Acidic regulation of junction channels between glomus cells in the rat carotid body. Possible role of [Ca]i

The purpose of this work was to characterize the gap junctions between cultured glomus cells of the rat carotid body and to assess the effects of acidity and accompanying changes in [Ca²⁺]_i on electric coupling. Dual voltage clamping of coupled glomus cells showed a mean macrojunctional conductance (G_j) of 1.16 nS±0.6 (S.E.), range 0.15–4.86 nS. At normal pH_o (7.43), a steady transjunctional voltage (ΔV_j=100.1±10.9 mV) showed multiple junction channel activity with a mean microconductance (g_j) of 93.98±0.6 pS, range 0.3–324.5 pS. Single-channel conductances, calculated as variance/mean g_j, gave a mean value of 16.7±0.2 pS, range 5.13–39.38 pS. Manual measurements of single-channel activity showed a mean g_j of 22.03±0.2 pS, range 1.3–160 pS. Computer analysis of the noise spectral density distribution gave a channel mean open time of 12.7±1.5 ms, range 6.37–23.42 ms. The number of junction channels, estimated in each experiment from G_j/single-channel g_j, showed a range of 7 to 258 channels (mean, 107.2). Optical measurements of [Ca²⁺]_i gave a mean value of 80.2±4.27 nM at pH_o of 7.43. Acidification of the medium with lactic acid (1 mM, pH 6.3) induced: 1) Variable changes in G_j (decreases and increases); 2) A significant decrease in mean g_j (to 80.36±0.34 pS) and in single-channel conductance (g_j=12.8±0.2 pS in computer analyses and 17.23±0.2 pS when measured by hand); 3) Variable changes in open times, resulting in a similar mean (12.8±1.5 ms) and 4) No change in the number of junction channels. When pH_o was lowered to 6.3 [Ca²⁺]_i did not change significantly (there were increases and decreases). However, when pH_o was lowered to 4.4, [Ca²⁺]_i increased significantly to 157.1±8.1 nM. It is concluded that saline acidification to pH 6.3 depresses the conductance of junction channels and this effect may be either a direct effect on channel proteins or synergistically enhanced by increases in [Ca²⁺]_i. However, there are no studies correlating changes of [Ca²⁺]_i and intercellular coupling in glomus cells. Stronger acidification (pH_o 4.4), producing much larger changes in [Ca²⁺]_i, may enhance this synergism. But, again, there are no studies correlating these effects.     

Modulation of junctional conductance between rat carotid body glomus cells by hypoxia, cAMP and acidity

Short-term cultures of glomus cells (up to seven days), were employed to study intercellular electrical communications. Bidirectional electric coupling was established under current clamping after impaling two adjacent glomus cells with microelectrodes, and alternate stimulation and recording. Their resting potential (V_m) and input resistance (R_o) were thus measured. Both coupled cells were then voltage clamped at a level between their V_ms. Current pulses applied to either cell elicited a transjunctional voltage (V_j) and current (I_j), used to calculate the junctional conductance (G_j). G_j was 1.52±0.29 nS (mean±S.E.; n=147). V_j linearly influenced G_j, suggesting ohmic junctions. G_j was not affected by V_m in 50% of the cases. However, there was V_m-dependence in the others, but voltage changes had to be large (>±40 mV from the V_m). Therefore, physiologically or pharmacologically induced glomus cell depolarization or hyperpolarization may not significantly affect intercellular coupling unless there are large variations in V_m. Hypoxia (induced by Na₂S₂O₄ 1 mM or 100% N₂) decreased G_j in 60–80% of the pairs while producing tighter coupling in the rest. Similar effects were obtained when the medium was acidified with lactic acid 1–10 mM. Cobalt chloride (3 mM) prevented, diminished or reversed the changes in G_j observed during low PO₂, suggesting that [Ca²⁺]_i changes are important in hypoxic uncoupling. However, non-specific cationic effects of Co²⁺ have not been ruled out. Applications of the membrane-permeant dB-cAMP 1 mM tightened coupling in almost all cell pairs. This is important because endogenous cAMP increases during hypoxia. Our results suggest that multiple factors modulate junctional conductance between glomus cells. Changes in G_j by ‘natural' stimuli and/or cAMP may play an important role in chemoreception, especially in titrating the release of transmitters toward the carotid nerve terminals.     

The firing patterns of spinal neurons: in situ patch‐clamp recordings reveal a key role for potassium currents

Neuron firing patterns underpin the detection and processing of stimuli, influence synaptic interactions, and contribute to the function of networks. To understand how intrinsic membrane properties determine firing patterns, we investigated the biophysical basis of single and repetitive firing in spinal neurons of hatchling Xenopus laevis tadpoles, a well‐understood vertebrate model; experiments were conducted in situ. Primary sensory Rohon–Beard (RB) neurons fire singly in response to depolarising current, and dorsolateral (DL) interneurons fire repetitively. RB neurons exhibited a large tetrodotoxin‐sensitive sodium current; in DL neurons, the sodium current density was significantly lower. High‐voltage‐activated calcium currents were similar in both neuron types. There was no evidence of persistent sodium currents, low‐voltage‐activated calcium currents, or hyperpolarisation‐activated currents. In RB neurons, the potassium current was dominated by a tetraethylammonium‐sensitive slow component (I_Ks); a fast component (I_Kf), sensitive to 4‐aminopyridine, predominated in DL neurons. Sequential current‐clamp and voltage‐clamp recordings in individual neurons suggest that high densities of I_Ks prevent repetitive firing; where I_Ks is small, I_Kf density determines the frequency of repetitive firing. Intermediate densities of I_Ks and I_Kf allow neurons to fire a few additional spikes on strong depolarisation; this property typifies a novel subset of RB neurons, and may activate escape responses. We discuss how this ensemble of currents and firing patterns underpins the operation of the Xenopus locomotor network, and suggest how simple mechanisms might underlie the similar firing patterns seen in the neurons of diverse species.     

INa and IKir are reduced in Type 1 hypokalemic and thyrotoxic periodic paralysis

We evaluated voltage‐gated Na⁺ (I_Na) and inward rectifier K⁺ (I_Kir) currents and Na⁺ conductance (G_Na) in patients with Type 1 hypokalemic (HOPP) and thyrotoxic periodic paralysis (TPP). We studied intercostal muscle fibers from five subjects with HOPP and one with TPP. TPP was studied when the patient was thyrotoxic (T‐toxic) and euthyroid. We measured: (1) I_Kir, (2) action potential thresholds, (3) I_Na, (4) G_Na, (5) intracellular [Ca²⁺], and (6) histochemical fiber type. HOPP fibers had lower I_Na, G_Na, and I_Kir and increased action potential thresholds. Paralytic attack frequency correlated with the action potential threshold, G_Na and I_Na, but not with I_Kir. G_Na, I_Na, and [Ca²⁺] varied with fiber type. HOPP fibers had increased [Ca²⁺]. The subject with TPP had values for G_Na, I_Na, action potential threshold, I_Kir, and [Ca²⁺] that were similar to HOPP when T‐toxic and to controls when euthyroid. HOPP T‐toxic TPP fibers had altered G_Na, I_Na, and I_Kir associated with elevation in [Ca²⁺]. Muscle Nerve, 2010     

Modulation of voltage-activated Ca currents by pain-inducing agents in a dorsal root ganglion neuronal line,F-11

Whole cell currents evoked by pain-inducing agents—bradykinin (Bk), capsaicin (Cap), and reciniferatoxin (RTX), and their modulation of voltage-activated Ca currents were examined in F-11 cells using a patch electrode voltage clamp technique. Most F-11 cells generated action potentials under current clamp if their membrane potentials were held sufficiently negative. Average peak inward Na current (I_Na) was 100 μA/cm² and the I_Na was abolished by 10^?6 M tetrodotoxin. At least two types of Ca currents could be clearly distinguished on the basis of voltage dependency and kinetics; a low threshold transient I_Ca(t) and a high threshold sustained I_Ca(I). In addition, another high threshold transient Ca current, presumably I_Ca(n), was observed. About 30% of the cells produced inward current for these pain-inducing agents, when activated at the membrane holding potential of ?70 mV. In some F-11 cells, the amplitude of action potential was observed to increase during 10^?6 M Cap-induced depolarization. Both low and high threshold Ca currents were reduced by 10^?6 M Bk in the majority of the cells. Similarly, both 10^?6 M Cap and 10^?9 M RTX reduced these Ca currents. However, a considerable number of cells showed an initial enhancement followed by reduction in the amplitude of these Ca currents. With higher concentrations of these ligands, all Ca currents were suppressed. Such modulation of voltage-activated Ca currents by pain-inducing agents occurred in both the presence and absence of apparent receptor-activated current flows in the cells. In pertussis toxin (PTX)-treated cells, the inhibitory modulation of Ca currents by pain-inducing agents was suppressed. In contrast, in cholera toxin (CTX)-treated cells, this inhibitory modulation appeared to be enhanced. These data indicate that the inhibitory modulation of Ca channel currents by Cap and RTX, similarly to that of Bk, involves a PTX-sensitive inhibitory G protein (G_i). © 1993 Wiley-Liss, Inc.     

Differential contributions of Ca2+‐activated K+ channels and Na+/K+‐ATPases to the generation of the slow afterhyperpolarization in CA1 pyramidal cells

In many types of CNS neurons, repetitive spiking produces a slow afterhyperpolarization (sAHP), providing sustained, intrinsically generated negative feedback to neuronal excitation. Changes in the sAHP have been implicated in learning behaviors, in cognitive decline in aging, and in epileptogenesis. Despite its importance in brain function, the mechanisms generating the sAHP are still controversial. Here we have addressed the roles of M‐type K⁺ current (I_M), Ca²⁺‐gated K⁺ currents (I_Ca(K)'s) and Na⁺/K⁺‐ATPases (NKAs) current to sAHP generation in adult rat CA1 pyramidal cells maintained at near‐physiological temperature (35 °C). No evidence for I_M contribution to the sAHP was found in these neurons. Both I_Ca(K)'s and NKA current contributed to sAHP generation, the latter being the predominant generator of the sAHP, particularly when evoked with short trains of spikes. Of the different NKA isoenzymes, α₁‐NKA played the key role, endowing the sAHP a steep voltage‐dependence. Thus normal and pathological changes in α₁‐NKA expression or function may affect cognitive processes by modulating the inhibitory efficacy of the sAHP.     

Effects of Acetazolamide on Transient K+ Currents and Action Potentials in Nodose Ganglion Neurons of Adult Rats

The aim of the present study was to determine whether acetazolamide (AZ) contributes to the inhibition of the fast inactivating transient K⁺ current (I_A) in adult rat nodose ganglion (NG) neurons. We have previously shown that pretreatment with either AZ or 4‐AP attenuated or blocked the CO₂‐induced inhibition of slowly adapting pulmonary stretch receptors in in vivo experiments. The patch‐clamp experiments were performed by using the isolated NG neurons. In addition to this, the RT‐PCR of mRNA and the expression of voltage‐gated K⁺ (Kv) 1.4, Kv 4.1, Kv 4.2, and Kv 4.3 channel proteins from nodose ganglia were examined. We used NG neurons sensitive to the 1 mM AZ application. The application of 1 mM AZ inhibited the I_A by approximately 27% and the additional application of 4‐AP (1 mM) further inhibited I_A by 48%. The application of 0.1 μM α‐dendrotoxin (α‐DTX), a slow inactivating transient K⁺ current (I_D) blocker, inhibited the baseline I_A by approximately 27%, and the additional application of 1 mM AZ further decreased the I_A by 51%. In current clamp experiments, AZ application (1 mM) increased the number of action potentials due to the decreased duration of the depolarizing phase of action potentials and/or due to a reduction in the resting membrane potential. Four voltage‐gated K⁺ channel proteins were present, and most (80–90%) of the four Kv channels immunoreactive neurons showed the co‐expression of carbonic anhydrase‐II (CA‐II) immunoreactivity. These results indicate that the application of AZ causes the reduction in I_A via the inhibition of four voltage‐gated K⁺ channel (Kv) proteins without affecting I_D.     

Functional characterization of the H-current in SCN neurons in subjective day and night: a whole-cell patch-clamp study in acutely prepared brain slices

Neurons of the rat suprachiasmatic nucleus (SCN) exhibit a circadian rhythm in spontaneous firing rate. In this whole-cell patch-clamp study in slices, we examined the possibility that H-current (I_H) contributes to the spontaneous firing rate of SCN neurons. Most of our experiments were performed during the subjective day, because this is the time epoch during which one would expect the largest excitatory effect of I_H if it were to fluctuate in a circadian rhythm. Current-clamp experiments showed that blockade of I_H by Cs⁺ (1 mM) did not influence the spontaneous firing rate and resting membrane potential. Voltage-clamp experiments revealed that I_H, when activated at the resting membrane potential, is probably too small in magnitude and too slow in activation to make a significant contribution to the spontaneous firing rate. Both results suggest that I_H does not significantly contribute to the spontaneous firing of SCN neurons. In addition, we investigated whether the kinetics and voltage dependence of I_H were modulated in a circadian manner. However, no substantial day–night differences in I_H were found. We conclude that I_H, as recorded in whole-cell mode, does not contribute significantly to spontaneous firing in most SCN neurons and that this current, is more likely to be involved in `rescuing' SCN neurons from large and long-lasting hyperpolarizations by depolarizing the membrane.     

Clonidine reduces hyperpolarization-activated inward current (Ih) in rat hypoglossal motoneurons

We used intracellular recording techniques to investigate the actions of clonidine on hypoglossal motoneurons (HMs) in rat brainstem slices. Clonidine (10–100 μM) produced a small (2–6 mV), dose-dependent hyperpolarization in HMs, accompanied by an increase in peak input resistance (R_N). It also slowed the time course of the depolarizing `sag' of the voltage response to constant hyperpolarizing current steps. These effects were mimicked by the α₂-adrenoceptor (α₂-AR) agonist guanabenz, but not by the I₁-imidazoline receptor agonists moxonidine or rilmenidine. Recorded in single-electrode voltage clamp mode, clonidine decreased input conductance of HMs and reduced the amplitude of a hyperpolarization-activated inward current (I_h). Clonidine's effect on I_h was three-fold: it shifted the half-activation voltage (V_1/2) in the hyperpolarizing direction (by 4.4±0.7 mV at a dose of 10 μM), decreased the maximal current (by ∼20%), and slowed the time course of I_h activation at all voltage steps. At the most hyperpolarized potential steps, clonidine slowed activation of I_h dramatically, yielding a striking increase in the activation time constant. The α₂-AR antagonists yohimbine and idazoxan reduced clonidine's effect on V_1/2 and on the I_h activation time course, but neither blocked clonidine's reduction of the maximal current, nor its strong slowing of I_h activation at the most hyperpolarized steps. We were unable to mimic or occlude clonidine's actions with the adenylate cyclase inhibitor SQ 22536 nor with the non-specific protein kinase inhibitor H-7. We conclude that clonidine hyperpolarizes HMs via a reduction of the amount of I_h that is active at rest, and that the response is mediated in part by α₂-ARs. Some effects of clonidine on these neurons do not appear to be receptor-mediated, and may be due to physical block by clonidine of I_h channels.     

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as I_DHPG, which was developmentally stable through postnatal day P56. I_DHPG had low temperature dependence in the range 25–34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K⁺ or Cl⁻. Thus, it is likely that more than one ion type contributes to I_DHPG and the mix may be voltage dependent. I_DHPG was resistant to the Na⁺ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). I_DHPG was inhibited 21% by Ba²⁺ (500 μM), 60% by ZD7288 (100 μM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of I_H indicated a small (6%) increase in G_max by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.     

Sevoflurane-induced ionic current in acutely dissociated CA1 pyramidal neurons of the rat hippocampus

The electrophysiological properties of sevoflurane (Sev)-induced current (I_Sev) were investigated in CA1 pyramidal neurons freshly dissociated from the rat hippocampus by using the nystatin perforated patch recording configuration under voltag-clamp condition. Within the range of Sev concentrations from 3 · 10⁻⁴ to 2 · 10⁻³ M, I_sev was an inward current which consisted of an initial transient peak component and a successive steady-state plateau component. The peak current component increased in a concentration-dependent manner with a conductance increase. The application of Sev over 2 · 10⁻³ M, however, suppressed the peak and steady-state current components with a concomitant decrease in conductance and elicited a transient inward current (‘hump’ current) immediately after wash out. The current-voltage relationship for I_Sev showed some outward rectification suggesting a slight voltage-dependency of the I_sev. The reversal potential of I_Sev (E_Sev) was close to the E_Cl and shifted by 52 mV for a 10-fold change in extracellular Cl⁻ concentrations, indicating that I_Sev is passing through Cl⁻ channels. The single channel conductance obtained from the analysis of the variance of I_Sev fluctuations was 15.3 ± 1.3pS.     

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca²⁺]_i, (2) a calcium and voltage dependent potassium channel (K_Ca). K_Ca channels had a conductance of (302±15) pS (n=5) and were activated by [Ca²⁺]_i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on K_Ca channels from rat cerebellum.     

Fluctuation analysis of nonselective cation currents induced by AlF complex in guinea-pig chromaffin cells

Properties of aluminium fluoride (AlF) complex-activated nonselective cation (NS) channels in guinea-pig chromaffin cells were investigated using the patch clamp technique. As the membrane potential was hyperpolarized from the holding potential of −55 mV, the AlF-induced nonselective cation current (I_NS) diminished progressively. With hyperpolarizations to −100 mV or more negative potentials, the AlF I_NS almost instantaneously disappeared. The apparent unit conductance of AlF I_NS was estimated to be 3 pS by fluctuation analysis. The open state probability of AlF-activated NS channels became large with a decrease in concentration of free Mg²⁺ ions inside the cell and was less than 0.5 at 12 μM Mg²⁺. It is concluded that NS channels in the chromaffin cell apparently differ from those in smooth muscle cells.     

Arachidonic acid potently inhibits both postsynaptic-type Kv4.2 and presynaptic-type Kv1.4 IA potassium channels

Arachidonic acid (AA) is a free fatty acid membrane‐permeable second messenger that is liberated from cell membranes via receptor‐ and Ca²⁺‐dependent events. We have shown previously that extremely low [AA]_i (1 pm ) inhibits the postsynaptic voltage‐gated K⁺ current (I_A) in hippocampal neurons. This inhibition is blocked by some antioxidants. The somatodendritic I_A is mediated by Kv4.2 gene products, whereas presynaptic I_A is mediated by Kv1.4 channel subunits. To address the interaction of AA with these α‐subunits we studied the modulation of A‐currents in human embryonic kidney 293 cells transfected with either Kv1.4 or Kv4.2 rat cDNA, using whole‐cell voltage‐clamp recording. For both currents 1 pm [AA]_i inhibited the conductance by > 50%. In addition, AA shifted the voltage dependence of inactivation by ?9 (Kv1.4) and +6 mV (Kv4.2), respectively. Intracellular co‐application of Trolox C (10 μm ), an antioxidant vitamin E derivative, only slowed the effects of AA on amplitude. Notably, Trolox C shifted the voltage dependence of activation of Kv1.4‐mediated I_A by ?32 mV. Extracellular Trolox for > 15 min inhibited the AA effects on I_A amplitudes as well as the effect of intracellular Trolox on the voltage dependence of activation of Kv1.4‐mediated I_A. Extracellular Trolox further shifted the voltage dependence of activation for Kv4.2 by +33 mV. In conclusion, the inhibition of maximal amplitude of Kv4.2 channels by AA can explain the inhibition of somatodendritic I_A in hippocampal neurons, whereas the negative shift in the voltage dependence of inactivation apparently depends on other neuronal channel subunits. Both AA and Trolox potently modulate Kv1.4 and Kv4.2 channel α‐subunits, thereby presumably tuning presynaptic transmitter release and postsynaptic somatodendritic excitability in synaptic transmission and plasticity.     

Ih “run‐up” in rat neocortical neurons and transiently rat or human HCN1‐expressing HEK293 cells

Hyperpolarization‐activated cyclic nucleotide‐gated ion channels (HCN) are key determinants of CNS functions. Here we describe an increase in hyperpolarization‐activated current (I_h) at the beginning of whole‐cell recordings in rat layer 5 cortical neurons. For a closer investigation of this I_h increase, we overexpressed the predominant layer 5 rat subunit HCN1 in HEK293 cells. We characterized the resulting I_h in the cell‐attached and whole‐cell configurations. Breaking into whole‐cell configuration led to about a 30% enhancement of rat HCN1‐mediated I_h accompanied by a depolarizing shift in voltage dependence and an accelerated time course of activation. This current enhancement is not species specific; for human HCN1, the current similarly increases in amount and kinetics. Although the changes were bound to cytosolic solution exchange, they were independent of cAMP, ATP, GTP, and the phosphate group donor phosphocreatine. Together, these data provide a characterization of heterologous expression of rat HCN1 and suggest that cytosolic contents suppress I_h. Such a mechanism might constitute a reserve in h‐channel function in vivo. © 2010 Wiley‐Liss, Inc.     

Generation of resonance‐dependent oscillation by mGluR‐I activation switches single spiking to bursting in mesencephalic trigeminal sensory neurons

The primary sensory neurons supplying muscle spindles of jaw‐closing muscles are unique in that they have their somata in the mesencephalic trigeminal nucleus (MTN) in the brainstem, thereby receiving various synaptic inputs. MTN neurons display bursting upon activation of glutamatergic synaptic inputs while they faithfully relay respective impulses arising from peripheral sensory organs. The persistent sodium current (I_NaP) is reported to be responsible for both the generation of bursts and the relay of impulses. We addressed how I_NaP is controlled either to trigger bursts or to relay respective impulses as single spikes in MTN neurons. Protein kinase C (PKC) activation enhanced I_NaP only at low voltages. Spike generation was facilitated by PKC activation at membrane potentials more depolarized than the resting potential. By injection of a ramp current pulse, a burst of spikes was triggered from a depolarized membrane potential whereas its instantaneous spike frequency remained almost constant despite the ramp increases in the current intensity beyond the threshold. A puff application of glutamate preceding the ramp pulse lowered the threshold for evoking bursts by ramp pulses while chelerythrine abolished such effects of glutamate. Dihydroxyphenylglycine, an agonist of mGluR1/5, also caused similar effects, and increased both the frequency and impedance of membrane resonance. Immunohistochemistry revealed that glutamatergic synapses are made onto the stem axons, and that mGluR1/5 and Nav1.6 are co‐localized in the stem axon. Taken together, glutamatergic synaptic inputs onto the stem axon may be able to switch the relaying to the bursting mode.     

Dopamine neuron membrane physiology: Characterization of the transient outward current (IA) and demonstration of a common signal transduction pathway for IA and IK

Dopamine neurons derived from the mesencephalon of embryonic rats were maintained in primary culture, identified and studied with whole-cell patch recording techniques. These neurons demonstrated a rapidly activating and inactivating voltage-dependent outward current which required the presence of K⁺ ions. This current was termed I_A because of its transient nature. It was elicited by step depolarizations from holding potentials more negative than -50 mV and exhibited steady-state inactivation at a membrane potential more positive than -40 mV and half-maximal inactivation observed at -65 mV. This current rapidly achieved peak activation in less than 8 msec and decayed with a time constant (τ) of 58±5 msec. This current was observed in the presence of tetraethylammonium but was readily blocked by 4-aminopyridine (2-4 mM). This current was also observed to be modulated by stimulation of D₂ dopamine receptors (DA autoreceptors) located on the dopamine neurons. Thus, both DA and the D₂ receptor agonist quinpirole enhanced the peak I_A observed, while the partial D₁ receptor agonist SKF 38393 was without effect. The enhancement of I_A was confirmed to be due to the activation of D2 receptors as the effects of either DA or quinpirole were blocked by the D₂ receptor antagonists eticlopride and sulpiride, but not by the D₁ receptor antagonist SCH 23390. Since we have previously demonstrated that the I_K present: in these cells is also enhanced by D₂ receptor stimulation, we investigated the signal transduction pathways involved in coupling DA autoreceptors to both I_A and I_K. The response of both these potassium currents to DA autoreceptor stimulation was completely abolished by the preincubation of cultures with pertussis toxin, indicating the possible involvement of the G proteins G_i and G_O. In an attempt to further characterize which G protein may be involved, additional experiments were performed. The ability of DA autoreceptor stimulation to augment both currents was also blocked completely when G protein activation was prevented by the intracellular application of GDPßS (100 μM). In contrast, irreversible activation of G proteins by intracellular application of the nonhydrolyzable GTP analog GTPγS (100 μM) mimicked the effects of DA autoreceptor stimulation on both I_A and I_K. In addition, the intracellular application of a polyclonal antibody that was selective for the β-subunit of G_O completely abolished the DA autoreceptor modulation of both currents while preimmune serum was without effect. Taken together, these data demonstrate that the enhancement of I_A and I_K in response to stimulation of DA autoreceptors is dependent upon the activation of G_O and appears to involve a G_Oα subunit. © 1994 Wiley-Liss, Inc.     

Loss of β1 accessory Na+ channel subunits causes failure of carbamazepine,but not of lacosamide,in blocking high‐frequency firing via differential effects on persistent Na+ currents

Purpose: In chronic epilepsy, a substantial proportion of up to 30% of patients remain refractory to antiepileptic drugs (AEDs). An understanding of the mechanisms of pharmacoresistance requires precise knowledge of how AEDs interact with their targets. Many commonly used AEDs act on the transient and/or the persistent components of the voltage‐gated Na⁺ current (I_NaT and I_NaP, respectively). Lacosamide (LCM) is a novel AED with a unique mode of action in that it selectively enhances slow inactivation of fast transient Na⁺ channels. Given that functional loss of accessory Na⁺ channel subunits is a feature of a number of neurologic disorders, including epilepsy, we examined the effects of LCM versus carbamazepine (CBZ) on the persistent Na⁺ current (I_NaP), in the presence and absence of accessory subunits within the channel complex. Methods: Using patch‐clamp recordings in intact hippocampal CA1 neurons of Scn1b null mice, I_NaP was recorded using slow voltage ramps. Application of 100 μm CBZ or 300 μm LCM reduced the maximal I_NaP conductance in both wild‐type and control mice. Key Findings: As shown previously by our group in Scn1b null mice, CBZ induced a paradoxical increase of I_NaP conductance in the subthreshold voltage range, resulting in an ineffective block of repetitive firing in Scn1b null neurons. In contrast, LCM did not exhibit such a paradoxical increase, and accordingly maintained efficacy in blocking repetitive firing in Scn1b null mice. Significance: These results suggest that the novel anticonvulsant LCM maintains activity in the presence of impaired Na⁺ channel β₁ subunit expression and thus may offer an improved efficacy profile compared with CBZ in diseases associated with an impaired expression of β sub‐units as observed in epilepsy.     

Na+ currents near and away from endplates on human fast and slow twitch muscle fibers

Fast and slow twitch muscle fibers have distinct contractile properties. Here we determined that membrane excitability also varies with fiber type. Na⁺ currents (I_NA) were studied with the loose-patch voltage clamp technique on 29 histochemically classified human intercostal skeletal muscle fibers at the endplate border and <200 μm from the endplate (extrajunctional). Fast and slow twitch fibers showed slow inactivation of endplate border and extrajunctional I_NA and had increased I_NA at the endplate border compared to extrajunctional membrane. The voltage dependencies of I_NA were similar on the endplate border and extrajunctional membrane, which suggests thatboth regions have physiclogically similar channels. Fast twitch fibers had larger I_NA on the endplate border and extrajunctional membrane and manifest fast and slow inactivation of I_NA at more negative potentials than slow twitch fibers. For normal muscle, the differences between I_NA on fast and slow twitch fibers might: (1) enable fast twitch fibers to operate at high firing frequencies for brief periods; and (2) enable slow twitch fibers to operate at low firing frequencies for prolonged times. Disorders of skeletal membrane excitability, such as the periodic paralyses and myotonias, may impact fast and slow twitch fibers differently due to the distinctive Na⁺ channel properties of each fiber type. © 1993 John Wiley & Sons, Inc.