The effect of subunit composition of rat brain GABAA receptors on channel function.

Different combinations of cloned rat brain subunit isoforms of the GABAA receptor channel were expressed in Xenopus oocytes. The voltage-clamp technique was then used to measure properties of the GABA-induced membrane currents and to study the effects of various modulators of the GABAA receptor channel (diazepam, DMCM, pentobarbital, and picrotoxin). This approach was used to obtain information on the minimal structural requirements for several functional properties of the ion channel. The combination alpha 5 beta 2 gamma 2 was identified as the minimal requirement reproducing consensus properties of the vertebrate GABAA receptor channel, including cooperativity of GABA-dependent channel gating with a Ka in the range of 10 microM, modulation by various drugs acting at the benzodiazepine binding site, picrotoxin sensitivity, and barbiturate effects.

Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site.

We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity. Grin1(D481N) mice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype of Grin1(K483Q) animals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.

Cytosolic ADP enhances the sensitivity to tolbutamide of ATP-dependent K+ channels from pancreatic B-cells.

The effects of intracellular purine nucleotides on tolbutamide-induced block of ATP-dependent K+ channels from mouse pancreatic B-cells were studied using the patch-clamp technique. When applied to the inside of excised patches, tolbutamide alone blocked channel activity half-maximally at 55 microM and the concentration-response curve for the inhibition of K+ channels by tolbutamide was flat. ADP (1 mM), but not other nucleotides (AMP, GTP or GDP) increased the steepness of the concentration-response curve and decreased the half-maximally effective tolbutamide concentration to 4.2 microM. It is suggested that the ATP-dependent K+ channel or a closely related structure contains a receptor which is accessible for cytosolic ADP and controls the sensitivity to tolbutamide.

Expression of K channels in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin-secreting beta-cell line, HIT T15.

Two types of exogenous K channel were identified in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin-secreting cell line HIT T15. One of these was the ATP-regulated K channel (G channel) as evidenced by its conductance and inhibition by tolbutamide. The other resembled the Ca-activated K channel from beta-cells.

Pharmacological and Electrophysiological Properties of Recombinant GABAA Receptors Comprising the alpha3, beta1 and gamma2 Subunits.

To assess the role of subunits for channel function and drug modulation in recombinant GABAA receptors, the alpha3beta1gamma2 subunits and the dual combinations alpha3beta1, beta1gamma2 and alpha3gamma2 were expressed by transfection of human embryonic kidney cells and by RNA injection in Xenopus oocytes (alpha3beta1gamma2 combination). GABA-induced chloride currents were recorded using the whole-cell configuration of the patch-clamp technique (transfected cells) or the voltage-clamp technique (oocytes). The currents recorded from the alpha3beta1gamma2 subunit combination in transfected cells were reduced by bicuculline and picrotoxin, enhanced by flunitrazepam in a flumazenil-sensitive manner and reduced by beta-carboline-3-carboxylic acid methyl ester (beta-CCM). The GABA-induced current was reduced by beta-CCM in all combinations containing the gamma2 subunit, but potentiation by flunitrazepam was only obtained when the gamma2 subunit was coexpressed in the presence of the alpha3 subunit (alpha3beta1gamma2 or alpha3gamma2). The GABA sensitivities of the receptors were similar when the alpha3beta1gamma2 combination was expressed in oocytes (half-maximum effective concentration=240 microM) or in the kidney cell line (270 microM). However, the currents were less potentiated by flunitrazepam in oocytes (129% of controls) than in transfected cells (189%). These results suggest that the alpha3beta1gamma2 subunit combination, which is coexpressed in various brain regions as shown by in situ hybridization histochemistry, may represent a building block of functional GABAA receptors in situ.

State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist.

1. Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-¿3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy ¿-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1(001)/NR2B than NR1(001)/NR2A receptors. 2. Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil. 3. The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 microM NMDA responses with IC50s of 2.3 and 0.36 microM, respectively. Currents elicited by 1 microM NMDA were slightly potentiated in the presence of 10 microM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors. 4. These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3 4 fold increase in receptor affinity for glutamate site agonists. 5. Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.

Effects of calcium "antagonists" on vertebrate skeletal muscle cells.

Clinically potent skeletal muscle relaxants are used primarily for their effects on the central nervous system. But they also have direct effects on muscle contraction that possibly involve Ca2+ channels. We compared the effects of dantrolene, an agent known to have a direct action on vertebrate skeletal muscle, with other substances used as (1) relaxants and (2) antagonists of Ca-dependent excitation-contraction coupling. Isolated intact frog muscle cells were injected with the photoprotein aequorin, and membrane potential changes, intracellular Ca2+ transients, and contractile force were measured. Dantrolene (10(-8) to 10(-5) M) decreased the amplitude of Ca2+ transients, did not affect their rates of decay, and reduced contractile force. We also used an integrated digital-imaging system to record microscopic changes, namely, active shortening in myofibrils and changes in striation spacing. Dantrolene did not increase the time between contraction in myofibrils near the surface compared with myofibrils near the center of a cell. Hence dantrolene does not suppress Ca2+ transients by disturbing current flow in the transverse tubular system. Each of the following actually increased Ca2+ transients and contractile force evoked by action potentials: baclofen (10(-7) to 10(-5) M) less than flordipine (10(-6) M) less than meprobamate (10(-7) to 10(-3) M) less than chlordiazepoxide (10(-5) X 10(-4) M) less than procaine (10(-5) to 5 X 10(-4] less than GABA (10(-5) M) less than D-600 (10(-6) M) less than nylidrin (10(-5) M)--in order of increasing potency. Ca2+ channels in the sarcoplasmic reticulum of intact skeletal muscle are evidently inhibited by dantrolene but not by Ca2+ antagonists.

Action of diazepam on the voltage-dependent Na+ current. Comparison with the effects of phenytoin, carbamazepine, lidocaine and flumazenil.

The influence of diazepam, an agonist, and flumazenil (Ro 15-1788), an antagonist of the benzodiazepine receptor, on repetitive firing of action potentials in cultured spinal neurons and on voltage-dependent Na+ currents in cultured N2A neuroblastoma cells was examined. The effects were compared to those of the antiepileptics phenytoin and carbamazepine and the local anesthetic lidocaine. The whole-cell configuration of the patch-clamp technique was used for potential and current recording. Diazepam (10 microM) or phenytoin (10 microM) reduced the duration of repetitive action potential discharges in 50 or 67% of the spinal neurons, respectively. At a concentration of 100 microM repetitive firing was completely blocked. Flumazenil (100 microM) had no effect. In N2A neuroblastoma cells diazepam, phenytoin, carbamazepine and lidocaine, but not flumazenil, at a concentration of 100 microM reduced the Na+ current to 60-67% of control. At 10 microM no or only a weak depression was seen with any drug. In the presence of diazepam (100 microM) the Na+ channel inactivation curve was shifted in the hyperpolarizing direction by -4.8 +/- 0.5 mV. Phenytoin, carbamazepine and lidocaine (all 100 microM) caused stronger shifts of -17.4 +/- 2.1, -10.6 +/- 0.9 and -17.0 +/- 2.1 mV, respectively. Inhibition of the Na+ current by diazepam increased use-dependently over 9 depolarizing pulses repeated at high frequency (200 Hz), whereas use-dependent effects of the other compounds developed less rapidly. At a low stimulation rate (7 Hz) use-dependent block was pronounced with lidocaine, but weak or absent with diazepam and carbamazepine.(ABSTRACT TRUNCATED AT 250 WORDS)

Dextromethorphan blocks N-methyl-D-aspartate-induced currents and voltage-operated inward currents in cultured cortical neurons.

The effect of dextromethorphan on several types of cation currents in cultured rat cortical neurons and PC12 cells was studied by using the whole-cell configuration of the patch-clamp technique. The Ba2+ current through L- and N-type Ca2+ channels was blocked with similar potencies (52-71 microM) in both types of cells. The effect was not voltage-dependent, in contrast to that of amlodipine (a dihydropyridine). Dextromethorphan was able to block the Ba2+ current completely unlike amlodipine and omega-conotoxin (an N-type channel blocker) which produced only partial inhibition. The voltage-activated Na+ and Ca2+ channels in cortical neurons were inhibited by similar concentrations of dextromethorphan (IC50 approximately 80 microM). The morphinan was at least 100 times more potent (IC50 = 0.55 microM) as a blocker of the current induced by N-methyl-D-aspartate (NMDA) in cortical neurons. Currents induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ((RS)-AMPA) or kainic acid were not significantly affected even at 1 mM. The results suggest that the neuroprotective effect of dextromethorphan, previously found to occur in a concentration range of 10-100 microM, may be due to a complete blockade of the NMDA receptor channel and a partial inhibition of voltage-dependent Ca2+ and Na+ channels.

How do sulfonylureas approach their receptor in the B-cell plasma membrane?

Since it was unknown whether the uncharged or the anionic form of hypoglycemic sulfonylureas and meglitinide is the effective modulator of ATP-dependent K+ channels and insulin secretion, we studied the inhibitory effects of tolbutamide and meglitinide on the ATP-dependent K+ current at different external pH. The whole-cell configuration of the patch-clamp technique was used in mouse pancreatic B-cells. When the concentrations of the undissociated forms of these drugs were kept constant at increasing pH of the bath solution (6.4 to 8.4), the rate of development and the degree of K+ channel block varied only slightly. Raising the pH-value in the bath solution at constant total concentration of tolbutamide diminished both the rate of development and the degree of K+ channel block. It is concluded that the undissociated forms of tolbutamide and related compounds are the effective forms. Examination of the K+ current records during the application and removal of different concentrations of tolbutamide, meglitinide, glipizide and glibenclamide at pH 7.4 indicated that the kinetics of the current records reflected not only association and dissociation of the drug-receptor complex but perhaps also the kinetic of drug distribution between bath and the lipid phase of the plasma membrane. As there is evidence against an interaction between sulfonylureas and their receptor via a binding site freely accessible from the cytoplasm, the drugs probably get access to their binding site on the receptor from the lipid phase of the B-cell plasma membrane.

Concentration-dependent effects of tolbutamide, meglitinide, glipizide, glibenclamide and diazoxide on ATP-regulated K+ currents in pancreatic B-cells.

The influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K+ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 mumol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 mumol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/l glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+ channels. Half maximally effective concentrations of diazoxide were 20 mumol/l with 0.3 mmol/l ATP and 102 mumol/l with 1 mmol/l ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K+ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K+ channel of the pancreatic B-cell or a structure closely related to this channel.

Contractions of skinned cardiac cells elicited by current pulses.

Cardiac muscle fragments with disrupted sarcolemma were prepared by homogenization of mouse ventricles. The preparations exhibited spontaneous contractions of a rate between 3 min-1 and 12 min-1 at 20 degrees C, when they were kept in an appropriate solution. 2. Fragments of about cellular size were attached to two stiff glass microelectrodes, and additional contractions between the spontaneous beats were elicited by current pulses. The duration and intensity of the stimuli were varied to obtain strength-duration curves. Rheobase was in the range of 1.5 muA to 10 muA (no isolation of surrounding bath), chronaxia at 35 degrees C between 30 ms and 80 ms. 3. One microelectrode could be glued to a photodiode-force-transducer for simultaneous recording of contractions and electrical stimulation or potential measurement. Duration of phasic contractions was nearly 1s, force was up to 4muN (20 mN/mm2 of tension).

Measurements of single-channel currents in the membrane of isolated cells: ATP-dependence of K+-channels.

Single-channel currents in ventricular cells of guinea-pig hearts were recorded by the patch-clamp technique. An inwardly rectifying K+-current was found in cell-attached membrane patches. Patches could be isolated from the cell exposing the cytoplasmic face of the membrane directly to the bathing solution. After isolation, the same current as in cell-attached patches was seen if the bath contained 4 mM ATP. Without ATP, this current disappeared and another channel of larger conductance and different kinetics was activated. Currents through the latter channel were also seen in cell-attached patches after poisoning the cells by DNP. It is suggested that the ATP-dependence of the observed membrane channels mediates the increase of potassium conductance after metabolic inhibition.

Anion dependence of the contractions of skinned cardiac cells.

Cardiac muscle fragments with disrupted sarcolemmas were prepared by homogenization of mouse ventricles. The rate of spontaneous contractions was increased when a solution containing isobutyrate as the main anion was substituted with a solution rich in chloride. At low calcium concentrations preparations which were quiescent in the isobutyrate solution responded to the chloride with a strong single contraction.

Dextromethorphan: cellular effects reducing neuronal hyperactivity.

Dextromethorphan is a dextrorotary morphinan without affinity for opioid receptors, commonly used as an antitussive medication. During the past 5 years, interest in the compound and its demethylated derivative, dextrorphan, has been revived because additional neuroprotective and antiepileptic properties were found in in vitro studies, animal experiments, and a few clinical cases. Both morphinans are able to inhibit N-methyl-D-aspartate (NMDA) receptor channels and voltage-operated calcium and sodium channels with different potencies. The inhibition of the NMDA receptor is believed to be the predominant mechanism of action responsible for the anticonvulsant and neuroprotective properties of the compounds.

Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions.

Pancreatic islets of NMRI mice were dissociated into single cells which were kept in tissue culture for 1-3 days. The whole-cell configuration of the patch-clamp technique was used to study inward and delayed outward currents of beta-cells under voltage-clamp conditions at 20-22 degrees C. Outward currents were suppressed by substituting the impermeant cation N-methyl-D-glucamine for intracellular K+. The remaining inward current had a V-shaped current-voltage relation reaching a peak value of 39 +/- 4 pA (mean +/- S.E. of mean) around -15 mV. It was identified as a Ca2+ current, because the peak amplitude was increased 1.6 times by increasing external [Ca2+] ([Ca2+]o) from 2.6 mM to 10 mM and it was blocked by Co2+ (5 mM) or nifedipine (5 microM) but not by TTX (20 microM). The activation time constant of the inward current at -10 mV was 1.28 +/- 0.08 ms. The relation between the degree of activation (estimated from the size of the tail currents) and membrane potential V followed the sigmoidal function f = 1/(1 + exp [(Vh-V)/k]) with half-maximal activation potential, Vh = 4 +/- 1 mV and slope factor, k = 14 +/- 1 mV (for [Ca2+]o 10 mM). The inward current inactivated only weakly during depolarizing pulses of 0.1-1 s duration. The delayed outward current (in experiments with 155 mM-internal [K+] ([K+]i)) had a linear voltage dependence at potentials above -20 mV; its amplitude at -10 mV was 210 +/- 30 pA. Tail currents related to the activation of the outward current had K+-dependent reversal potentials. The current was blocked by extracellularly applied tetraethylammonium (20 mM) and 4-aminopyridine (2 mM). It was not affected by glibenclamide (3 microM), tolbutamide (0.2 mM) and alterations of intracellular [Ca2+] (1 nM-1 microM). The activation time constant of the outward current at -10 mV was 21 +/- 3 ms. The voltage dependence of activation could be described by the sigmoidal function (see above) with Vh = 19 +/- 1 mV and k = 5.6 +/- 0.4 mV. The outward current inactivated during long (15 s) depolarizing pre-pulses (time constant at -10 mV: 2.6 +/- 0.6 s). 50% inactivation occurred at Vh = -36 +/- 2 mV, k was -4.1 +/- 0.2 mV. Inward and outward currents during depolarizing voltage pulses in beta-cells are similar to Ca2+ and delayed K+ currents in other cell types. These currents seem sufficient to generate the action potentials of the beta-cell.

Inward-rectifying channels in isolated patches of the heart cell membrane: ATP-dependence and comparison with cell-attached patches.

Inward rectifying potassium single-channel currents were studied in the membrane of guinea pig cardiac myocytes. In isolated inside-out patches two different channels were observed: a channel of 25 pS conductance ([K+]o = 147 mM, T = 21 degrees C), if the solution at the cytoplasmic face of the patch contained 4 mM ATP and a channel of 80 pS conductance without ATP. The 25-pS-channel was also regularly seen in cell-attached patches (Sakmann and Trube 1984a,b), but the 80-pS-channel appeared only after inhibiting cellular metabolism by DNP. The percentage of time which the 25-pS-channel spent in the open state was 3.3 times larger in isolated patches compared to cell-attached patches. However, both types of single channel currents disappeared several minutes after the isolation of the patches. In contrast to the 25-pS-channel, the 80-pS-channel, which is activated by the lack of ATP, carried measurable outward currents saturating at 1.5 pA (inward rectification). It is suggested that the 80-pS-channel mediates part of the increase in potassium current during metabolic inhibition. The openings of this channel appeared in bursts. The mean open time was 1.6 ms and the mean duration of the gaps within bursts 0.33 ms at -80 mV.