Recording Configuration Research Articles

The relationship between acetylcholine-evoked Ca(2+)-dependent current and the Ca2+ concentrations in the cytosol and the lumen of the endoplasmic reticulum in pancreatic acinar cells.

In a study of isolated mouse pancreatic acinar cells, we used the patch-clamp whole-cell recording configuration to monitor the Ca(2+)-dependent inward ionic current and simultaneously measured the Ca2+ concentration in either the cytosol ([Ca2+]i) or the lumen of the endoplasmic reticulum ([Ca2+]Lu), using appropriate Ca(2+)-sensitive fluorescent probes. A high concentration of acetylcholine (ACh, 10 microM) evoked an increase in [Ca2+]i, which resulted in the activation of Ca(2+)-dependent inward current. Continued ACh application for several minutes led to a marked reduction in both the current and the [Ca2+]i response and after about 4-10 min of sustained ACh stimulation, the inward current response had disappeared and [Ca2+]i was back to the pre-stimulation level. Repeated stimulation with shorter pulses of ACh (10 microM) resulted in responses of declining magnitude both in terms of inward current and [Ca2+]i rises. The ACh-activated inward current was entirely dependent on the elevation of [Ca2+]i, but at a relatively high [Ca2+]i the current was saturated. ACh caused a rapid release of Ca2+ from the lumen of the endoplasmic reticulum and after discontinuation of stimulation, [Ca2+]Lu was only very slowly (10-15 min) fully restored to the pre-stimulation level. Repeated applications of ACh did not change the relationships between the Ca(2+)-dependent current and [Ca2+]i or the current and [Ca2+]Lu. When [Ca2+]Lu was greater than 100 microM, the ACh-evoked Ca2+ release from the store was so large that the current response was initially saturated. We conclude that the ACh-evoked current response essentially depends on the release of stored Ca2+. Desensitization is mainly due to the relatively slow reloading of the intracellular stores with Ca2+.

A small sodium channel blocking factor in the cerebrospinal fluid is preferentially found in Guillain-Barré syndrome: a combined cell physiological and HPLC study.

The cerebrospinal fluid (CSF) of patients with Guillain-Barré syndrome (GBS) contains a low molecular weight factor with sodium channel blocking activity. This study investigated whether such activity also exists in the CSF of patients with other neurological diseases. Further, using high-performance liquid chromatography (HPLC) we tested whether the electrophysiological effect of the CSF is correlated with the size of the corresponding peak in the chromatograms. The existence of sodium channel blocking activity was tested in 27 native CSF samples of three groups of patients (group 1: GBS, n = 13; group 2: other inflammatory diseases, n = 8; group 3: controls, n = 6). NH15-CA2 neuroblastoma x glioma cells in the whole-cell recording configuration was used as a system for assaying the sodium channel blocking activity of CSF specimens. CSF shifted the steady-state inactivation curve of the sodium channels reversibly by -10.2 +/- 4.4 mV in group 1, -6.7 +/- 3.9 mV in group 2, and - 3.5 +/- 2.8 mV in group 3 (P < 0.01). The shift was greater in demyelinating (9.3 +/- 4.7 mV) than in nondemyelinating (5.6 +/- 3.9 mV) diseases (P < 0.04). HPLC analysis of CSFs showed a well separated peak containing the substance responsible for the electrophysiological effect at about 41 min elution time. The peak covered the molecular weight range of 600-800 Da. Sodium channel blocking activity of CSFs and areas of the corresponding peak in the chromatograms were well correlated. We conclude that sodium current inhibition by a low molecular weight factor is generally present but increased in GBS.

κ- and μ-Opioids Reverse the Somatostatin Inhibition of Ca2+Currents in Ciliary and Dorsal Root Ganglion Neurons

Neuromodulators, including transmitters and peptides, modify neuronal excitability. In most neurons, multiple neuromodulator receptors are present on a single cell. Previous work has demonstrated either occlusive or additive effects when two neuromodulators that target the same ion channel are applied together. In this study, we characterize the modulation of Ca2+ and K+ channels in embryonic chick ciliary ganglion neurons by somatostatin (Som) and opioids, including the effects of these neuromodulators when applied in combination. We report a modulation of calcium current by kappa- or mu-opioids that can prevent Som effects when applied before Som and can replace Som effects when applied after Som. We term these effects demodulation because they do not have the characteristics of simple occlusion but rather represent a dominant effect of opioid-mediated modulation of calcium channels over Som-mediated modulation. These opioid effects persist in the presence of kinase and phosphatase inhibitors, as well as after alteration of the intracellular Ca2+ concentration. Furthermore, they are present in both whole-cell and perforated-patch recording configurations. These effects of opioids on Som-mediated modulation do not seem to be mediated by a general uncoupling of Som receptors from G-protein-coupled signaling systems because K+ current modulation by Som can persist in the presence of opioids. Demodulation by opioids was also observed in dorsal root ganglion neurons on the modulation of calcium current by GABA and norepinephrine (NE). In both preparations, this demodulatory interaction occurred between voltage-independent (opioids) and voltage-dependent (Som, GABA, and NE) modulatory pathways.

Role of muscarinic receptors, G-proteins, and intracellular messengers in muscarinic modulation of NMDA receptor-mediated synaptic transmission.

Previously, we reported that activation of muscarinic receptors modulates N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission in auditory neocortex [Aramakis et al. (1997a) Exp Brain Res 113:484-496]. Here, we describe the muscarinic subtypes responsible for these modulatory effects, and a role for G-proteins and intracellular messengers. The muscarinic agonist oxotremorine-M (oxo-M), at 25-100 microM, produced a long-lasting enhancement of NMDA-induced membrane depolarizations. We examined the postsynaptic G-protein dependence of the modulatory effects of oxo-M with the use of the G-protein activator GTP gamma S and the nonhydrolyzable GDP analog GDP beta S. Intracellular infusion of GTP gamma S mimicked the facilitating actions of oxo-M. After obtaining the whole-cell recording configuration, there was a gradual, time-dependent increase of the NMDA receptor-mediated slow-EPSP, and of iontophoretic NMDA-induced membrane depolarizations. In contrast, intracellular infusion of either GDP beta S or the IP3 receptor antagonist heparin prevented oxo-M mediated enhancement of NMDA depolarizations. The muscarinic receptor involved in enhancement of NMDA iontophoretic responses is likely the M1 receptor, because the increase was prevented by pirenzepine, but not the M2 antagonists methoctramine or AF-DX 116. Oxo-M also reduced the amplitude of the pharmacologically isolated slow-EPSP, and this effect was blocked by M2 antagonists. Thus, muscarinic-mediated enhancement of NMDA responses involves activation of M1 receptors, leading to the engagement of a postsynaptic G-protein and subsequent IP3 receptor activity.

Inhibition of Ca 2+-activated K + current by clotrimazole in rat anterior pituitary GH 3 cells

The ionic mechanism of clotrimazole, an imidazole antimycotic P-450 inhibitor, was examined in rat anterior pituitary GH 3 cells. In perforated-patch whole-cell recording experiments, clotrimazole reversibly caused an inhibition of the Ca 2+-activated K + current in a dose-dependent manner. The IC 50 value of the clotrimazole-induced inhibition of I K(Ca) was 3 μM. In the outside-out configuration of single channel recording, application of clotrimazole (10 μM) into the bath medium did not change the single channel conductance of large conductance Ca 2+-activated K +(BK Ca) channels, but it suppressed the channel activity significantly. The change in the kinetic behavior of BK Ca channels caused by clotrimazole in these cells is found to be due to a decrease in mean open time and an increase in mean closed time. Other structurally distinct P-450 inhibitors (e.g. ketoconazole or econazole) also effectively suppressed the amplitude of I K(Ca). Clotrimazole (10 μM) blocked both the inactivating and non-inactivating components of the voltage-dependent K + outward current ( I K(V)), but it produced a slight reduction of l-type Ca 2+inward current ( I Ca,L) without altering the current–voltage relationship of I Ca,L. Clotrimazole (10 μM) also increased the firing rate of action potentials. These results provide direct evidence that clotrimazole is capable of suppressing the activity of BK Ca channel in GH 3 cells. Because of the non-selective inhibitory effect of clotrimazole on I K(Ca) and I K(V), this inhibition is mainly, if not entirely, due to a direct channel blockade. Thus, the present study implies that the blockade of these ionic channels by clotrimazole would affect hormonal secretion and neuronal excitability.

Inhibitory and excitatory responses of olfactory receptor neurons of xenopus laevis tadpoles to stimulation with amino acids

Recordings were made from olfactory receptor neurons of Xenopus laevis tadpoles using the patch-clamp technique to investigate the responses of these cells to odorants. Four amino acids (glutamate, methionine, arginine and alanine) both individually and as a mixture were used as stimuli. Of the 156 olfactory neurons tested, 43 showed a response to at least one of the stimuli. Of the cells tested, 19 % responded to glutamate, 16 % to methionine, 12 % to arginine and 10 % to alanine. Each amino acid was able to induce both excitatory and inhibitory responses, although these occurred in different cells. Each amino acid produced approximately equal numbers of inhibitory and excitatory responses. Inhibitory responses could best be observed in the perforated-patch configuration using gramicidin as an ionophore and a recording configuration that is a current-clamp for fast signals and a voltage-clamp for slow signals. The diversity of the odorant responses, in particular the existence of excitatory and inhibitory responses, is not consistent with a single transduction pathway in olfactory neurons of Xenopus laevis tadpoles.

Functional properties of ionotropic glutamate receptor channels in rat sacral dorsal commissural neurons

Nystatin perforated patch and conventional whole-cell recording configurations were used to characterize the properties of ionotropic glutamate receptor (GluR) channels in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). l-Glutamate (Glu), N-methyl- d-aspartate (NMDA), quisqualate (QA), α-amino-3-hydroxy-5methyl-4-isoxazoleprop ionate (AMPA) and kainate (KA) applied via a Y-tube produced inward currents at −44 mV which increased in a concentration-dependent manner; they desensitized when induced at higher concentrations except for the KA-induced current ( I KA). (1 S-3 R)1-amino-cyclopentane-1,3-dicarboxylate (1 S-3 R-ACPD) evoked no response. The EC 50 and Hill coefficient ( n H) values of the GluR responses were 3.3×10 −5 M, 0.74 for Glu; 9.0×10 −5 M, 0.83 for NMDA; 6.4×10 −7 M, 1.30 for QA; 1.3×10 −4 M, 1.10 for AMPA and 9.6×10 −5 M, 1.30 for KA, respectively. The reversal potentials of the GluR responses were all near 0 mV. The 6-Cyano-7-nitroquinoxaline-2-3-dione (CNQX) and d-2-amino-5-phosphonovalerate ( d-APV) suppressed the non-NMDA and NMDA responses in a concentration-dependent manner, respectively. Cyclothiazide strongly potentiated both KA- and AMPA-induced responses while concanavalin A potentiated both the responses to a much lesser degree. NS-102 produced no significant effect on either KA- or AMPA-activated currents, while GYKI 52466 reversibly blocked both the currents. The Ca 2+ permeabilities ( P Ca/ P Cs) of the NMDA and AMPA receptor channels were 8.33 and 1.23, respectively. In addition, the current–voltage ( I– V) relationship of I KA showed little rectification. There was a poor correlation between the Ca 2+ permeability and the shape of the I– V curves of I KA. These results suggest that rat SDCN neurons possess NMDA and non-NMDA receptor channels, and express AMPA type receptors with unique properties (slow desensitization to AMPA, high Ca 2+ permeability but lack of inward rectification). These ionotropic receptor charnels may play important roles in mediating and regulating pelvic visceral information including nociception.

α2-adrenoceptor-mediated enhancement of glycine response in rat sacral dorsal commissural neurons

The effect of noradrenaline on the glycine response was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus using nystatin perforated patch recording configuration under voltage-clamp conditions. Noradrenaline reversibly potentiated the 10 −5M glycine-induced Cl − current in a concentration-dependent manner. Single channel recordings in a cell-attached mode revealed that noradrenaline decreased the closing time of the glycine-activated channel activity. Noradrenaline neither changed the reversal potential of the glycine response nor affected the affinity of glycine to its receptor. Clonidine mimicked and yohimbine blocked the noradrenaline action on glycine response. N-[2(methylamino)ethyl]-5-misoquinoline sulfonamide dihydrochloride, protein kinase A inhibitor, mimicked the effect of noradrenaline on glycine response. Noradrenaline failed to affect the glycine response in the presence of these intracellular cyclic AMP and protein kinase A modulators. However, noradrenaline further enhanced the glycine response even in the presence of phorbol-12-myristate-13-acetate and chelerythrine, a protein kinase C inhibitor. Pertussis toxin treatment for 6–8 h blocked the noradrenaline facilitatory effect on the glycine response. In addition, noradrenaline potentiated the strychnine-sensitive postsynaptic currents evoked in a slice preparation of sacral dorsal commissural nucleus. These results suggest that the activation of α2-adrenoceptor by noradrenaline coupled with pertussis toxin-sensitive G-proteins reduces intracellular cyclic AMP formation through the inhibition of adenyl cyclase. The reduction of cyclic AMP decreases the protein kinase A activity, thus resulting in the potentiation of the glycinergic inputs to the sacral dorsal commissural neurons. It is thus feasible that the noradrenergic input to the sacral dorsal commissural nucleus modulates such nociceptive signals as pain by intracellular enhancing the glycine response.

Outward photocurrent component in chloroplasts of Peperomia metallica and its assignment to the `closed thylakoid' recording configuration

The photoinduced electrical events at energy-conserving chloroplast membranes can be studied in whole plastids using suction electrodes. In chloroplasts of Peperomia metallica the kinetic profile of photocurrent contains a minor outward component that occurs prior to and differs in polarity from the main component. The origin of this outward current was analyzed using single-turnover flashes in combination with prolonged light exposures and differential physicochemical treatments of tip-located (internal) and the exposed parts of a chloroplast. The outward current signal was higher after 10- to 20-s preillumination and gradually reduced in darkness. The relative amplitude of the outward peak current was enhanced when photosystem II (PS II) was excited by flashes given in the presence of far-red background light ( λ=712 nm). The outward current was small or absent under conditions promoting activity of photosystem I (cyclic electron transport supported by artificial redox mediators in the presence of diuron) and was particularly high in the presence of PS II electron acceptors (e.g., p-phenylenediamine). This indicates the predominant association of the outward current with activity of PS II. The external application of diuron strongly inhibited the inward current, giving rise to a temporal increase in the outward current. On the contrary, when diuron was added into the suction pipette, the outward current was inhibited soon after sealing. The data suggest that the outward current originated in the tip-located portions of the thylakoid membrane that have orientation opposite to the exposed part of `whole thylakoid'. These tip-located membrane portions are least accessible for inhibitors added into the outer medium and are highly sensitive to inhibitors (diuron), ionophores (gramicidin D), and detergents (Triton X-100) added into the pipette. Differential involvement of two photosystems in generation of the outward current may be caused by uneven structural distribution of photosystems I and II between appressed (granal) and nonappressed (stromal) thylakoids and by different recording configurations for these thylakoids.

γ-Aminobutyric acid-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus

The electrophysiological and pharmacological properties of GABA-activated Cl − currents ( I GABA) were investigated in enzymatically dissociated rat sacral dorsal commissural nucleus (SDCN) neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. Exogenous application of GABA to SDCN neurons induced Cl − currents which increased in a concentration-dependent manner. Bicuculline (BIC) and strychnine (STR) antagonized the I GABA in a concentration-dependent manner. Zn 2+ suppressed the I GABA with an IC 50 of 2.8×10 −5 M. Muscimol mimicked the I GABA, while baclofen evoked no response. Pentobarbital (PB) and 5β-pregnan-3α-ol-20-one (pregnanolone, PGN) also induced GABA A-mimic Cl − currents. Diazepam (DZP), PB and PGN all enhanced the I GABA by increasing the apparent affinity of the GABA A receptors to GABA. Moreover, spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) were observed in mechanically dissociated SDCN neurons attached with synaptic boutons, so called `synaptic bouton preparation'. These results indicate that SDCN neurons express GABA A receptors with relatively low sensitivity to Zn 2+ inhibition, and that GABA may have a functional role as an inhibitory transmitter in the SDCN regulating nociceptive, analgesic, and autonomic functions.

Rapid action of 17beta-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors.

17Beta-estradiol can potentiate kainate-induced currents in isolated hippocampal CA1 neurons. The action of estrogen was rapid in onset, steroid and stereospecific, and reversible. The potentiation could be mimicked by 8-bromo-cAMP, an activator of protein kinase A. As the hippocampus expresses both isoforms of the intracellular estrogen receptor (ER alpha and ER beta), the role of ERs in the rapid action of 17beta-estradiol remains elusive. Here we report that the rapid action of 17beta-estradiol is independent from the classical ER activation in the modulation of membrane excitability. Under whole cell voltage clamp recording configuration, 17beta-estradiol-induced potentiation was observed in both wild-type and the ER alpha gene knockout mice. The perfusion or incubation of ICI 182,780, which blocks both ER alpha and ER beta, did not affect estrogen potentiation in either group. Further study showed that adenosine 3',5'-cyclic-monophosphothioate Rp-isomer, a specific inhibitor of protein kinase A, completely blocked the potentiation observed with the application of 17beta-estradiol in ER alpha gene knockout mice. Our results provide evidence that a distinct estrogen-binding site exists, which appears to be coupled to alpha-amino-3-hydroxyl-5-methyl-4-isoxazole proprionic acid/kainate receptors by a cAMP-dependent phosphorylation process.

Modulation of glycine-induced currents by zinc and other metal cations in neurons acutely dissociated from the dorsal motor nucleus of the vagus of the rat

Effects of Zn 2+ and other polyvalent cations on glycine-induced currents in the freshly dissociated rat dorsal motor nucleus of the vagus neurons were investigated under voltage-clamp conditions by the use of the nystatin-perforated patch recording configuration. Glycine evoked a Cl − current in a concentration-dependent manner with a half-maximum effective concentration (EC 50) of 3.3×10 −5 M. Strychnine inhibited the 3×10 −5 M glycine-induced current in a concentration-dependent manner with a half-maximal inhibitory concentration (IC 50) of 6.8×10 −7 M. At low concentrations (3×10 −8 M–3×10 −6 M), Zn 2+ potentiated the current elicited by 3×10 −5 M glycine. On the other hand, at concentrations higher than 10 −5 M, Zn 2+ inhibited the glycine response. The biphasic action of Zn 2+ was mimicked by Ni 2+, but La 3+ and Co 2+ had only potentiating effect. Zn 2+ shifted the concentration–response curve for the glycine-induced current without changing the maximum response, and the EC 50 values for the glycine response in the absence and presence of 10 −6 M and 10 −4 M Zn 2+ were 3.3×10 −5 M, 1.3×10 −5 M and 1.3×10 −4 M, respectively. These results suggest that the biphasic modulation of glycine response by Zn 2+ results from changes in apparent glycine affinity.

Gramicidin perforated patch recording technique

Cl- is one of the major ionic constituents of cells and extracellular spaces. Intracellular Cl- plays an important role in regulating the cell volume and pH, in both salt secretion and reabsorption, in membrane excitability, and G-protein-dependent intracellular signal transduction. GABA and glycine are the primary inhibitory neurotransmitters. Such agonist-stimulated responses are affected by the intracellular Cl- concentration ([Cl-]i). However, it was difficult to make an electrical recording of the physiological Cl- response from cells with native intracellular Cl- activity because of the limitations of present recording techniques using conventional glass-microelectrode, whole-cell patch and nystatin perforated patch recording modes. Recently, this difficulty was overcome by developing the gramicidin perforated patch recording mode in our laboratory. Gramicidin is a polypeptide antibiotic that forms pores in the cell membrane as well as nystatin but allows only monovalent cations to permeate the membrane, enabling both [Cl-]i and the second messenger system to remain undisturbed. Here, I would like to primarily focus on the GABA- and glycine-induced Cl- responses to in mammalian CNS neurons maintaining native cellular [Cl-]i under normal and pathological neuronal conditions by use of gramicidin perforated patch recording configuration. Age-related and developmental changes in neuronal [Cl-]i are also described in detail.

Software-based correction of single compartment series resistance errors

Resistance across a patch pipette following seal formation and attainment of the whole cell recording configuration can introduce considerable and sometimes non-intuitive errors into voltage clamp recordings. These errors can be corrected actively on most commercially available amplifiers, although decisions during the experiment often must be made concerning the degree of correction to be employed, and the decision is essentially irreversible once the data is recorded. Amplifier-based corrections assume a single compartment, and thus any degree of compensation could be applied after data collection with a delay of only a single digitization interval. This report describes computer algorithms that correct capacitative filtering that results from pipette series resistance as well as the voltage error for current responses with either linear or non linear current–voltage curves. The algorithms are designed to operate on data that are recorded at a single holding potential rather than in response to voltage steps. The simplest algorithm for correction of responses with linear IV consists of about a dozen lines of C code and can easily be incorporated into data analysis programs, spreadsheets, and mathematical analysis packages. Code, sample programs, and spreadsheets that implement these algorithms are available from ftp.pharm.emory.edu.

ATP-dependent regulation of inwardly rectifying K+ current in bovine retinal pigment epithelial cells.

Inwardly rectifying K+ current (IKir) in freshly isolated bovine retinal pigment epithelial (RPE) cells was studied in the whole cell recording configuration of the patch-clamp technique. When cells were dialyzed with pipette solution containing no ATP, IKir ran down completely in <10 min [half time (t1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained IKir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM ATP was used, IKir ran down by approximately 71%. Mg2+ was a critical cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+ (t1/2 = 1.8 min). IKir also ran down when the pipette solution contained 4 mM Mg2+ + 4 mM 5'-adenylylimidodiphosphate (t1/2 = 2.7 min) or 4 mM adenosine 5'-O-(3-thiotriphosphate) (t1/2 = 1.9 min), nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We conclude that the sustained activity of IKir in bovine RPE requires intracellular MgATP and that the underlying mechanism may involve ATP hydrolysis.

Voltage-activated sodium currents in a cell line expressing a Cu,Zn superoxide dismutase typical of familial ALS.

The whole-cell configuration of the patch-clamp recording was used to study the voltage-dependent Na+ currents in a model system for the familial form of amyotrophic lateral sclerosis (ALS) associated with mutations in Cu,Zn superoxide dismutase. Here we report that the amplitude of voltage-gated Na+ currents is significantly reduced in cell lines expressing mutant Cu,Zn superoxide dismutase G93A when compared with the parental, untransfected cell line and to a cell line expressing the wild-type enzyme. This effect is associated with a shift toward positive values of the steady-state inactivation curve of the Na+ currents. These results indicate that expression of a Cu,Zn superoxide dismutase typical of patients affect with familial ALS influence the functionality of the voltage-dependent Na+ channels; this effect may contribute to the pathogenesis of the disease.

Effects of sulphonylureas on the volume-sensitive anion channel in rat pancreatic beta-cells.

1. The volume-sensitive anion conductance in rat pancreatic beta-cells was studied directly using the conventional whole-cell and perforated patch recording techniques, and indirectly by measuring 3H-taurine efflux from pre-loaded, perifused islets. 2. Using the conventional whole-cell recording configuration, activation of the outwardly-rectifying, DIDS-sensitive conductance was induced by glibenclamide (10 microM) but not by tolbutamide (100 microM) nor by meglitinide (20 microM). A high concentration of glibenclamide (100 microM) caused a voltage-and time-dependent inhibition of the conductance. Tolbutamide had a modest inhibitory effect on swelling-induced inward currents. 3. In perforated patch recordings, glibenclamide, tolbutamide and meglitinide were all without effect on the conductance, although activation could be induced under these conditions by exposure to a hypotonic bath solution. 4. The rate of efflux of 3H-taurine, a marker for activity of the volume-sensitive anion channel, from preloaded, perifused islets was markedly stimulated by exposure to a hypotonic solution. However, glibenclamide and tolbutamide were both without effect. 5. Electrical activity of beta-cells in response to glibenclamide or tolbutamide was not inhibited by 4,4'-dithiocyanatostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of the volume-sensitive anion channel. 6. It is concluded that activity of the volume-sensitive anion conductance in rat pancreatic beta-cells is not modulated by the sulphonylurea receptor. The activation of the conductance by glibenclamide in whole-cell recordings could be the result of a non-specific interaction of the drug with plasma membrane lipids.

Temperature-dependent effect of zolpidem on the GABA A receptor-mediated response at recombinant human GABA A receptor subtypes

The effects of zolpidem on the two forms of recombinant human GABA A receptors (α1β2γ2s and α3β2γ2s) at different temperatures were functionally investigated, using the whole-cell patch recording configuration. In both forms, zolpidem potentiated the response to GABA in a concentration-dependent manner. At 16°C, the apparent dissociation constant ( K D) values for the α1β2γ2s and α3β2γ2s forms were 3.7×10 −8 and 5.6×10 −7 M, respectively. When the temperature was increased to 36°C, the K D values for the α1β2γ2s and α3β2γ2s forms were 2.1×10 −7 and 1.5×10 −6 M, respectively. Although the affinity ratio was reduced from 15.1 to 7.1-fold the selectivity of zolpidem for the α1β2γ2s still remained at 36°C.

Phosphorylation of the GABA A receptor γ2L subunit in rat sensory neurons may not be necessary for ethanol sensitivity

The effect of ethanol on the current activated by 2.5 to 40 μM γ-aminobutyric acid (GABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons under voltage clamp in the whole-cell and perforated-patch recording configurations. Our results confirmed that GABA A-activated current in these neurons was insensitive to ethanol at concentrations from 2.5 to 100 mM [G. White, D.M. Lovinger, F.F. Weight, Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron, Brain Res. 507 (1990) 332–336.]. In addition, the ethanol sensitivity of GABA receptors was studied under conditions that promote phosphorylation of the PKC site on the γ2L subunit. The presence of the γ2L and other subunit mRNAs was detected by reverse transcription (RT) of total RNA purified from adult DRG followed by polymerase chain reaction (PCR) using subunit specific primer sets. We found that the GABA response remained insensitive to 2.5–100 mM ethanol despite: (i) the extracellular preapplication of 5, 20 or 500 nM phorbol 12-myristate 13-acetate (PMA); (ii) raising free intracellular Ca 2+ ([Ca 2+] i) from 7 to 100 or 600 nM by altering the intracellular Ca 2+/EGTA ratio; (iii) intracellular application of PKC (0.247 U ml −1 ); and (iv) combining the intracellular application of 1 μM okadaic acid and 30 μM peptide 3 with the extracellular application of 20 nM PMA. These results suggest that phosphorylation of the γ2L subunit is not the only requirement for ethanol sensitivity of GABA A receptors.

Insulin occludes leptin activation of ATP-sensitive K+ channels in rat CRI-G1 insulin secreting cells.

1. Using whole-cell and cell-attached recording configurations, the effects of insulin on leptin activation of ATP-sensitive K+ (KATP) channels were examined in the CRI-G1 insulinoma cell line. 2. Whole-cell recordings demonstrated that the leptin-induced hyperpolarization and increased potassium conductance are completely occluded by prior exposure to insulin (1-50 nM). In cell-attached recordings, insulin prevented leptin activation of tolbutamide-sensitive KATP channels. Furthermore, insulin (50 nM) slowly and completely reversed the effects of leptin (10 nM), an action not attributable to direct inhibition of KATP channels per se. 3. Low concentrations of insulin-like growth factor-1 (IGF-1; 10-100 nM) failed to prevent leptin activation of KATP channels, although higher concentrations (1 microM) did inhibit leptin actions. 4. The action of insulin was specific for leptin, as the hyperglycaemic agent diazoxide activated KATP channels following prior exposure to insulin. 5. Wortmannin (1-10 nM) and LY 294002 (10 microM) prevented leptin activation of KATP channels, indicating an involvement of phosphoinositide 3-kinase (PI 3-kinase). 6. In conclusion, leptin activation of KATP channels is counter-regulated by insulin in the CRI-G1 insulinoma cell line. This feedback mechanism may be important in the local integration of hormonal signals which regulate insulin secretion and in alterations of metabolic homeostasis associated with obesity and non-insulin dependent diabetes mellitus (NIDDM).