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PubMed1992

Nerve membrane Na+ channels as targets of insecticides.

The mechanisms of action of neuroactive insecticides on the nervous system has been studied for many years. It is now well established that severe neurological symptoms of poisoning with pyrethroids and DDT in mammals and insects are the result of modification of Na+ channel activity. Toshio Narahashi discusses the history, approaches and results of the studies leading to this conclusion. Advanced electrophysiological experiments using voltage clamp and patch clamp, together with ligand-binding and ionic flux experiments, have unveiled unique actions of pyrethroids and DDT of keeping the Na+ channel in the open state for an extremely long period, sometimes as long as several seconds. This modification of Na+ channel properties leads to hyperactivity of the nervous system. These insecticides have also been shown to suppress GABA and glutamate receptor-channel complexes and voltage-activated Ca2+ channels, but the toxicological significance of these actions remains to be seen. The results of these studies provide clues for developing newer insecticides with higher selectivity between mammals and insects and for coping with the problem of insecticide resistance.

Animals↗

PubMed1986

Two modes of gating during late Na+ channel currents in frog sartorius muscle.

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.

Action Potentials↗

PubMed1989

Voltage-gated and agonist-mediated rises in intracellular Ca2+ in rat clonal pituitary cells (GH3) held under voltage clamp.

1. Intracellular free calcium (Ca2+i) was estimated in single GH3 cells by dual wavelength emission spectrofluorimetry using the Ca2+ indicator dye Indo-1, while cells were held under voltage clamp using patch clamp techniques. 2. Depolarization of cells evoked a transient rise in Ca2+i that increased with increasing duration of depolarization to a peak at about 10 s. 3. Calcium transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. They were abolished in the absence of extracellular calcium and by application of 10 microM-nifedipine. 4. Thyrotrophin-releasing hormone (TRH) evoked a transient rise in Ca2+i that was followed by a more sustained period of elevated Ca2+i in some cells. The transient phase of the response but not the sustained phase was seen in the absence of extracellular calcium. 5. Ca2+i transients evoked by depolarization were not affected by pre-release of internal Ca2+ stores with TRH. 6. The results demonstrate that voltage-gated Ca2+ entry and Ca2+ store release can each elevate cytoplasmic free calcium in GH3 cells and may both be important for stimulus-secretion coupling. Non-voltage-gated Ca2+ entry is not a major source of Ca2+ under these conditions.

Action Potentials↗

PubMed1994

Block by 4-aminopyridine of a Kv1.2 delayed rectifier K+ current expressed in Xenopus oocytes.

1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34 degrees C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4-AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4-AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C-type inactivation and 4-AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4-AP on the Kv1.2 channel is proposed based on experimental data.

4-Aminopyridine↗

PubMed2005

Direct inhibition of substantia gelatinosa neurones in the rat spinal cord by activation of dopamine D2-like receptors.

Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs+ or GDP-beta-S in the pipette solution, and was inhibited in the presence of Ba+. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K+ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K+ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.

Action Potentials↗

PubMed1993

Membrane potential oscillation from a novel combination of ion channels.

Single pigmented epithelial cells from the ciliary body of the eye were studied using the whole cell voltage and current clamp, permeabilized patch recording, and patch-clamp recording. These cells can produce two types of oscillation. Both are slow, with a period in the range of 1-2 min; one has a low amplitude and oscillates between -60 and -80 mV, and the second is larger, with biphasic hyperpolarizing and depolarizing phases. The latter was seen when the membrane potential was driven negative by a constant current and results from the interplay between the inward rectifier K+ channel and a hyperpolarizing-activated cation channel. The hyperpolarization is caused by the constant current acting on a decreasing conductance as the inward rectifier inactivates, and the depolarization drive results from the activation of cation channels. It is suggested that the constant current would be provided by the Na+ pump in vivo, and such an interplay of channels and pumps could drive the uptake of cations in absorbing epithelia or provide an increased driving force for chloride exit in secretory epithelia.

Animals↗

PubMed1998

Angiotensin II Receptor Regulates Ionic Currents in Guinea Pig Ventricular Myocytes.

BACKGROUND: Previous studies have shown that angiotensin II (Ang II) receptors are preset in a wide variety of target tissues and that Ang II regulates the target tissue functions through Ang II receptors. However, the action of Ang II receptors on transsarcolemmal currents in ventricular myocytes has not been elucidated. METHODS AND RESULTS: We performed whole-cell voltage clamp and patch clamp experiments to determine the effects of Ang II-receptor agonists and antagonists on ionic currents in single isolated guinea pig ventricular myocytes. We found that extracellular perfusion of Ang II (30 nM) increased the L-type Ca(2+) current from 581 +/- 27 to 837 +/- 42 pA (n = 5, P <.01). Ang II also prolonged the Ca(2+) current activation and inactivation time constants. These were reversible by losartan (100 nM), a type 1 Ang II receptor (AT(1)) blockade. On the other hand, perfusion of 30 nM Ang II decreased K(+) current (I(K)) from 1543 +/- 28 to 1194 +/- 50 pA (n = 5, P <.05) and K(+) tail current (I(K-tail)) from 275 +/- 24 to 206 +/- 29 pA (n = 5, P <.05). These effects were also abolished by perfusion of losartan. However, perfusion of Ang II resulted in an increase of inward rectified K(+) current (I(K1)) in whole-cell recordings. Single channel recordings showed that the increase in I(K1) was attributed to a burst opening current with a larger unit of amplitude. These effects were reversed by saralasin but not losartan, indicating possible type 2 Ang II receptor (AT(2)) involvement. CONCLUSIONS: Our results provide evidence that Ang II receptors regulate the transsarcolemmal currents in single guinea pig ventricular myocytes. Therefore, Ang II regulation of ionic currents is mediated through the different subtypes of Ang II receptors.

Journal Article↗

PubMed1989

Activations of the Ca dependent K channel by Ca released from the sarcoplasmic reticulum of mammalian smooth muscles.

In mammalian smooth muscles, the outward K current recorded using the whole cell voltage clamp or patch clamp methods can be classified into the Ca-dependent and independent K currents. The former is sub-classified into the extra- and intra-cellular Ca dependent K current. The intra-cellular Ca dependent K current has a close relation to Ca released from the sarcoplasmic reticulum, i.e. Ca released by inositol 1,4,5-trisphosphate (InsP3), ryanodine or Ca ionophores (A23187 or ionomycin) modify the appearance of the K current. The transient (Ca dependent) outward current evoked by depolarization pulses, as measured using the whole cell voltage clamp method, is closely related with after-hyperpolarization of the action potential as recorded using the microelectrode method and is postulated to be due to activations of the Ca-induced Ca release mechanism in the sarcoplasmic reticulum. The oscillatory (Ca dependent) outward K current is closely related with the amount of Ca released from the sarcoplasmic reticulum during the long depolarization induced by electrical stimulation (command pulse) or applications of Ca releasers such as InsP3 or ryanodine. In this review, the Ca dependent K current recorded from smooth muscle cells is compared with the influx and release of Ca.

Animals↗

PubMed1994

Molecular pore structure of voltage-gated sodium and calcium channels.

1. Voltage-gated ion channels have a high homology in their primary structure but yet show quite distinct ion selection properties. Therefore, ion selection was studied in voltage-gated wild-type and mutant sodium (Na RBII) and calcium (Ca BI) channels, which were heterologously expressed in Xenopus oocytes, using two-electrode voltage clamp and patch clamp methods. 2. In order to facilitate the electrophysiological experiments, fast sodium channel inactivation was abolished by introducing the point mutation F1489Q into the linker between the homologous repeats III and IV. 3. The ion pore in rat brain sodium channel II was located by testing single-point mutants for their sensitivity to externally applied tetrodotoxin (TTX), leading to a model in which part of the linker S5-S6 folds into the membrane to form at least part of the ion pore; two amino acids from each domain are thereby of major importance for TTX association and ion permeation, presumably forming two rings of predominantly charged residues in three-dimensional space (outer ring: E387, E945, M1425, D1717; inner ring: D384, E942, K1422, A1714). 4. When the residue F385 in repeat I of Na RBII channel is mutated to a cysteine, the residue being at the homologous position in cardiac sodium channels, the channel lost its high sensitivity for TTX and gained a high sensitivity for external Cd2+ and Zn2+, properties found in cardiac sodium channels. Mutation of the other cysteine in the pore region, C940, had no effect on channel block by TTX or by external divalent cations. 5. The inner ring in sodium channels constitutes a selectivity filter: after mutation of residues K1422 and A1714 into glutamates, the homologous residues in calcium channels, the sodium channels show ion selection properties similar to calcium channels: a) no selectivity among monovalent cations, b) strong block of monovalent current by divalent cations, c) permeation of divalent cations. 6. Reverse mutations in the BI calcium channel, where glutamate residues were mutated into neutral glutamines, showed that also in this channel these amino acids form a selectivity filter. The individual residues interact differently with the permeating ions suggesting an asymmetric spatial arrangement of the four pore-forming regions of the ion channel pore.

Amino Acid Sequence↗

PubMed2000

Pressure polishing: a method for re-shaping patch pipettes during fire polishing.

The resolution of patch-clamp recordings is limited by the geometrical and electrical properties of patch pipettes. The ideal whole-cell patch pipette has a blunt, cone-shaped tip and a low resistance. The best glasses for making patch pipettes are low noise, low capacitance glasses such as borosilicate and aluminasilicate glasses. Regrettably, nearly all borosilicate glasses form pipettes with sharp, cone-shaped tips and relatively high resistance. It is possible, however, to reshape the tip during fire polishing by pressurizing the pipette lumen during fire polishing, a technique we call 'pressure polishing.' We find that this technique works with pipettes made from virtually any type of glass, including thick-walled aluminasilicate glass. We routinely use this technique to make pipettes suitable for whole-cell patch-clamp recording of tiny neurons (1-3 microm in diameter). Our pipettes are made from thick-walled, borosilicate glass and have submicron tip openings and resistances <10 MOmega. Similar pipettes could be used to record from subcellular neuronal structures such as axons, dendrites and dendritic spines. Pressure polishing should also be useful in patch-clamp applications that benefit from using pipettes with blunt tips, such as perforated-patch whole-cell recordings, low-noise single channel recordings and experiments that require internal perfusion of the pipette.

Animals↗

PubMed1995

Cyclic AMP prevents activation of a swelling-induced chloride-sensitive conductance in chick heart cells.

1. Changes in myocardial cell volume and whole-cell currents were measured simultaneously during hyposmotically induced cell swelling. In the conventional patch clamp configuration, hyposmotic challenge caused myocytes to swell continuously and was associated with the development of a sustained, swelling-induced chloride conductance (ICl). In contrast, perforated patch-clamped myocytes demonstrated regulatory volume decreases (RVD) during hyposmotic challenge, and ICl was not generated. 2. The swelling-induced ICl in conventionally patch-clamped myocytes was inhibited by application of forskolin (15 microM) and was prevented when the pipette filling solution contained cAMP (10 microM) and isobutylmethylxanthine (IBMX, 1 mM). ICl could also be prevented by inhibition of protein phosphatase activity, using okadaic acid (100 nM). Conversely, a swelling-induced current could be generated in myocytes under perforated patch clamp by inhibition of protein kinase A, using the antagonist Rp-cAMPS (10 microM). These data demonstrate that cAMP-dependent protein phosphorylation is both necessary and sufficient to prevent development of ICl during cell swelling. 3. Unlike other chloride currents described previously in heart muscle, generation of the novel swelling-induced ICl requires dephosphorylation of a cAMP-dependent protein phosphorylation site; hence it can be prevented by stimulation of cAMP-dependent protein phosphorylation or by inhibition of protein phosphatase activity.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons.

Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau >/= 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time approximately 0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.

Animals↗

PubMed2002

A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions.

This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.

Animals↗

PubMed2000

Molecular cloning and expression of an inwardly rectifying K(+) channel from bovine corneal endothelial cells.

PURPOSE: To determine the presence of a putative inwardly rectifying K(+) channel in bovine corneal endothelial (BCE) cells and to characterize its molecular and electrophysiological properties. METHODS: An RT-PCR strategy was used to clone an IRK1 channel sequence from BCE mRNA. Northern blot analysis was used to confirm expression of this sequence in cultured BCE cells. Two-electrode voltage-clamp and whole-cell patch-clamp recordings were used to characterize the cloned channel expressed in Xenopus oocytes and the native channels in cultured BCE cells, respectively. RESULTS: A full-length (1284 bp) coding sequence that shares 99.7% nucleotide sequence and 100% amino acid sequence identity to bovine lens IRK1 (Kir2.1) was cloned. The authors designate this sequence BCE IRK1 or BCIRK1. Northern blot analysis indicated that BCIRK1 mRNA is expressed in cultured BCE cells with two major transcripts of 7.5 and 5.5 kb. BCIRK1 cDNA was subcloned into the vector, pcDNA3.1(-), and cRNA transcribed from the BCIRK1 cDNA clone was injected into Xenopus oocytes. Two-electrode voltage-clamp recordings from injected oocytes revealed inwardly rectifying K(+) currents that were blocked by external Ba(2+) and Cs(+) in a concentration- and voltage-dependent manner. Whole-cell patch-clamp recordings from dissociated cultured BCE cells revealed strongly inwardly rectifying K(+) currents with similar properties. CONCLUSIONS: Corneal endothelial cells express IRK1 (Kir2.1) inwardly rectifying K(+) channels. Consistent with the properties of IRK1 channels, BCIRK1 is likely involved in regulating membrane potential and possibly other cellular functions in corneal endothelial cells.

Amino Acid Sequence↗

PubMedDate not supplied

Automated electrophysiology in the preclinical evaluation of drugs for potential QT prolongation.

INTRODUCTION: The human ether-a-go-go-related gene (hERG) potassium channel plays a major role in the electrical conductances involved in human heart repolarization. Drugs that decrease hERG K(+) currents are at risk to produce a prolongation of the cardiac action potential, resulting in an increase of the QT interval. Drug-induced QT prolongation or acquired long QT (aLQT) can lead to a fatal arrhythmia known as Torsade de Pointes (TdP). Electrophysiological methods are the best approach to evaluate potential drug candidates for hERG current inhibition. Here we identify limitations with the PatchXpress 7000A automated electrophysiology instrument and describe hERG protocol optimizations necessary for reliable preclinical assessment. METHODS: The PatchXpress 7000A automated electrophysiology system was used to evaluate a group of drugs with known hERG activity under voltage clamp conditions. We used a recombinant cell line expressing hERG, and assessed the inhibition of hERG K(+) currents at different drug concentrations. These data were used to determine hERG IC(50) values and compare assay parameters under different recording conditions. RESULTS: We found that due to limitations with the PatchXpress 7000A instrument, repeated compound additions were critical for achieving steady state drug concentrations that generated data comparable to standard patch clamp methods, particularly when similar voltage pulse protocols were implemented. Some discrepancies were observed between the PatchXpress 7000A and standard patch clamp techniques including shifts in IC(50) values for very hydrophobic compounds. Most hERG IC(50) values were within 3-fold of standard patch clamp IC(50) values. DISCUSSION: Automation of electrophysiology technologies has greatly improved the throughput of assessing lead drug candidates for hERG liability. To maintain hERG data quality comparable to standard patch clamp techniques, the PatchXpress 7000A instrument limitations should be recognized and protocols optimized accordingly to ensure accuracy.

Animals↗

PubMed1990

Complex regulation of calcium current in cardiac cells. Dependence on a pertussis toxin-sensitive substrate, adenosine triphosphate, and an alpha 1-adrenoceptor.

We investigated regulation of the cardiac L-type calcium channel by intracellular ATP and by alpha 1-adrenergic agonism using single adult guinea pig ventricular cells and the whole-cell patch clamp method. Inclusion of 5 mM ATP in the patch clamp pipette prevented calcium current rundown but did not increase the maximal magnitude of the slow inward calcium current (ICa). During beta 1-adrenergic blockade with 10 microM (-)-propranolol, cells preincubated with 1 microgram/ml pertussis toxin for 2-5 h exhibited a rapid twofold increase in ICa after rupture of the membrane patch when 5 mM ATP was present in the patch clamp pipette. In the absence of ATP, the increase in ICa did not occur. In pertussis toxin-treated cells, 100 microM (-)-phenylephrine inhibited the augmentation of ICa. This inhibitory effect was blocked by 100 nM terazosin, a selective alpha 1-antagonist. The inhibitory effect of alpha 1-adrenergic agonism was not mediated by cAMP-dependent phosphodiesterase since incubation with 100 microM (-)-phenylephrine did not augment the activity of this enzyme. We conclude that regulation of the L-type calcium channel in cardiac cells is complex, and is dependent on a pertussis toxin-sensitive substrate, ATP, and an alpha 1-adrenergic receptor. The marked increase in ICa after pertussis toxin treatment in the presence of ATP indicates significant inhibition of ICa by a pertussis toxin substrate, presumably the guanine nucleotide inhibitory protein (Gi) in the basal state. The inhibitory action of (-)-phenylephrine in pertussis toxin-treated cells is consistent with modulation of ICa by an alpha 1-adrenergic receptor not coupled to Gi.

Adenosine Triphosphate↗

PubMed1994

Involvement of Na(+)-H+ antiporter in regulation of L-type Ca2+ channel current by angiotensin II in rabbit ventricular myocytes.

The present study investigated the possible involvement of a Na(+)-H+ antiporter in the regulation of L-type Ca2+ channels by angiotensin II (Ang II) in isolated rabbit ventricular cardiac myocytes by using both cell-attached and whole-cell patch-clamp current recording techniques. In cell-attached patch-clamp current recordings, an increase in the open-state probability of the Ca2+ channel (144.8 +/- 9.8% [mean +/- SEM], n = 11) was seen after exposure of the cells to Ang II (100 nmol/L). This effect was inhibited by pretreatment with losartan (10 mumol/L), a synthetic antagonist of the AT1 receptor. 5(N,N-Dimethyl)amiloride (100 mumol/L), an amiloride analogue, as well as Na(+)-deficient bath solution abolished Ang II-induced stimulation of the Ca2+ channel activities. In whole-cell patch-clamp current recordings, Ang II also increased the L-type Ca2+ current when a pipette solution of pH 7.1 containing 5 mmol/L HEPES (139 +/- 5%, n = 4) was used but did not significantly increase the current when a pipette solution of pH 7.5 containing 5 mmol/L HEPES or a pipette solution of pH 7.1 containing 30 mmol/L HEPES was used. These results suggest that Ang II-induced stimulation of the Ca2+ channels is mediated by a Na(+)-H+ antiporter and therefore provide a novel insight into signal transduction of Ang II receptor stimulation in cardiac myocytes.

Angiotensin II↗

PubMed2001

Combined pre- and postsynaptic action of IgG antibodies in Miller Fisher syndrome.

BACKGROUND: Miller Fisher syndrome (MFS), a variant of the Guillain-Barré syndrome, is associated with the presence of neuromuscular blocking antibodies, some of which may be directed at the ganglioside GQ1b. MATERIALS AND METHODS: The authors investigated the in vitro effects of serum and purified immunoglobulin (Ig) G in a total of 11 patients with typical MFS during active disease, and in three of those patients after recovery. From one patient's serum, we prepared an IgG fraction enriched in anti-GQ1b antibodies by affinity chromatography. For combined pre- and postsynaptic analysis, endplate currents were recorded by a perfused macro-patch clamp electrode. Postsynaptic nicotinic acetylcholine receptor channels were investigated by an outside-out patch clamp technique in cultured mouse myotubes. RESULTS: AllMFS-sera depressed evoked quantal release and reduced the amplitude of postsynaptic currents. Five of the 11 sera were additionally examined by outside-out patch clamp analysis and caused a concentration-dependent and reversible decrease in acetylcholine-induced currents. The time course of activation and desensitization of nicotinic acetylcholine receptor channels was not altered by MFS-IgG. Nine patients (82 %) were positive for anti-GQ1b antibodies in ELISA and dot-blot. The enriched anti-GQ1b antibody fraction had a similar effect as whole serum. After recovery from MFS, blocking activity was lost and sera originally positive for anti-GQ1b antibodies became negative. CONCLUSION: Circulating IgG antibodies induce both pre- and postsynaptic blockade and may play a pathogenic role in acute MFS.

Acetylcholine↗