Christian Franke

Ketamine blocks currents through mammalian nicotinic acetylcholine receptor channels by interaction with both the open and the closed state

Single channel recordings have shown that ketamine (Ket) decreases the open time of the nicotinic acetylcholine receptor channel (nAChR).The present experiments on simultaneous openings of the nAChRs of mouse myotubes investigate the interaction of Ket with the open as well as with the closed state of the channels. The patch-clamp technique was used to record currents activated by 10-4 M acetylcholine (ACh) in the outside-out mode. ACh together with increasing concentrations of Ket was applied with a piezo-driven system. In a second protocol, the patches were preexposed to Ket before activation with ACh. With addition of Ket, the currents showed a biexponential decay, indicating an open-channel block. The peak current amplitude decreased reversibly and in a concentration-dependent manner. The rate constants of block (b+1) and of unblock (b-1) were modeled by computer simulation and were found to be: b (+1) = 3 times 106 M/s, b-1 = 100/s. Preexposure of the patches to Ket revealed an additional block with a KD of approximately 2 times 10-6 M, which is below clinical concentrations. These data suggest that Ket also interacts with the closed state of the nAChR. (Anesth Analg 1996;83:830-6)

Isoflurane and Sevoflurane Interact with the Nicotinic Acetylcholine Receptor Channels in Micromolar Cconcentrations

Background This study was performed to elucidate and compare the effects of sevoflurane and of isoflurane on the nicotinic acetylcholine receptor of mouse myotubes. The experiments were done with special reference to anesthetic concentrations considerably less than those used for clinical anesthesia. Methods The patch‐clamp technique was used to record acetylcholine‐activated currents from the embryonic type of the nicotinic acetylcholine receptor in the outside‐out mode. A piezo‐driven liquid filament switch was used for the ultrafast application of acetylcholine alone or in combination with isoflurane or sevoflurane. In addition, the patches were preexposed to either anesthetic, preceding the activation with acetylcholine. Results The current elicited by acetylcholine was reduced reversibly and in a concentration‐dependent manner by both anesthetics, which were equally effective. Preexposure of the patches to isoflurane or sevoflurane showed an additional inhibition that was present at micromolar concentrations. The time courses of current decay could be fitted by single exponentials for isoflurane. At higher concentrations of sevoflurane, the current decay became biexponential. In contrast to isoflurane, sevoflurane increased the time constants of desensitization when applied in low concentrations. Conclusion At the nicotinic acetylcholine receptor, isoflurane and sevoflurane act primarily through the same mechanisms: Both affect the open and the closed state of the channels in concentrations equal to and less than those encountered clinically. The kinetics of desensitization, however, are altered in a different manner. Thus there may be several different sites of interaction.

Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps.

1. Pulses of acetylcholine (ACh) in concentrations between 0.1 and 1000 microM were applied repetitively to outside‐out patches of enzymatically denervated (14 days) mouse muscle with the liquid filament switch. Solutions superfusing the patch could be changed rapidly (within 0.2 ms). 2. Single‐channel activity was studied under steady‐state conditions in the outside‐out and in the cell‐attached mode. The single‐channel conductance was 26 pS in outside‐out patches, characteristic for embryonic‐like channels. Apparent mean open time was about 2.5 ms, a shorter component of closed times was 800 microseconds and burst length was about 5 ms. 3. Channel currents elicited by pulses of ACh were averaged. The time‐to‐peak current was concentration dependent and decreased from a level of about 10 ms below 10 microM to about 400 microseconds at 100 microM‐ACh. 4. For a typical experiment, the average peak current, imax, increased from ‐0.4 pA with 0.1 microM to ‐82 pA with 1000 microM‐ACh, close to the value at saturation. The half‐maximal response was at 60 microM‐ACh. The dose‐response curves for imax had double‐logarithmic slopes of 1.1‐1.3, consistent with two binding sites at the embryonic nicotinic acetylcholine receptor (nAChR). 5. The current elicited by ACh pulses decreased rapidly after the peak. The time constant of desensitization increased from 20‐50 ms with 1000 microM‐ACh to up to more than a second with 1 microM‐ACh. 6. The current in steady state (fully desensitized) increased up to 10 microM‐ACh, but decreased slightly to values of imax/100 to imax/500 when higher concentrations were applied. 7. In addition to the well‐known differences between adult and embryonic nAChR concerning the apparent mean open time and burst length, we found differences in the slope of the dose‐response curve for imax, in the ratio of peak to steady‐state response, and in the rise time of the response.

Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis

Objective: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies. Background: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG). Methods: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used. Results: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with α-bungarotoxin, a similar mechanism of block was found. Conclusions: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.

Molecular modulation of recombinant rat α1β2γ2 GABAA receptor channels by diazepam

Recombinant gamma-aminobutyric acid (GABA(A)) receptor channels containing alpha1beta2gamma2-subunits were transiently expressed in HEK293 cells. Modulation by diazepam (DZ) was investigated using the patch-clamp technique with a device for ultra-fast solution exchange. GABA activated Cl(-)-currents were potentiated when DZ > 0.1 microM was added to non-saturating concentrations of GABA (< 0.1 mM GABA). Maximal potentiation of the peak current amplitude by a factor of 2.5 was observed when 1 microM DZ was added to the test-solution. Deactivation of GABA-activated currents after the end of GABA pulses was best fitted with two time constants. After application of DZ + GABA, increase of time constants of deactivation was measured. It was independent on GABA concentration. We conclude that prolongation of deactivation after application of GABA + DZ may be an important mechanism of the modulatory action of DZ at GABA(A) receptor channels.

Dual action of isoflurane on the γ-aminobutyric acid (GABA)-mediated currents through recombinant α1β2γ2L-GABAA-Receptor channels

Isoflurane (ISO) increased the agonist-induced chloride flux through the &ggr;-aminobutyric acid A receptor (GABAAR). This may reflect an anesthetic-induced increase in the apparent agonist affinity. A dual effect of anesthetics was postulated for both the nicotinic acetylcholine receptor (nAChR) and the GABAAR. We tested the hypothesis that, in addition to a blocking effect, ISO increases &ggr;-aminobutyric acid (GABA)-gated currents through recombinant GABAAR channels. HEK293 cells were transfected with rat cDNA for &agr;1,&bgr;2,&ggr;2L subunits. Currents elicited by 1 mM or 0.01 mM GABA, respectively, alone, or with increasing concentrations of ISO, were recorded by using standard patch clamp techniques. ISO reduced the peak current elicited by 1 mM GABA. Currents induced by 0.01 mM GABA were potentiated by small ISO (twofold at 0.5 mM ISO) and inhibited by larger concentrations. Withdrawal of ISO and GABA induced rebound currents, suggesting an open-channel block by ISO. These currents increased with increasing concentrations of ISO. At large concentrations of ISO, the inhibitory effect predominated and was caused by, at least partly, an open-channel block. At small concentrations of ISO, potentiation of the GABA-gated currents was more prominent. This dual action of ISO indicates different binding sites at the GABAAR. The balance between potentiation and block depends on the concentrations of both ISO and GABA. Implications Isoflurane (ISO) interacts with the inhibitory &ggr;-aminobutyric acid (GABA) receptor. This patch clamp study demonstrates that it may block or potentiate the type A of GABA receptor studied, depending on the concentrations of ISO and of GABA used. At clinically relevant concentrations, ISO considerably potentiates this receptor. This may partly explain its clinical effect.

Activation kinetics and single channel properties of recombinant α1β2γ2L GABAΑ -receptor channels

RECOMBINANT α1δ2α2L GABAA receptor channels, transiently expressed in HEK 293 cells, were investigated using the patch-clamp technique in combination with a device for ultra-fast solution exchange. The dose–response relationship revealed an EC50 of 11.6 ± 0.9 μM and saturated with 3 mM GABA. The slope between 0.001 and 0.01 mM GABA was 2.2 ± 0.4, indicating at least three binding sites for GABA. The rise time decreased from about 120 ms at 0.001 mM GABA to about 0.8 ms at 10 mM GABA. Single channel openings were grouped in bursts with an average duration of 10.3 ± 3.0 ms. More than 95% of the current was represented by a single channel slope conductance of about 29 pS.

Interaction of midazolam with the nicotinic acetylcholine receptor of mouse myotubes

The effects of midazolam on the peripheral embryonic nicotinergic acetylcholine receptor (nAChR) of mouse myotubes were studied to elucidate the mechanism of its effect on neuromuscular transmission.Standard patch clamp techniques on outside-out patches were used. Pulses of 10-4 M acetylcholine (ACh) applied by a liquid filament switch technique elicited macroscopic channel currents with a peak current amplitude of approximately 40 pA within <1 ms. The current decayed with a time constant of 30-100 ms due to desensitization. When midazolam was added in stepwise increased concentrations (10-7 M to 7 x 10-4 M) to the pulses, the current decay became bi-exponential, and a concentration-dependent decrease of the fast component of decay was observed. The current amplitude, however, was reduced slightly, and only at high concentrations of midazolam. This may indicate that midazolam binds to the open channel to cause the block. The rate constant of block (b+1) was found to be 1.8 x 106 M/s. Recovery experiments revealed a rate of unblocking (b-1) of approximately 2 x 10-1 s-1. After preincubation of the patches with midazolam, a substantial reduction of the current amplitude was seen at very low midazolam concentrations (<10-7 M), which suggests an additional closed channel block with a Kd of approximately 10-6 M. This closed channel block may be responsible for the muscle-relaxing effects of midazolam. (Anesth Analg 1997;85:174-81)

Block of nicotinic acetylcholine-activated channels of cultured mouse myotubes by isoflurane

It is well known that volatile anesthetics cause muscle relaxation. A block of nicotinic acetylcholine-activated receptors (nAChRs) in staedy state by isoflurane was recently reported. Pulses of acetylcholine (ACh) were applied to outside-out patches from mouse myotubes using a system for ultra-fast solution exchange allowing the study of the block of nAChRs by isoflurane under conditions similar to the situation during synaptic transmission. Isoflurane in concentrations used during general anesthesia blocked approximately 50% of the receptors within 0.5 ms after application. The block of nAChRs could be partially relieved by application of high concentrations of ACh. Therefore, muscle relaxation and the reduction of the amplitude of postsynaptic currents by isoflurane may be caused by the block of nAChRs reported here.

The NO synthase inhibitors L-Name and L-NMMA, but not L-arginine, block the mammalian nicotinic acetylcholine receptor channel.

(1) Nitric oxide (NO) synthase inhibitors (NOS-I) such as L-Name (N(G)-nitro L-arginine methyl ester) and L-NMMA (N(G)-monomethyl L-arginine) may enhance anesthesia indirectly by inhibiting the NO pathway. Moreover, NOS-I interact directly with receptor proteins. In an animal study, L-NMMA potentiated muscle relaxants. (2) The present experiments investigate the effects of L-NMMA, L-Name, and L-arginine on the nicotinic acetylcholine receptor channel (nAChR) using patch clamp techniques and a piezo-driven application system. Both NOS-I appear to directly interact with the nAChR in the open as well as in the closed conformation. L-Arginine has no effect.