3). Highly acidic solution (pH 5.4) applied to cultured DRG neurons evoked prolonged currents that were associated with a net increase in membrane conductance. Consistent with the involvement of TRPV1, these proton-evoked currents were blocked by capsazepine and were only found in neurons that responded to capsaicin with an increase in membrane conductance. Less acidic extracellular solution (pH 6.0) evoked such currents only rarely, but was able to strongly enhance the currents evoked by capsaicin. Capsazepine (1 microm) blocked the currents evoked by capsaicin at pH 7.35, as well as the potentiated responses to capsaicin at pH 6.0. In neurons that were not excited by capsaicin, moderate extracellular acidification (pH 6.0) caused a sustained decrease in resting membrane conductance. The decrease in membrane conductance by protons was associated with inhibition of background potassium channels. This excitatory effect of protons was not blocked by capsazepine. We conclude that in most neurons the sustained depolarization in response to moderately acidic solutions is the result of blocked background potassium channels. In a subset of neurons, TRPV1 also contributes.     

Effect of a water-soluble carbodiimide on the gating mechanism of "fast" sodium channels of the sensory neuron membrane of the rat

Currents through "fast" (tetrodotoxin-sensitive) sodium channels in dorsal root ganglion of rat were measured before and after the 5-6 min. external application of solutions containing 100 mM 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide-HCl (WSC). The WSC treatment (pH 4.8-5.0) resulted in a decrease of the sodium conductance, slowing down of the activation kinetics by the factor of 1.5-2.5 and a decrease of the steepness of the activation curve. Effective charge of activation determined from the limiting logarithmic slope of the activation curve at potentials where channels just begin to open was reduced by the factor of 2.0. At normal pH (7.6) the WSC induced no changes in activation parameters. The results suggest that observed effects are due to the WSC interaction with carboxyl groups, situated at the external membrane surface. These groups may be incorporated in the gating mechanism of the sodium channel.     

Hydrogen currents through aconitine-modified sodium channels in nerve fiber membranes

Ionic currents in nodal membrane treated with aconitine were measured under voltage clamp conditions when nodes were bathed in Na-free solutions. At pH lower than 4.6 inward ionic currents were detected which had kinetics and voltage range of activation analogous to those of aconitine-modified sodium channels at low pH. These currents were blocked by benzocaine (2 mM). Experiments with various concentrations of Ca2+, tris+, TEA+, choline+ ions showed that these ions are essentially impermeable both at normal and acidic pH. It is concluded that the inward currents observed are carried by H+ (or H3O+) ions through aconitine-modified sodium channels. From reversal potential measurements relative permeability (PH/PNa) of sodium channels is estimated to be 1059 +/- 88. The results suggest that the aconitine-modified channel is a rather wide water-filled pore and the rate of H+ passing through the channel is limited by its binding to an acidic group.     

Potential-dependent changes in the ionic selectivity of batrachotoxin-modified sodium channels of a frog nerve fiber

Currents through batrachotoxin-modified sodium channels in frog myelinated fibres were measured under voltage-clamp conditions. Reversal potential (Erev) of steady-state currents is shown to be about 5 mV less positive than Erev of initial (peak) currents. Control experiments with procaine and tetrodotoxin in external solutions showed that this shift of Erev during depolarizing pulse cannot be accounted for by the presence of unmodified sodium channels, unblocked potassium channels, nonlinearity of the leakage or any changes in transmembrane gradients of current-carrying cations. "Instantaneous" current measurements showed that Erev becomes less positive as amplitude and duration of preliminary depolarization increase. The results obtained are consistent with assumption that sodium-potassium selectivity of the batrachotoxin-modified channels depends on potential.     

Hydrogen ion currents through sodium channels in myelinated nerve fiber membranes

Ionic currents in the nodal membrane of myelinated frog nerve fibre were measured under voltage clamp conditions when the Ranvier node was bathed in solutions containing impermeant cations instead Na. At pH lower than 4.0 small (less than 0.1 nA) currents were detected which rose to peak and then decayed more slowly. Kinetics and voltage range of activation of these currents were similar to those of usual sodium currents at low pH. These currents were reversibly blocked by benzocaine (1 mM). All this permitted identifying them as currents through sodium channels. Experiments in which concentrations of substituting cations (tris+, choline+), Ca2+ and H+ ions were varied showed that the inward currents observed are carried by hydrogen (or hydronium) ions. According to reversal potential measurements the relative permeability of the channels (PH/PNa) is equal to 203 +/- 14 on the average. It is concluded that the energy barriers for H+ in sodium channel are much lower than for Na+, but their passage through the channel is slow because of binding to an acidic group in the channel.     

Inward current responses to urinary substances in rat vomeronasal sensory neurons

No study has yet demonstrated an inward current in response to pheromonal substances in vomeronasal sensory neurons. Using female rat vomeronasal sensory neurons, we here successfully recorded inward currents in response to urine from various sources. Of the neurons that responded to urine, 77% responded to only one type of urine. Male Wistar urine induced responses preferentially in the apical layer of the sensory epithelium, whilst male Donryu and female Wistar urine induced responses mainly in the basal layer of the epithelium. The amplitude of inward currents induced by application of male Wistar urine was voltage-dependent with average amplitude of -47.1+/-6.2 pA at -74 mV. The average reversal potential for male Wistar urine was -9.3 +/-6.1 mV, which was not apparently different from the reversal potentials for urine from different species. It is likely that the urine-induced inward currents in response to different types of urine are mediated via a similar channel. The simultaneous removal of Na+ and Ca2+ from extracellular solution eliminated the response. The magnitude of the urine-induced inward current in Cl--free external solution was similar to that in normal solution, suggesting that the urine-induced current is cation selective. Removal of external Ca2+ enhanced the amplitude of the urine-induced current and prolonged the response. Application of the constant-field equation indicated a very high permeability coefficient for Ca2+. This study first demonstrated that substances contained in urine elicited inward currents, which induce an excitatory response in vomeronasal sensory neurons, through cation-selective channels.     

Properties of NMDA receptors in rat spinal cord motoneurons

Postnatal development and properties of N-methyl-d-aspartate (NMDA) receptors were studied with whole-cell and outside-out patch-clamp techniques in interneurons and fluorescence-labelled motoneurons in rat spinal cord slices. Both the absolute amplitude of NMDA-induced currents and currents normalized with respect to the motoneuron capacitance increased significantly at postnatal days 10-13 when compared to the responses evoked at postnatal days 2-3. The mean amplitude of the responses to kainate also increased in motoneurons of postnatal days 10-13. Single-channel currents induced by low concentrations of glutamate, exhibited four distinct amplitude levels corresponding to 19.2 +/- 2.4 pS, 38.4 +/- 3.5 pS, 56.3 +/- 2. 4 pS and 69.6 +/- 3.7 pS. In contrast, the conductance of single channels, recorded under identical conditions, in rat spinal cord interneurons was less, 15.3 +/- 3.2 pS, 29.9 +/- 5.4 pS, 46.7 +/- 4. 8 pS and 62.4 +/- 3.9 pS. The high (56/70 pS) conductance single-channel openings in motoneuron patches were sensitive to NMDA receptor inhibitors D-2-amino-5-phosphonovalerate, 7-chlorokynurenic acid and ifenprodil. Whole-cell NMDA-evoked currents were blocked in a voltage-dependent manner by extracellular Mg2+ with an apparent dissociation constant for Mg2+ binding at 0 mV of 1.8 +/- 0.5 mm. The conductance and relative distribution of NMDA receptor channel openings induced by 1 micrometer glutamate in patches isolated from the motoneurons were independent of age from postnatal day 4 to 14. The results suggest that the properties of NMDA receptor channels in motoneurons differ from those in spinal cord interneurons and cells transfected with NR1/NR2 subunits.     

The anticonvulsant SGB-017 (ADCI) blocks voltage-gated sodium channels in rat and human neurons: comparison with carbamazepine

PURPOSE: SGB-017 (ADCI) is a novel anticonvulsant that blocks both voltage-activated sodium channels and N-methyl-D-aspartate (NMDA)-receptor-gated channels. Results by Rogawski et al. suggested that SGB-017 produces its anticonvulsant action primarily by inhibition of NMDA-receptor channels. However, SGB-017 is effective in several animal models of epilepsy that are unresponsive to NMDA antagonists. These results indicate that block of NMDA-receptor channels is not the only mechanism contributing to its anticonvulsant activity. Thus the effects of SGB-017 on neuronal sodium channels were investigated. METHODS: Whole cell voltage-clamp techniques were used to record sodium currents in freshly dissociated rat superior cervical ganglion (SCG) and hippocampal neurons and cultured human NT2 neurons. The effects of SGB-017 on the amplitude of sodium currents, elicited by a depolarizing pulse to 0 mV from different holding potentials, were measured and compared with those of carbamazepine (CBZ). RESULTS: SGB-017 inhibited sodium currents in rat SCG and hippocampal neurons with a similar potency to CBZ. Like CBZ, the inhibition of sodium channels by SGB-017 was voltage dependent. Its median inhibitory concentration (IC50) for inhibition of sodium channels at depolarized holding potentials is similar to that for its inhibition of NMDA receptor channels. In human hNT2 neurons, SGB-017 was more potent than CBZ at inhibiting sodium currents. CONCLUSIONS: SGB-017 produces its anticonvulsant activity by blocking both sodium- and NMDA-receptor channels in a voltage- and use-dependent manner. The combination of these two mechanisms of action makes SGB-017 an effective AED in several different animal models of epilepsy.     

The effect of allapinine on the sodium currents of isolated trigeminal ganglion neurons and cardiomyocytes of rats

Block of sodium currents by allapinine (diterpene alkaloid with strong antiarrhythmic properties) was investigated in isolated, voltage-clamped trigeminal neurons of a rat and single ventricular myocytes of a neonatal rat. Allapinine (in micromolar concentrations) produced a 70-90% decrease of the sodium current amplitude without any changes in voltage-dependent properties of INa in both neurons and cardiomyocytes. Allapinine also blocked the aconitine-modified sodium current. An increase of depolarization frequencies (0.5 to 5.0 Hz) produced no additional block of sodium currents in allapinine-bathed neurons and ventricular myocytes.     

Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Ion currents through batrachotoxin-modified sodium channels of node of Ranvier membranes at high positive and negative potentials

Ionic currents through batrachotoxin-modified sodium channels in frog nerve fibres were measured over a wide range of membrane potentials. At potentials above +80 mV currents decay in time and their steady-state level decreased as potentials increased. "Instantaneous" current measurements have shown that this phenomenon was due to the decrease in net channel conductance. Scorpion toxin affected current kinetics only slightly at these potentials, which suggested that these decays were not caused by usual inactivation process. Externally applied procaine induced slow (tens of ms) potential-dependent block of batrachotoxin-modified channels at large positive potentials. At large negative potentials (above -100 mV) "instantaneus" currents decreased due to fast voltage-dependent block of the channels by calcium ions.     

pH-dependent actions of aluminum on voltage-activated sodium currents in snail neurons

The pH-dependent actions of aluminum(III) hydroxides (Al(III))on the voltage-activated sodium currents (VASCs) in the giant neurons of the pond snail Lymnaea stagnalis L. were studied by means of a conventional two-electrode voltage-clamp technique. The final concentration of Al(III) was 5-500 microM at pH 7.7, 6.9 or 6.0. A significant and concentration-dependent increase in the peak amplitude of the VASCs was recorded over the entire voltage range at pH 7.7 (EC50 = 100.7 +/- 33.7 microM, n = 9), without alteration of the gating properties. A concentration-dependent decrease in the peak amplitude (IC50 = 175.9 +/- 73.6 microM, n = 6) and concomitant increases in the time constants of activation and inactivation of the VASCs were recorded in slightly acidic media (pH 6.0), whereas there were no changes in the investigated parameters at pH 6.9. A significant increase in the V1/2 of the half-maximal current of the steady-state inactivation resulted on Al(III) application at pH 7.7, but not at pH 6.9 or 6.0. These results suggest that Al(III) can differentially up- and down-modulate the sodium current and related physiological functions to extents dependent on the pH-determined speciation of the Al(III) hydroxides present.     

Feedback Inhibition of Ca2+ Currents by Dopamine in Glomus Cells of the Carotid Body

The effect of dopamine on the voltage-dependent ionic channels of enzymatically dispersed glomus cells from rabbit carotid bodies was studied. Whole-cell currents were recorded on isolation with patch electrodes and dopamine applied to the bath solution. Dopamine at nanomolar concentrations produced a reversible attenuation of the calcium current whereas sodium and potassium currents remained unaltered. Dopamine inhibition of Ca2+ current was observed in all cells tested (n=48) and at a saturating concentration (1 microM) the average reduction was of 40 +/- 6.5% (n=8). The effect of dopamine was probably caused by a decrease in the number of channels activatable on depolarization since it did not modify the voltage-dependent parameters of the current. These results indicate that dopamine, which is the major transmitter secreted by glomus cells, regulates further transmitter release by feedback inhibition of Ca2+ channels.     

Modulation of calcium currents in mouse ventral horn neurons by extracellular pH

Neuronal activity has been shown to modulate the pH of the extracellular environment. Since neuronal circuits in the ventral horn of the spinal cord are highly active during patterned movements, and voltage-gated calcium channels play an important role in the production of spinal motoneuron output, the effects of changes in extracellular pH (pH(e)) on calcium currents in ventral horn neurons of the mouse spinal cord were examined. It is demonstrated that these channels are sensitive to modulation by pH(e). The amplitude of the current mediated by these channels increased as the pH(e) was elevated. The elevated pH(e) also led to a hyperpolarizing shift in the voltage dependence of both activation and inactivation. The opposite effects were seen for a decrease in pH(e). It was also noted that a decrease in pH(e) was associated with a faster inactivation of the current. It is concluded that voltage-gated calcium currents in ventral horn neurons are modulated by changes in pH(e), and that this modulation may play a physiologically important role in determining motoneuronal excitability during behaviors such as locomotion.     

Origin of Aspartate-induced Responses in Rat Cerebellar Purkinje Cells

The effects of glutamate, aspartate and N-methyl-d -aspartate (NMDA) on Purkinje cells and interneurons were investigated in cerebellar slice cultures using the whole-cell configuration of the patch-clamp technique. l -Glutamate and l -aspartate induced inward currents in Purkinje cells voltage-clamped at -60 mV. In standard external solution, the amplitude of the responses induced by these two amino-acids was a linear function of the membrane potential. l -Aspartate-induced currents were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a selective antagonist of non-NMDA receptors. NMDA, a selective agonist of NMDA receptors, had no effect of its own on the excitability of Purkinje cells, but was effective in blocking the responses induced by aspartate in Purkinje cells in a voltage-independent manner. In contrast, d -(–)-2-amino-5-phosphonovaleric acid (d -APV), a selective antagonist of NMDA receptors, had no effect on aspartate-induced responses. d -Aspartate also induced responses in Purkinje cells, and the amplitude of these responses was a linear function of the membrane potential. Currents induced by l - and d -aspartate were inhibited by dihydrokainate, a glutamate uptake blocker. In sodium-free external solution, glutamate still induced outward currents in Purkinje cells, whereas l - and d -aspartate no longer evoked any current. When sodium was replaced by lithium in the external medium, no change in the holding current could be detected in Purkinje cells maintained at -60 mV; moreover, in this bathing medium l -aspartate no longer evoked any current whereas glutamate-induced responses were still present. In contrast, interneurons were sensitive to both NMDA and aspartate applications, and these responses were antagonized by d -APV. In addition, aspartate still induced an outward current in sodium-free external solution. This study presents rather direct evidence in favour of l -aspartate as being a very selective NMDA receptor agonist in the cerebellum. l -Aspartate-induced currents in Purkinje cells are not due to activation of mixed NMDA/non-NMDA receptors, but are probably due to the release of l -glutamate induced by aspartate through glutamate uptake.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Modification of the gating mechanism of incurrent sodium channels by an osmotic pressure gradient on a perfused neuron

The influence of the osmotic pressure gradient was investigated by the voltage clamp method. It was shown that the osmotic gradient changed both activation and inactivation of the sodium conductivity and decreased the sodium current amplitude. Conductivity of single sodium channels was not affected by the osmotic pressure gradient. It is concluded that the decrease in sodium current amplitude is due to a decrease in the number of open channels.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

Do neurons have a reserve of sodium channels for the generation of action potentials? A study on acutely isolated CA1 neurons from the guinea‐pig hippocampus

The density of voltage-gated sodium channels is high in several regions of the neuronal membrane. It is unclear if this density of channels represents a reserve for the neuron, or if it fulfils a special role in action potential firing. This problem was addressed by studying sodium currents and action potentials in acutely isolated hippocampal CA1 neurons whose number of active sodium channels was acutely changed by applying the sodium channel blocker tetrodotoxin (TTX) at different concentrations. The results show that more than a third of the sodium channels can fail without affecting the single action potential. Thus, the neurons have a remarkable surplus of sodium channels. The surplus, however, is necessary for repetitive action potential firing, as every decrease in the fraction of sodium channels reduces the maximal frequency of action potentials that can be generated by the neuron.