Whole-cell Voltage-clamp Mode Research Articles

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and 10Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while 10Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NPo) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NPo of Cx43 HCs, while 200 µM 10Panx1 was necessary to obtain a half-maximal NPo reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NPo by 26% while reducing the NPo of the Cx43 HCs by 84%. In contrast 10Panx1 significantly reduced the NPo of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that 10Panx1 inhibits Cx43 HCs over the 100-500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.

Group I metabotropic glutamate receptor-triggered temporally patterned action potential-dependent spontaneous synaptic transmission in mouse MNTB neurons

Rhythmic action potentials (AP) are generated via intrinsic ionic mechanisms in pacemaking neurons, producing synaptic responses of regular inter-event intervals (IEIs) in their targets. In auditory processing, evoked temporally patterned activities are induced when neural responses timely lock to a certain phase of the sound stimuli. Spontaneous spike activity, however, is a stochastic process, rendering the prediction of the exact timing of the next event completely based on probability. Furthermore, neuromodulation mediated by metabotropic glutamate receptors (mGluRs) is not commonly associated with patterned neural activities. Here, we report an intriguing phenomenon. In a subpopulation of medial nucleus of the trapezoid body (MNTB) neurons recorded under whole-cell voltage-clamp mode in acute mouse brain slices, temporally patterned AP-dependent glycinergic sIPSCs and glutamatergic sEPSCs were elicited by activation of group I mGluRs with 3,5-DHPG (200 µM). Auto-correlation analyses revealed rhythmogenesis in these synaptic responses. Knockout of mGluR5 largely eliminated the effects of 3,5-DHPG. Cell-attached recordings showed temporally patterned spikes evoked by 3,5-DHPG in potential presynaptic VNTB cells for synaptic inhibition onto MNTB. The amplitudes of sEPSCs enhanced by 3,5-DHPG were larger than quantal size but smaller than spike-driven calyceal inputs, suggesting that non-calyceal inputs to MNTB might be responsible for the temporally patterned sEPSCs. Finally, immunocytochemical studies identified expression and localization of mGluR5 and mGluR1 in the VNTB-MNTB inhibitory pathway. Our results imply a potential central mechanism underlying the generation of patterned spontaneous spike activity in the brainstem sound localization circuit.

VPAC2 receptor mediates VIP-potentiated insulin secretion via ion channels in rat pancreatic β cells

Vasoactive intestinal peptide (VIP), a small neuropeptide composing of 28 amino acids, functions as a neuromodulator with insulinotropic effect on pancreatic β cells, in which it is of vital importance in regulating the levels of blood glucose. VIP potently agonizes VPAC2 receptor (VPAC2-R). Agonists of VPAC2-R stimulate glucose-dependent insulin secretion. The purpose of this study was to further investigate the possible ion channel mechanisms in VPAC2-R-mediated VIP-potentiated insulin secretion. The results of insulin secretion experiments showed that VIP augmented insulin secretion in a glucose-dependent manner. The insulinotropic effect was mediated by VPAC2-R rather than VPAC1 receptor (VPAC1-R), through the adenylyl cyclase (AC)/protein kinase A (PKA) signalling pathway. The calcium imaging analysis demonstrated that VIP increased intracellular Ca2+ concentration ([Ca2+]i). In addition, in the whole-cell voltage-clamp mode, we found that VIP blocked the voltage-dependent potassium (Kv) channel currents, while this effect was reversed by inhibiting the VPAC2-R, AC or PKA respectively. Taken together, these findings suggest that VIP stimulates insulin secretion by inhibiting the Kv channels, activating the Ca2+ channels, and increasing [Ca2+]i through the VPAC2-R and AC/PKA signalling pathway. These findings provide theoretical basis for the research of VPAC2-R as a novel therapeutic target.

Presynaptic mechanisms underlying GABAB-receptor-mediated inhibition of spontaneous neurotransmitter release.

SUMMARYInhibition of neurotransmitter release by neurotransmitter substances constitutes a fundamental means of neuromodulation. In contrast to well-delineated mechanisms that underlie inhibition of evoked release via suppression of voltage-gated Ca2+ channels, processes that underlie neuromodulatory inhibition of spontaneous release remain unclear. Here, we interrogated inhibition of spontaneous glutamate and GABA release by presynaptic metabotropic GABAB receptors. Our findings show that this inhibition relies on Gβγ subunit action at the membrane, and it is largely independent of presynaptic Ca2+ signaling for both forms of release. In the case of spontaneous glutamate release, inhibition requires Gβγ interaction with the C terminus of the key fusion machinery component SNAP25, and it is modulated by synaptotagmin-1. Inhibition of spontaneous GABA release, on the other hand, is independent of these pathways and likely requires alternative Gβγ targets at the presynaptic terminal.

Charge Selectivity in Plgics: An Aspect of Channel Function that Remains Elusive Even When Multiple Structures are Known

The structural determinants of opposite charge selectivities in pentameric ligand-gated ion channels have been mapped to the first turn of the pore-lining M2 alpha-helices. There, the presence of a glutamate in the cation-selective members and the presence of an extra residue in the preceding M1-M2 linker of the anion-selective members are usually proposed to underlie these contrasting phenotypes. Although a number of hypotheses have been put forward to explain how these differences in primary sequence give rise to such pronounced permeation effects, none of them have found support from the structural models that have recently become available. To further our understanding of the relationship between primary sequence and charge selectivity, we studied in parallel the muscle AChR, the alpha1 GlyR, and the 5HT3AR. Permeability ratios of mutant channels were calculated from reversal potentials estimated under KCl-dilution conditions in whole-cell voltage-clamp mode. Also, we computed PMF profiles and KCl-dilution current-voltage relationships using Brownian-dynamics and molecular-dynamics simulations on some of the structural models. Some of the conclusions we have drawn are: a) Glutamates at position -1' are neither necessary nor sufficient for cation selectivity; b) A longer M1-M2 linker is neither necessary nor sufficient for anion selectivity; c) Identical sequences engineered in the first turn of the M2 alpha-helix of different receptors may give rise to channels with opposite charge selectivities; d) The effects of mutations on charge selectivity are not necessarily additive; e) Neither the peptide-unit dipoles of the M2 alpha-helix nor its “free” N-terminal backbone-amide -NH groups play a major role in charge selectivity; and f) The implied charge-selectivity of open-channel structural models and the effect of mutations on it are not always captured by molecular simulations.

Recreating Ion Channel IV Curves using Specific Frequency Components

INTRODUCTION: Impedance spectroscopy cannot distinguish between ion channel families. We hypothesized that amplitudes of specific characteristic frequencies will correlate with the current amplitude passed by a specific ion channel families. Previously, we demonstrated the feasibility of this technique using the inward rectifying potassium channel, KIR2.1. In this study, NaV1.5 is used to demonstrate that the technique is applicable to other families of ion channels. METHODS: IV curves were generated using a standard voltage step protocol performed in whole-cell voltage clamp mode on HEK293 cells transiently transfected with SCN5A (encodes NaV1.5). Noise functions containing 1-50 kHz frequencies were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) was then calculated for each trace. Each frequency magnitude as a function of voltage step was correlated with the IV curve. RESULTS: The magnitude of 22.5 and 24.5 kHz correlated well with the IV curve of NaV1.5 in the presence of the noise function (R>0.8), and poorly in the absence of noise (|R|<0.3). Two nodes of zero correlation were also found (11.36 +/- .08 kHz and 36.23 +/- 4.79 kHz. For KIR2.1, current and frequency amplitudes did not correlate well between 11 and 36 kHz, suggesting that this correlation may be unique to NaV1.5. On the other hand, frequencies were identified below 10 kHz whose amplitudes highly correlate with either one or both channels. CONCLUSIONS: These data suggest that specific frequencies exist which can re-create the shape of both KIR2.1 and NaV1.5 IV curves. Furthermore, the correlation at some frequencies is channel specific, while others are not. This methodology could be a powerful tool for assessing the behavior of multiple ionic currents simultaneously during a freely running action potential.

Characteristic Frequency Analysis of Inward Rectifier Kir 2.1

INTRODUCTION: Impedance spectroscopy cannot distinguish between ion channel subtypes. We hypothesized that amplitudes of specific characteristic frequencies will correlate with the current amplitude passed by a specific ion channel subtype (characteristic frequency). We chose to test this hypothesis using the human inward rectifying potassium channel, Kir 2.1. METHODS: IV-relationships were generated using a standard voltage step protocol (−140 to 0mV, 7mV steps) performed in whole-cell voltage clamp mode on HEK293 cells stably transfected with KCNJ2, which encodes Kir 2.1. Noise functions containing equal magnitudes of 1-15 kHz frequencies (amplitudes: 25, 50, 75 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the IV-relationship. RESULTS: In the absence of noise (control), magnitudes of all frequencies correlated poorly (|R|<0.15) with the IV relationship. With noise, magnitudes of frequencies between 0.2-1 and 2-4 kHz demonstrated high negative (R < −0.9) and positive correlation (R > 0.9) respectively, with the IV-relationship. Two nodes of zero correlation were also found (1.39 +/- 0.10 kHz and 8.49 +/- 0.74 kHz). Increasing noise amplitude increased the absolute value of the correlation for the aforementioned frequencies without significantly changing the nodes of zero correlation. CONCLUSIONS: These data suggest that the observed frequency response reflects current passing through Kir 2.1 channels. However it remains unknown whether any of these characteristic frequencies are unique to Kir2.1. Identifying characteristic frequencies of other ion channel subtypes could allow simultaneous measurement of multiple ionic currents.

Glial cell line-derived neurotrophic factor acutely modulates the excitability of rat small-diameter trigeminal ganglion neurons innervating facial skin

Glial cell line-derived neurotrophic factor (GDNF) plays an important role in adult sensory neuron function. However, the acute effects of GDNF on primary sensory neuron excitability remain to be elucidated. The aim of the present study was to investigate whether GDNF acutely modulates the excitability of adult rat trigeminal ganglion (TRG) neurons that innervate the facial skin by using perforated-patch clamping, retrograde-labeling and immunohistochemistry techniques. Fluorogold (FG) retrograde labeling was used to identify the TRG neurons innervating the facial skin. The FG-labeled small- and medium-diameter GDNF immunoreactive TRG neurons, and most of these neurons also expressed the GDNF family receptor α-1 (GFRα-1). In whole-cell voltage-clamp mode, GDNF application significantly inhibited voltage-gated K + transient (I A) and sustained (I K) currents in most dissociated FG-labeled small-diameter TRG neurons. This effect was concentration-dependent and was abolished by co-application of the protein tyrosine kinase inhibitor, K252b. Under current-clamp conditions, the repetitive firing during a depolarizing pulse were significantly increased by GDNF application. GDNF application also increased the duration of the repolarization phase and decreased the duration of the depolarization phase of the action potential, and these characteristic effects were also abolished by co-application of K252b. These results suggest that acute application of GDNF enhances the neuronal excitability of adult rat small-diameter TRG neurons innervating the facial skin, via activation of GDNF-induced intracellular signaling pathway. We therefore conclude that a local release of GDNF from TRG neuronal soma and/or nerve terminals may regulate normal sensory function, including nociception.

Adenosine A2A Receptors Are Essential for Long-Term Potentiation of NMDA-EPSCs at Hippocampal Mossy Fiber Synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.

Streptomycin and gentamicin have no immediate effect on outer hair cell electromotility

The cochlear outer hair cell (OHC), which plays a crucial role in mammalian hearing through its unique voltage-dependent motility, has been established as a primary target of the ototoxicity of aminoglycoside antibiotics. These polycationic drugs are also known to block a wide variety of ion channels, purinergic ionotropic channels, and nicotinic ACh receptors in hair cells in vitro. The OHC motor protein, prestin, is a voltage-sensitive transmembrane protein containing several negatively charged residues on both intra- and extracellular surface. The acidic sites may be susceptible to polycationic-charged aminoglycoside binding, which may result in disruption of motility. We attempted to examine whether aminoglycosides such as streptomycin and gentamicin could affect OHC motility and its electrical signature, the nonlinear capacitance (NLC) in adult gerbil OHCs. Somatic motility and NLC were measured under the whole-cell voltage-clamp mode. Streptomycin and gentamicin were applied extracellularly or intracellularly. Results show that streptomycin and gentamicin did not change either the magnitude of motility or the NLC. Theses results suggest that, although streptomycin and gentamicin can block mechanotransduction channels as well as ACh receptors in hair cells, they have no direct affect on OHC somatic motility.

Regulation of N-Methyl-D-aspartate Receptors by Calpain in Cortical Neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.

Brain-derived neurotrophic factor increases inhibitory synapses, revealed in solitary neurons cultured from rat visual cortex

To elucidate chronic actions of brain-derived neurotrophic factor (BDNF) on GABAergic synapses, we examined effects of a long-term application of BDNF for 10–15 days on autapses (synapses) of solitary GABAergic neurons cultured from rat visual cortex. Solitary neuron preparations were used to exclude a possible contamination of BDNF actions on excitatory neurons in dissociated neuron culture or slice preparations. Neurons were confirmed to be GABAergic pharmacologically with bicuculline, a selective antagonist for GABA A receptors and immunocytochemically with antibody against glutamic acid decarboxylase 65, a GABA synthesizing enzyme. To evaluate GABAergic synaptic function, evoked and/or miniature inhibitory postsynaptic currents (IPSCs) were recorded in the whole-cell voltage-clamp mode. The treatment with BDNF at a concentration of 100 ng/ml enhanced the amplitude of evoked IPSCs and the frequency of miniature IPSCs. In contrast, BDNF did not have a detectable effect on the amplitude of miniature IPSCs and the paired pulse ratio of IPSCs evoked by two, successive activations. To evaluate morphological changes, neurons were immunocytochemically stained with antibodies against microtubule-associated protein 2, to visualize somatodendritic region and synapsin I, to visualize presynaptic sites. The quantitative analysis indicated that BDNF increased the area of soma, the numbers of primary dendrites and dendritic branching points, the total length of dendrites and the number of synaptic sites. Such an action of BDNF was seen in both subgroups of GABAergic neurons, parvalbumin-positive and -negative neurons. To visualize functionally active presynaptic sites, neurons were stained with a styryl dye, FM1-43. BDNF increased the number of stained sites that was correlated with the frequency of miniature IPSCs. These results suggest that the chronic treatment with BDNF promotes dendritic and synaptic development of GABAergic neurons in visual cortex.

Prestin and the Dynamic Stiffness of Cochlear Outer Hair Cells

The outer hair cell (OHC) lateral wall is a unique trilaminate structure consisting of the plasma membrane, the cortical lattice, and subsurface cisternae. OHCs are capable of altering their length in response to transmembrane voltage change. This so-called electromotile response is presumed to result from conformational changes of membrane-bound protein molecules, named prestin. OHC motility is accompanied by axial stiffness changes when the membrane potential of the cell is altered. During length changes, intracellular anions (mainly Cl-) act as extrinsic voltage sensors. In this study, we inquired whether the motor proteins are responsible for the voltage-dependent axial stiffness of OHCs, and whether ACh, the neurotransmitter of efferent neurons, modulates the stiffness of the cortical lattice and/or the stiffness of the motor protein. The experiments were done on isolated guinea pig OHCs in the whole-cell voltage-clamp mode. Axial stiffness was determined by loading a fiber of known stiffness onto the apical surface of the cells. Voltage-dependent stiffness and cell motility disappeared, and the axial stiffness of the cells significantly decreased after removal of intracellular Cl-. The result suggests that the stiffness of the motor protein is a major contributor to the global axial stiffness of OHCs. ACh was found to affect both the motor protein and other lateral wall stiffness components.

Time Course and Permeation of Synaptic AMPA Receptors in Cochlear Nuclear Neurons Correlate with Input

AMPA receptors mediate rapid glutamatergic synaptic transmission. In the mammalian cochlear nuclei, neurons receive excitatory input from either auditory nerve fibers, parallel fibers, or both fiber systems. The functional correlates of differences in the source of input were examined by recording AMPA receptor-mediated, miniature EPSCs (mEPSCs) in whole-cell voltage-clamp mode from identified neurons. Bushy, octopus, and T-stellate cells of the ventral cochlear nucleus (VCN) and tuberculoventral cells of the dorsal cochlear nucleus (DCN) receive most of their excitatory input from the auditory nerve; fusiform cells receive excitatory inputs from both the auditory nerve and parallel fibers; cartwheel cells receive excitatory input from parallel fibers alone. mEPSCs from bushy, octopus, T-stellate, and tuberculoventral cells had significantly faster decay time constants (0.35-0.40 msec) than did those from fusiform and cartwheel cells (1.32-1.79 msec). Some fusiform cells had two populations of mEPSCs with distinct time courses. mEPSCs in cells with auditory nerve input alone were inhibited by philanthotoxin, a blocker of calcium-permeable AMPA receptors, whereas mEPSCs in cells with parallel fiber input were not. Thus AMPA receptors postsynaptic to the auditory nerve differ from those postsynaptic to parallel fibers both in channel-gating kinetics and in their permeability to calcium. These results confirm the conclusion that synaptic AMPA receptors are specialized according to the source of input (Hunter et al., 1993; Rubio and Wenthold, 1997; Wang et al., 1998).

Brain-Derived Neurotrophic Factor Prevents Low-Frequency Inputs from Inducing Long-Term Depression in the Developing Visual Cortex

Brain-derived neurotrophic factor (BDNF) is reported to enhance synaptic transmission and to play a role in long-term potentiation in hippocampus and neocortex. If so, a shortage or blockade of BDNF might lead to another form of synaptic plasticity, long-term depression (LTD). To test this possibility and to elucidate mechanisms if it is the case, EPSCs evoked by test stimulation of layer IV were recorded from layer II/III neurons in visual cortical slices of young rats in the whole-cell voltage-clamp mode. LTD was induced by low-frequency stimulation (LFS) at 1 Hz for 10-15 min if each pulse of the LFS was paired with depolarization of neurons to -30 mV but was not induced if their membrane potentials were kept at -70 mV. Such an LTD was blocked by exogenously applied BDNF, probably through presynaptic mechanisms. Suppression of endogenous BDNF activity by the anti-BDNF antibody or an inhibitor for BDNF receptors made otherwise ineffective stimuli (LFS without postsynaptic depolarization) effective for LTD induction, suggesting that endogenous BDNF may prevent low-frequency inputs from inducing LTD in the developing visual cortex.

Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line

Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na + and K + and symmetric concentrations of Cl − (150 mM) across the plasma membranes. K + currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only ∼20 mV was seen with a change of extracellular K + concentration from 3 to 150 mM. In contrast, the reversal potential shifted ∼25 mV when extracellular Cl − was reduced to 50 mM, indicating that most of the outward currents are carried by Cl −. In inside-out patches, several distinct Cl − currents were identified. They were: (1) 350 pS Cl − current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl − current with a weak outward rectification; and (3) 55 pS Cl − current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl − currents did not require an elevation of free Ca 2+ level in the cytosol. Besides these three currents, we observed two other Cl − currents with much smaller conductances (25 and 16 pS, respectively). Two different K + currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.

Agonist and antagonist effects of apomorphine enantiomers on 5-HT 3 receptors

Direct effects of the enantiomers of the classical dopamine receptor ligand apomorphine on 5-HT 3 receptors were examined in N1E-115 neuroblastoma cells in whole-cell voltage clamp mode. R(−)-apomorphine (R(−)APO; 3–300 μM) evokes a small, transient inward ion current. At 30 μM, R(−)APO induces its maximum inward current, which is approximately 3% of the amplitude of the inward current induced by 10 μM 5-HT. The R(−)APO-induced current is completely and reversibly inhibited after superfusion of 50 nM of the selective 5-HT 3 receptor antagonist MDL 72222 and after desensitization of the 5-HT 3 receptors by 10 μM 5-HT. The results indicate that R(−)APO is a partial agonist at the 5-HT 3 receptor. R(−)APO (30 μM) evokes a depolarization of the membrane potential with an amplitude which is 26% of the 10 μM 5-HT-induced depolarization. In addition, the 5-HT-induced depolarization is reduced (from 29 to 15 mV) after prolonged exposure of the cell to R(−)APO. S(+)-apomorphine (S(+)APO; 3–300 μM) does not evoke an ion current. Instead, S(+)APO antagonizes the 5-HT 3 receptor with an IC 50 of 32 μM. The combined results indicate that enantiomers of apomorphine act directly on 5-HT 3 receptors, and suggest that the in vivo effects of apomorphine are partially attributable to a direct interaction with 5-HT 3 receptors.

Carbachol induces inward current in rat neostriatal neurons through a G-protein-coupled mechanism

Our recent study demonstrated that carbachol can act at M l-like muscarinic receptors to reduce the membrane K + conductance and excite the neostriatal neurons. In the present study, we further studied the molecular mechanism by which carbachol induced inward currents in neostriatal neurons. In acutely isolated neostriatal neurons held at −60 mV, pressure application of carbachol (30 μM) induced a transient inward current underlying whole-cell voltage-clamp mode. In cells loaded with the stable GDP analogue guanosine 5'-0-(2-thiodiphosphate) (GDP- β-S, 1 mM), the carbachol-induced inward current was significantly diminished. However, the carbachol response was not affected by intracellular dialysis of the neostriatal neurons with either protein kinase C (PKC) inhibitors, PKCI 19–36 (5 μM) or NPC-15437 (20 μM), or a potent cAMP-dependent protein kinase (PKA) inhibitor, Rp-cAMPS (25 μM). These results show that a G-protein-coupled mechanism mediates carbachol-induced inward current in the neostriatal neurons and that neither PKC- nor PKA-dependent intracellular transduction pathways are involved in the carbachol response.

Temporal specificity of muscarinic synaptic modulation of the Ca(2+)-dependent K+ current (ISAHP) in rat hippocampal neurones.

1. We examined synaptic modulation of the Ca(2+)-dependent K+ current (ISAHP), which underlies the slow after-hyperpolarization (sAHP) in hippocampal CA1 neurones of rat brain slices. ISAHP was evoked in whole-cell voltage-clamp mode by depolarizing pulses, and synaptic afferents to CA1 neurones were stimulated electrically with a paired-pulse protocol. 2. Afferent stimulation delivered 200-1500 ms prior to be depolarizing pulse produced a profound reduction of ISAHP by 58%, but not other Ca(2+)-dependent outward currents that preceded ISAHP. Perfusion of slices with atropine significantly attenuated the synaptic reduction of ISAHP, indicating an event mediated largely by muscarinic receptor activation. When delivered < 400 ms after the depolarizing pulse, similar synaptic stimuli produced no substantial reduction in ISAHP, even in neurons where the duration of ISAHP was prolonged to 8-10 s either by lowering the recording temperature or by intracellular application of a calcium chelator. 3. To examine the effect of cholinergic stimulation of the depolarization-activated Ca2+ influx, high-threshold voltage-activated Ca2+ currents were recorded in the conventional or perforated whole-cell mode. Perfusion of slices with 5-10 microM carbachol for 5-10 min caused no substantial decrease in these Ca2+ currents, suggesting that the synaptic reduction of ISAHP is unlikely to be due to a blockade of depolarization-induced Ca2+ influx which triggers the generation of ISAHP. 4. The present data demonstrate that afferent stimulation reduces ISAHP only if it occurs prior to the depolarization-induced Ca2+ influx. We propose that modulation of inactive sAHP channels by muscarinic stimulation may decrease their sensitivity to the influx of Ca2+, whereas sAHP channels activated by Ca2+ may compete with the receptor-coupled modulation thus rendering the sAHP channels unresponsive to cholinergic afferent stimulation.

Characterization of whole-cell currents in mucosal and connective tissue rat mast cells using amphotericin-B-perforated patches and temperature control.

Rat mucosal type mast cells are thought to possess only a K+-selective inwardly rectifying (IRK) current in the resting state. We used rat-bone-marrow-derived mast cells (BMMCs) as a model of mucosal mast cells and recorded whole-cell membrane currents from cells perforated with amphotericin B. Under these conditions, both inwardly rectifying (IR) and outwardly rectifying (OR) currents were observed. The reversal potential and conductance of the IR current depended on the extracellular K+ concentration, indicating that the channel was K+ selective. The OR current was not affected by changes in extracellular K+ concentration, but lowering extracellular Cl- concentration reduced the conductance and shifted the reversal potential in a positive direction. The OR current was not affected by K+ channel blockers, but was reversibly blocked by the chloride channel blocker 4,4'-diisothiocyanato-2,2'-stilbenedisulphonate (DIDS), again indicating a Cl- conductance. The IRK current was also detected in the majority of cells using the conventional whole-cell recording configuration at room temperature. In contrast, the ORCl current was only observed in 7% of recordings made at room temperature with the conventional whole-cell voltage-clamp mode, but was detected in 66% of cells if the bath temperature was increased and the integrity of the cell's cytoplasm was preserved by using the perforated-patch technique. Under similar conditions, the ORCl current was also present in rat peritoneal mast cells, a connective tissue phenotype previously thought to have no whole-cell currents in the resting state. The role of this current and factors affecting its activation are discussed.