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Results for “patch clamp”

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PubMed2002

Anticonvulsant actions of lamotrigine on spontaneous thalamocortical rhythms.

PURPOSE: This study examined the actions of lamotrigine (LTG) on epileptiform discharges resembling generalized absence (GA) and primary generalized tonic-clonic (GTC) seizures in rat thalamocortical (TC) brain slices and attempted to characterize further the cellular mechanisms of action of LTG on neuronal ionic conductances. METHODS: Rat TC slices generated spontaneous generalized epileptiform discharges after perfusion with a medium containing no added Mg(2+). Using multiple channel extracellular field-potential recordings in thalamus and cortex, the effects of LTG were characterized on two principal variants of activity that are similar to spike-wave discharges (SWDs) of GA epilepsy and GTC seizure discharges. These were termed simple TC burst complexes (sTBCs) and complex TC burst complexes (cTBCs), respectively. With whole-cell patch-clamp recording techniques in acutely dissociated TC neurons, the effects of LTG on GABA (gamma-aminobutyric acid)(A)-receptor-mediated currents and the low-threshold calcium current (I(T)) were examined. RESULTS: In field-potential recording studies in TC slices, both sTBCs and cTBCs were blocked by clinically relevant concentrations of LTG. In patch-clamp recording studies, LTG was found to be ineffective in the modulation of both GABA(A) receptors (GABARs) and I(T) in TC neurons. CONCLUSIONS: The efficacy of LTG on both variants of epileptiform discharges in TC slices clearly parallels its broad human clinical spectrum of action. This demonstrates that neurons within the TC system constitute one probable therapeutic target of LTG. However, LTG did not block either GABAR-mediated responses or I(T) in TC neurons. Modulation of these conductances represent likely cellular mechanisms of action of other antiepileptic drugs effective in the control of GA epilepsy. This suggests that LTG may have as yet uncharacterized effects that could combine with its previously defined sodium channel-blocking actions to explain its clinical utility in the control GA seizures.

Animals↗

PubMed1995

Electrophysiological effect of BRL-32872, a novel antiarrhythmic agent with potassium and calcium channel blocking properties, in guinea pig cardiac isolated preparations.

The effects of N-(3,4-dimethoxyphenyl)-N-[3[[2-(3,4-dimethoxyphenyl) ethyl] propyl]-4-nitrobenzamide hydrochloride (BRL-32872), a novel antiarrhythmic agent, were studied in guinea pig cardiac preparations using standard microelectrode and patch-clamp techniques. In papillary muscle, BRL-32872 did not change resting membrane potential and maximum rate of depolarization but prolonged action potential duration (APD) by 24% +/- 2% at 1.0 microM. When the concentration was increased to 3.0 and 10.0 microM, the effect on APD was not further enhanced, and a bell-shaped dose-response curve resulted. Patch-clamp experiments in isolated myocytes showed that BRL-32872 inhibited the rapidly activating component of the delayed rectifier potassium current (EC50 = 0.028 microM) and the L-type calcium current (EC50 = 2.8 microM) but had a limited effect on the inward rectifier potassium current. In papillary muscles stimulated at 300, 500, 1000 and 2000 msec, the effect of BRL-32872 in prolonging APD did not vary (P = .717). By contrast, N-(4-(1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidyl)- carbonyl]phenyl)methanesulfonamide dihydrochloride dihydrate (E-4031), a pure class III antiarrhythmic agent, increased APD more at slower than at faster stimulation rates (P = .001), which illustrated the reverse frequency-dependence of this agent. Among the 35 experiments performed with BRL-32872, only one fiber showed early afterdepolarizations (EADs), and these, which occurred at 1.0 microM, were suppressed at higher concentration (3.0 microM). Moreover, EADs induced by E-4031 were suppressed by BRL-32872 (3.0 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗

PubMed2000

Mechanical injury alters volume activated ion channels in cortical astrocytes.

UNLABELLED: Although astrocytic swelling is likely to mediate brain edema and high ICP after traumatic brain injury, the mechanism is not understood. We employed whole cell patch clamp electrophysiology and a stretch injury model to understand whether volume regulating ion currents are altered following cell injury. Mixed rat astrocytes and neurons were co-cultured on deformable silastic membranes. Mild-moderate cell injury was produced using a timed pulse of pressurized air to deform the silastic substrates by 6.5 mm. Then, ion currents were recorded with patch clamp methods. Cells were held at -65 mV and were stepped to +10 mV to monitor current changes. RESULTS: In unstretched astrocytes, small amplitude currents were obtained under isotonic conditions. Hypotonic solution activated an outwardly-rectifying current which reversed near -40 mV. This current resembled a previously reported anion current whose activation may restore cell volume by mediating a net solute efflux. In contrast, stretch injured cells exhibited a large amplitude, nonrectifying current. This current was not due to non-specific ionic leakage, since it was fully suppressed by the cation channel blocker gadolinium. Activation of novel stretch-activated cation currents may exacerbate cell swelling in injured astrocytes. Stretch injured astrocytes thus express a dysfunctional cation current as opposed to an osmoregulatory anion current. This mechanism, if present in vivo, may contribute to the cytotoxic swelling seen after traumatic brain injury.

Animals↗

PubMed2005

Modulation of ICa-L by alpha1-adrenergic stimulation in rat ventricular myocytes.

We found when L-type calcium current (ICa-L) was recorded with the perforated patch-clamp method in rat ventricular myocytes that bath application of phenylephrine (with propranolol) evoked a biphasic response characterized by an initial transient suppression followed by a sustained potentiation. The transient suppression occurred 30-60 s after phenylephrine perfusion and reached peak inhibition at approximately 2 min. The biphasic modulation of ICa-L was also elicited by methoxamine, and the effects of phenylephrine were blocked by prazosin, indicating that the responses were mediated through alpha1-adrenoceptors. Pretreatment of cells with H7 (100 micromol/L), a broad-spectrum protein kinase inhibitor that inhibits both protein kinase C and A, eliminated potentiation but did not affect transient suppression. The transient suppression occurred concurrently with the acceleration of the fast component of ICa-L inactivation. Depletion of intracellular Ca2+ stores by ryanodine plus caffeine or thapsigargin eliminated the transient suppression. When ICa-L was recorded with whole-cell patch-clamp and with 0.05 mmol/L EGTA in the pipette solution to allow intracellular Ca2+ to fluctuate, phenylephrine evoked a transient suppression as in the perforated patch recordings. Heparin, a specific blocker of IP3 (inositol 1,4,5-trisphosphate) receptors, eliminated the phenylephrine-induced transient suppression of ICa-L when added to the pipette solution. Intensive chelation of intracellular Ca2+ by 5 mmol/L BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) in the pipette solution also eliminated the phenylephrine-induced transient suppression of ICa-L. We conclude that transient increase in the concentration of intracellular calcium ([Ca2+]i) caused by Ca2+ release from intracellular stores underlies the transient suppression of ICa-L, whereas the potentiation of ICa-L is a result of activation of protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1990

Electrophysiological characterization of contact sites in brain mitochondria.

From morphological and biochemical studies it has been recognized that the regions where the outer and inner membranes of mitochondria come in close contact (contact sites) can be the route mechanism through which mitochondria interact directly with the cytoplasm. We have studied these regions electrophysiologically with the patch clamp technique, with the aim of understanding if this direct interaction is mediated by high conductance ion channels similar to the channel already detected in the inner membrane of mitochondria (Sorgato M. C., Keller, B. U., and Stühmer, W. (1987) Nature 330, 498-500). Contact sites isolated from rat brain mitochondria were thus incorporated into liposomes subsequently enlarged sufficiently to be patch clamped. This study shows that these particular fractions contain ion channels with conductances ranging from approximately 5 picosiemens to 1 nanosiemens (in symmetrical 150 mM KCl). Most of these channels are not voltage-dependent and can be open at physiological potentials sustained by respiring mitochondria. The lack of voltage sensitivity seems not to be the outcome of methodological artifacts, as voltage-gated channels are detected in giant liposomes containing either the outer mitochondrial membrane or a partially purified fraction of the inner mitochondrial membrane. These data therefore indicate that channels present in mitochondrial contact sites have properties which render them amenable to perform several of the functions hypothesized for these regions, particularly that of translocating macromolecules from the cytoplasm to the matrix of mitochondria.

Animals↗

PubMed2005

Metamizol acts as an ATP sensitive potassium channel opener to inhibit the contracting response induced by angiotensin II but not to norepinephrine in rat thoracic aorta smooth muscle.

Clinically metamizol (MZ) has been related to alteration on haemodynamic parameters and modifications on blood pressure in humans when administered intravenously. These effects have been observed at MZ therapeutic doses. Experimentally, MZ is able to induce relaxation on several types of vascular smooth muscles and modulates the contraction induced by phenylephrine. However, the mechanism underlying the MZ effects on vascular reactivity is not clear. Potassium channels (K) present on vascular smooth muscle cells closely regulate the vascular reactivity and membrane potential. There are four described types of K in vascular tissue: K voltage sensitive (K(V)), K calcium sensitive (K(Ca)2+), K ATP sensitive (K(ATP) and K inward rectification (K(IR), voltage sensitive). The aim of this work was to investigate MZ effects on angiotensin II (AT II) and noradrenaline (NA) induced contraction and to evaluate the K participation on MZ modulating effect on vascular smooth muscle contraction, using isometric and patch clamp techniques. MZ induces relaxation in a concentration dependent manner. Furthermore, MZ strongly inhibits in a concentration dependent fashion the contraction induced by AT II. However, MZ inhibition on NA induced contraction was moderated compared with that observed on AT II. MZ effects on AT II induced contraction was blocked by glybenclamide (a specific K(ATP) blocker, 3 microM, *p < 0.01). In patch clamp experiments, MZ (3 mM) induces an increase on potassium current (K+) mediated by K(ATP) in similar way as diazoxide (a specific K(ATP) opener, 3 microM). Our results suggest that MZ induces relaxation and inhibits contraction induced by AT II acting as a K(ATP) opener.

Adenosine Triphosphate↗

PubMed2002

The virally encoded fungal toxin KP4 specifically blocks L-type voltage-gated calcium channels.

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. Previous studies demonstrated that this toxin inhibits growth of the target fungal cells by blocking calcium uptake rather than forming channels, as had been suggested previously. Unexpectedly, this toxin was also shown to inhibit voltage-gated calcium channel activity in mammalian cells. We used whole-cell patch-clamp techniques to further characterize this activity against mammalian cells. KP4 is shown to specifically block L-type calcium channels with weak voltage dependence to the block. Because KP4 activity is abrogated by calcium, KP4 probably binds competitively with calcium to the channel exterior. Finally, it is shown that chemical reagents that modify lysine residues reduce KP4 activity in both patch-clamp experiments on mammalian cells and in fungal killing assays. Because the only lysine residue is K42, this residue seems to be crucial for both mammalian and fungal channel activity. Our results defining the type of mammalian channel affected by this fungal toxin further support our contention that KP4 inhibits fungal growth by blocking transmembrane calcium flux through fungal calcium channels, and imply a high degree of structural homology between these fungal and mammalian calcium channels.

Animals↗

PubMed1990

Lack of direct evidence for a functional role of voltage-operated calcium channels in juxtaglomerular cells.

In this study we have examined the role of voltage-gated calcium channels in the regulation of calcium in juxtaglomerular cells. Using a combination of patch-clamp and single-cell calcium measurement we obtained evidence neither for voltage-operated calcium currents nor for changes of the intracellular calcium concentration upon acute depolarizations of the cell membrane. Increases of the extracellular concentration of potassium to 80 mmol/l depolarized the juxtaglomerular cells close to the potassium equilibrium potential, but did not alter the intracellular calcium concentration neither in patch-clamped nor in intact Furaester-loaded cells. Moreover, basal renin secretion from a preparation enriched in mouse juxtaglomerular cells and from rat glomeruli with attached juxtaglomerular cells was not inhibited when extracellular potassium was isoosmotically increased to 56 mmol/l. In mouse kidney slices, however, depolarizing potassium concentrations caused a delayed inhibition at 56 mmol/l and a delayed stimulation of renin secretion at 110 mmol/l. Taken together, our study does not provide direct evidence for a role of voltage-activated calcium channels in the regulation of calcium and renin secretion in renal juxtaglomerular cells.

Animals↗

PubMed1992

A large conductance ion channel in the nuclear envelope of a higher plant cell.

To detect and characterize ion channel activity in the nuclear envelope of a higher plant cell, we performed patch clamp experiments on nuclei isolated from coconut endosperm cells and on giant liposomes containing nuclear envelope fragments prepared from the same cells. An ion channel exhibiting a number of conductance substates, with a maximum of ca. 1,000 pS, was observed. Above an applied potential of +/- 100 mV, the behavior of the channel was similar in isolated nuclei and liposomes, indicating that both patch clamp modes were detecting the same channel. That such a channel has now been identified in members of both the animal and plant kingdoms reinforces the notion that the nuclear pores are not always open to ions.

Ion Channels↗

PubMed1992

Ionic currents in single cells from human cystic artery.

The patch-clamp technique was used to study the electrophysiological properties of single smooth muscle cells obtained from the human cystic artery. These cells contracted on exposure to high K+ and had a mean resting potential of -36 +/- 7 mV. Under current clamp, regenerative responses could not be elicited when depolarizing pulses were applied. Voltage-clamp measurements demonstrated that a large fraction of the outward current was inhibited by tetraethylammonium (5-10 mM) or Ca2+ channel blockers and that it was enhanced by increasing [Ca2+]o, suggesting that it is a Ca(2+)-activated K+ current. In addition, spontaneous transient outward currents that were sensitive to extracellular Ca2+ were observed in some cells. In cell-attached patch-clamp recordings, Ca(2+)-activated K+ channels that had a conductance of 117 pS were consistently identified. At negative potentials (approximately -60 mV), these single-channel events deactivated completely and very quickly, suggesting that they do not control the resting membrane potential in healthy cystic artery cells. Ca2+ currents that were recorded using Ba2+ (10 mM) as the charge carrier were enhanced by the dihydropyridine agonist, Bay K 8644, and blocked by nifedipine (0.1 microM). Only one type of Ca2+ current, the L-type, could be identified in these cells. These results demonstrate that the major ionic currents in the human cystic artery are similar to other mammalian arteries and indicate that this tissue will be a useful model for studying the metabolic and pharmacological modulation of ionic currents in human vascular smooth muscle.

Adult↗

PubMed1992

Electrophysiological studies of anion secretion in cultured human epididymal cells.

1. Primary monolayer cultures from adult human epididymis were grown on Petri dishes and previous supports. The epithelia so formed were used for whole-cell patch clamp recording and short-circuit current (ISC) measurement. 2. After 50 days of culture, the cells formed a tight epithelium with transepithelial potential of 5.5 +/- 1.3 mV (mean +/- S.E.M.., n = 16), apical side negative, and a basal ISC of 6.9 +/- 0.9 microA cm-2 (mean +/- S.E.M., n = 16). 3. Adrenaline, when added to the basolateral side, at a concentration of 0.23 mumol l-1 increased the ISC by 3.0 +/- 1.2 microA cm-2 (mean +/- S.E.M., n = 4). This increase was blockable by diphenylamine-2-carboxylate (DPC, 1 mmol l-1). Forskolin (10 mumol l-1) also evoked a similar response to adrenaline. 4. In whole-cell patch clamp experiment, the resting membrane potential of the cells after dialysis with pipette solution containing 135 mmol l-1 KCl was found to be -30 +/- 14 mV (mean +/- S.E.M., n = 15). 5. About 90% of the cells successfully forming patches responded to 1 mumol l-1 adrenaline by an increase in inward current at -70 mV holding potential (delta I = -1600 +/- 900 pA, mean +/- S.E.M., n = 15). This increase in current was accompanied by a shift in reversal potential to -2 +/- 1 mV (mean +/- S.E.M., n = 15). 6. The adrenaline-induced inward current was found to be blockable by the Cl- channel blocker, DPC (0.25 mmol l-1). Ion substitution experiments showed that the adrenaline-evoked current was carried mainly by Cl-. 7. The effect of adrenaline on the whole-cell current was found to be mimicked by forskolin and could be abolished by including GDP beta S or a protein kinase A inhibitor in the pipette solution. Propranolol, but not phentolamine, completely abolished the effect of adrenaline. 8. Inclusion of 20 mmol l-1 EGTA or 2 mmol l-1 BAPTA + 100 mumol l-1 TMB-8 (to inhibit intracellular Ca2+ release) in the pipette did not seem to have any marked effect on adrenaline-evoked whole-cell current. Lowering the pipette Ca2+ concentration to 1 nmol l-1 or raising it to 10 mumol l-1 had no effect on the whole-cell current response to adrenaline. 9. This study shows that adrenaline stimulates Cl- secretion in cultured human epididymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic Agonists↗

PubMed1999

beta3-adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway.

In human cardiac myocytes, we have previously identified a functional beta3-adrenoceptor in which stimulation reduces action potential duration. Surprisingly, in cardiac biopsies obtained from cystic fibrosis patients, beta3-adrenoceptor agonists produced no effects on action potential duration. This result suggests the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) chloride current in the electrophysiological effects of beta3-adrenoceptor stimulation in non-cystic fibrosis tissues. We therefore investigated the control of CFTR activity by human beta3-adrenoceptors in a recombinant system: A549 human cells were intranuclearly injected with plasmids encoding CFTR and beta3-adrenoceptors. CFTR activity was functionally assayed using the 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescent probe and the patch-clamp technique. Injection of CFTR-cDNA alone led to the expression of a functional CFTR protein activated by cAMP or cGMP. Co-expression of CFTR (but not of mutated DeltaF508-CFTR) with high levels of beta3-adrenoceptor produced an increased halide permeability under base-line conditions that was not further sensitive to cAMP or beta3-adrenoceptor stimulation. Patch-clamp experiments confirmed that CFTR channels were permanently activated in cells co-expressing CFTR and a high level of beta3-adrenoceptor. Permanent CFTR activation was not associated with elevated intracellular cAMP or cGMP levels. When the expression level of beta3-adrenoceptor was lowered, CFTR was not activated under base-line conditions but became sensitive to beta3-adrenoceptor stimulation (isoproterenol plus nadolol, SR 58611, or CGP 12177). This later effect was not prevented by protein kinase A inhibitors. Our results provide molecular evidence that CFTR but not mutated DeltaF508-CFTR is regulated by beta3-adrenoceptors expression through a protein kinase A-independent pathway.

Animals↗

PubMed1999

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.

Amino Acid Sequence↗

PubMed1992

Ion conductances of isolated cortical collecting duct cells.

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were -74 +/- 1 mV (n = 13) and -68 +/- 3 mV (n = 22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (-79 +/- 1 mV, n = 23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K(+)-concentration and did not demonstrate a measurable Cl- conductance. A large-conductance K+ channel (174 pS, n = 5, cell-attached, 145 mmol/l K+ in the pipette; 140 pS, n = 12, cell-free, 3.6 mmol/l K+ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca2+ on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1997

Interferon gamma gene expression in sensory neurons: evidence for autocrine gene regulation.

We explored expression and possible function of interferon-gamma (IFN-gamma) in cultured fetal (E15) rat dorsal root ganglion neurons combining whole cell patch-clamp electrophysiology with single cell reverse transcriptase polymerase chain reaction and confocal laser immunocytochemistry. Morphologically, we located IFN-gamma protein in the cytoplasm of the neurons in culture as well as in situ during peri- and postnatal development. Transcripts for classic IFN-gamma and for its receptor were determined in probes of cytoplasm sampled from individual cultured neurons, which had been identified by patch clamp electrophysiology. In addition, the cultured neurons expressed both chains of the IFN-gamma receptor. Locally produced IFN-gamma acts back on its cellular source. Phosphorylation and nuclear translocation of the IFN-inducible transcriptional factor STAT1 as well as IFN-gamma-dependent expression of major histocompatibility complex class I molecules on the neuronal membrane were noted in untreated cultures. However, both processes were substantially blocked in the presence of antibodies neutralizing IFN-gamma. Our findings indicate a role of IFN-gamma in autocrine regulation of sensory neurons.

Animals↗

PubMed2003

Inhibitory effects of recombinant neurotoxin BmK IM on seizures induced by pentylenetetrazol in Rats.

OBJECTIVE: To elucidate the inhibitory effects of recombinant Chinese scorpion neurotoxin BmK IM on seizures induced by pentylenetetrazol (PTZ) and the possible mechanism. METHODS: After purifying recombinant BmK IM from an E. coli cell line, its toxicity (both LD50 and minimum lethal dose) on rats was determined. BmK IM was then microinjected into the CA3 region of the right hippocampus and its ability to inhibit the effects of an intraperitoneal injection of PTZ was assessed. The effects of BmK IM on the electrophysiological properties of isolated CA3 pyramidal neurons were then studied using whole-cell patch clamp techniques. RESULTS: BmK IM can significantly prolong the latent period of epileptic seizures, decrease the degree of seizures, and decrease the frequency of epileptiform discharges induced by PTZ. At the same time, 24h after injection of BmK IM into the hippocampal tissue, BmK IM significantly reduces the concentration of the neurotransmitter glutamate and alleviates PTZ-induced lesions in the hippocampus. Whole-cell patch clamp recordings indicate that BmK IM inhibits INa of rat hippocampal neurons in a dose-dependent manner. BmK IM significantly shifts the activation curve of INa in a positive direction, indicating that BmK IM enhances the threshold potential of INa. CONCLUSIONS: BmK IM has significant anti-epileptic properties, and may prove useful as a drug in the therapy of epilepsy. The inhibitory effects of BmK IM on seizures caused by pentylenetetrazol might depend on reductions in the release of presynaptic glutamate via the blocking of Na+ channels.

Animals↗

PubMed1992

Structural and functional crosstalk between acetylcholine receptor and its membrane environment.

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or "annular" lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells.

Acetylcholine↗

PubMed1988

Theoretical study of the voltage and concentration dependence of the anomalous mole fraction effect in single calcium channels. New insights into the characterization of multi-ion channels.

Several recent independent studies on macroscopic Ca currents have demonstrated the anomalous mole fraction effect in mixtures of Ca and Ba at concentrations of 10 mM or less. Recently, Hess and Tsien (1984; Nature 309) proposed a dual binding site model, based upon Eyring rate theory, to account for this effect in L-type cardiac Ca channels. This model predicts that the anomalous mole fraction effect can be accounted for solely in terms of open single channel permeation properties; it was able to adequately reproduce the effect for macroscopic Ca currents recorded in 10 mM solutions. However, the electrochemical gradients under which single Ca channel current recordings are routinely made with the patch clamp technique vary dramatically from those used for macroscopic Ca currents. To properly assess the general validity of the Hess and Tsien model at the single Ca channel level, the effects of both large electrical potentials and elevated divalent concentrations must be understood. Computer simulations were therefore carried out using the original parameters used by Hess and Tsien under conditions designed to mimic those used in patch clamp studies. The permeation behavior generated by this model is quite complex. In particular, hyperpolarization and increased divalent concentration combine to reduce and ultimately abolish the anomalous mole fraction effect. It may therefore be very difficult to observe the anomalous mole fraction effect at the single Ca channel level; the dual-site model displays a relationship between current and mole fraction generally associated with a single-site model under the conditions frequently employed to resolve single Ca channel activity. Nonetheless, analysis of such monotonic mole fraction behavior can still be used as a test for the general validity of the dual-site model. Apparent Kms for Ca and Ba can be extracted from such monotonic behavior, and may not only be functions of membrane potential but may also depend upon the total divalent cation concentration. This is a unique prediction which is incompatible with the simple single-site model. Our analysis provides (a) a possible resolution for the apparent discrepancies presently existing in the experimental literature regarding the existence of the anomalous mole fraction effect at the single Ca channel level, (b) a mechanistic description of previously unexplained observations on the voltage-dependence of the anomalous mole fraction effect, and (c) a useful theoretical framework for future experimentation designed to test the general validity of the dual binding site model of the Ca channel.

Animals↗