Currents In Cell-attached Patches Research Articles

Stretch response of the mechano-gated channel TMEM63A in membrane patches and single cells.

The recently discovered ion channel TMEM63A has biophysical features distinctive for mechano-gated cation channels, activating at high pressures with slow kinetics while not inactivating. However, some biophysical properties are less clear, including no information on its function in whole cells. The aim of this study is to expand the TMEM63A biophysical characterization and examine the function in whole cells. Piezo1-knockout HEK293T cells were cotransfected with human TMEM63A and green fluorescent protein (GFP), and macroscopic currents in cell-attached patches were recorded by high-speed pressure clamp at holding voltages from -120 to -20 mV with 0-100 mmHg patch suction for 1 s. HEK293 cells cotransfected with TMEM63A and GCaMP5 were seeded onto polydimethylsiloxane (PDMS) membrane, and the response to 3-12 s of 1%-15% whole cell isotropic (equi-biaxial) stretch induced by an IsoStretcher was measured by the change in intracellular calcium ([Ca2+]i) and presented as (ΔF/F0 > 1). Increasing patch pressures activated TMEM63A currents with accelerating activation kinetics and current amplitudes that were pressure dependent but voltage independent. TMEM63A currents were plateaued within 2 s, recovered quickly, and were sensitive to Gd3+. In whole cells stretched on flexible membranes, radial stretch increased the [Ca2+]i responses in a larger proportion of cells cotransfected with TMEM63A and GCaMP5 than GCaMP5-only controls. TMEM63A currents are force activated and voltage insensitive, have a high threshold for pressure activation with slow activation and deactivation, and lack inactivation over 5 s. TMEM63A has the net polarity and kinetics that would depolarize plasma membranes and increase inward currents, contributing to a sustained [Ca2+]i increase in response to high stretch.NEW & NOTEWORTHY TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01–10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema.SIGNIFICANCE STATEMENTATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Brain astrocyte‐derived EETs and 20‐EHTE elicit opposing actions on calcium movement and KCa channel current activities in astrocytes

Cultured brain astrocytes incubated with arachidonic acid (AA) produce four CYP epoxygenase metabolites epoxyeicosatrienoic acids (EETs) 5,6‐, 8,9‐, 1,12‐and 14,15‐EET. The formation and action of 20‐hydroxyeicosatrienoic acid (20‐HETE) in brain astrocytes is not known. We investigated the capacity of brain astrocytes to express CYP‐4A‐ù‐hydroxylase protein and to convert AA to 20‐HETE. We also investigated the effects of 20‐HETE on membrane potential, KCa single‐channel activity, and on intracellular Ca2+ ([Ca2+]i) levels in cultured astrocytes. We found that astrocytes express CYP‐4A‐ù‐hydroxylase at the transcript and protein levels and produce 20‐HETE when incubated with AA. Treatment of astrocytes with 20‐HETE (100 nM) inhibited the openings of KCa single‐channel currents in cell‐attached patches and caused astrocyte membrane potential depolarization, whereas it had no effect on [Ca2+]i levels in astrocytes loaded with the Ca2+ probe Fura‐2AM. In contrast, treatment of cultured astrocytes with one of the EETs, 11.12‐EET increased [Ca2+]i level and activated astrocyte KCa channel currents. These findings indicate that the EETs and 20‐HETE produced and released by astrocytes could have opposing physiological actions at astrocyte end feet, which might be crucial for the maintenance of constant hyperemic cerebral blood flow during increased neuronal activation.

Phospholipase C not protein kinase C is required for the activation of TRPC5 channels by cholecystokinin

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the brain where it interacts with two G protein-coupled receptors (CCK1 and CCK2). Both types of CCK receptors are coupled to Gq/11 proteins resulting in increased function of phospholipase C (PLC) pathway. Whereas CCK has been suggested to increase neuronal excitability in the brain via activation of cationic channels, the types of cationic channels have not yet been identified. Here, we co-expressed CCK2 receptors and TRPC5 channels in human embryonic kidney (HEK) 293 cells and studied the effects of CCK on TRPC5 channels using patch-clamp techniques. Our results demonstrate that activation of CCK2 receptors robustly potentiates the function of TRPC5 channels. CCK-induced activation of TRPC5 channels requires the functions of G-proteins and PLC and depends on extracellular Ca2+. The activation of TRPC5 channels mediated by CCK2 receptors is independent of IP3 receptors and protein kinase C. CCK-induced opening of TRPC5 channels is not store-operated because application of thapsigargin to deplete intracellular Ca2+ stores failed to alter CCK-induced TRPC5 channel currents significantly. Bath application of CCK also significantly increased the open probability of TRPC5 single channel currents in cell-attached patches. Because CCK exerts extensive effects in the brain, our results may provide a novel mechanism to explain its roles in modulating neuronal excitability.

Analyses of gating thermodynamics and effects of deletions in the mechanosensitive channel TREK-1

TREK-1, a mechanosensitive K channel from the two-pore family (K2P), is involved in protective regulation of the resting potential in CNS neurons and other tissues. The structure of TREK-1 and the basis of its sensitivity to stretch and variety of lipid-soluble factors remain unknown. Using existing K channel structures as modeling templates, TREK-1 was envisioned as a two-fold symmetrical complex with the gate formed primarily by the centrally positioned TM2b helices of the second homologous repeat. Opening was modeled as a conical expansion of the barrel separating TM2b’s accompanied by extension of TM2a helices with the cytoplasmic TM2a-TM1b connector. Seeking first experimental support to the models we have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm2 in models was supported by the experimental ΔA of ~4 nm2 derived from the slope of open probability versus membrane tension in HEK-293T cells and their cytoskeleton-depleted blebs. In response to steps of suction, wild-type channel produced transient currents in cell-attached patches and mostly sustained currents upon patch excision. TREK-1 motifs not present in canonical K channels include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. Deletion of the extracellular loop (Δ76–124) reduced the average current density in patches, increased spontaneous activity and generated a larger sub-population of high-conductance channels, while activation by tension augmented by arachidonic acid was fully retained. Further deletion of the C-terminal end (Δ76–124/Δ334–411) removed voltage dependency but otherwise produced no additional effect. In an attempt to generate a cysteine-free version of the channel, we mutated two remaining cysteines 159 and 219 in the transmembrane region. C219A did not compromise channel activity, whereas the C159A/S mutants were essentially inactive. Treatment with β-mercaptoethanol suggested that none of these cysteines form functionally-important disulfides.

Deletion Analysis of the Mechanosensitive TREK-1 Channel

TREK-1, the first functionally characterized mechanosensitive K channel from the two-pore family (K2P) is involved in protective regulation of resting potential in CNS neurons and many other tissues. The structural basis of TREK-1 sensitivity to stretch and other factors such as arachidonic acid (AA) and anesthetics remains unknown. Attempts to use existing K channel structures as templates for TREK-1 modeling have identified several motifs that are not present in canonical K channels, which include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. To characterize functional roles of these domains, we analyzed TREK-1 deletion and cysteine mutants in patch-clamp experiments. In response to steps of suction, the control TREK-1-EGFP fusion protein expressed in HEK-293 cells produced transient currents in cell-attached patches and non-inactivating sustained currents upon patch excision. Responses in both configurations were augmented by AA. Deletion of the extracellular loop (Δ76-124) reduced functional surface expression of channels and increased background activity, but the activation by tension augmented by AA was fully retained. Further deletion of the C-terminal end (Δ76-124, Δ334-411) produced no additional effect. In an attempt to generate cysteine-free version of the channel, we additionally mutated two cysteines in the transmembrane domain. C219A did not compromise channel activity, whereas C159A/S was essentially inactive. Experiments in the presence of mercaptoethanol suggested that none of these cysteines form functionally-important disulfides. The functional deletion mutant without C219 is now topologically closer to other K channels and makes an amenable system for homology modeling and testing by disulfide cross-linking.

A cytosolic factor that inhibits KATP channels expressed in Xenopus oocytes by impairing Mg-nucleotide activation by SUR1.

ATP-sensitive K(+) (K(ATP)) channels couple cell metabolism to cell electrical activity. Wild-type (Kir6.2/SUR1) K(ATP) channels heterologously expressed in Xenopus oocytes give rise to very small inward currents in cell-attached patches. A large increase in the current is observed on patch excision into zero ATP solution. This is presumably due to loss of intracellular ATP leading to unblock of K(ATP) channels. In contrast, channels containing Kir6.2 mutations associated with reduced ATP-sensitivity display non-zero cell-attached currents. Unexpectedly, these cell-attached currents are significantly smaller (by approximately 40%) than those observed when excised patches are exposed to physiological ATP concentrations (1-10 mm). Cramming the patch back into the oocyte cytoplasm restores mutant K(ATP) current amplitude to that measured in the cell-attached mode. This implies that the magnitude of the cell-attached current is regulated not only by intracellular ATP but also by another cytoplasmic factor/s. This factor seems to require the nucleotide-binding domains of SUR1 to be effective. Thus a mutant Kir6.2 (Kir6.2DeltaC-I296L) expressed in the absence of SUR1 exhibited currents of similar magnitude in cell-attached patches as in inside-out patches exposed to 10 mm MgATP. Similar results were found when Kir6.2-I296L was coexpressed with an SUR1 mutant that is insensitive to MgADP or MgATP activation. This suggests the oocyte contains a cytoplasmic factor that reduces nucleotide binding/hydrolysis at the NBDs of SUR1. In conclusion, our results reveal a novel regulatory mechanism for the K(ATP) channel. This was not evident for wild-type channels because of their high sensitivity to block by ATP.

Effects of Dopamine on ATP-Sensitive Potassium Channels in Porcine Coronary Artery Smooth-Muscle Cells

Dopamine is reported to be a coronary vasodilator; however, the exact mechanism of dopamine action in the coronary circulation remains unclear. In this study, we hypothesized that dopamine-induced activation of coronary ATP-sensitive potassium (KATP) channels may be associated with coronary vasodilation. We therefore investigated the direct effects of dopamine on coronary KATP-channel activity. We used patch-clamp configurations to investigate the effects of dopamine on coronary KATP-channel activity. Application of dopamine (10 to 10 M) to the bath solution during cell-attached recordings induced a concentration-dependent increase in KATP-channel activity. In contrast, dopamine failed to activate KATP channels in inside-out patches. Dopamine-induced coronary KATP-channel currents in cell-attached patches were inhibited by pretreatment with the selective D1-like antagonist, Sch-23390, but they were not influenced by the selective D2-like antagonist, domperidone, or the beta-adrenergic receptor antagonist, propranolol. The selective D1-like agonist, SKF-38393, and the adenylyl cyclase activator, forskolin, mimicked the dopamine effects on coronary KATP channels. Furthermore, pretreatment with an inhibitor of protein kinase A, Rp-cAMPS, abolished the dopamine-induced KATP-channel activation. This study demonstrates that dopamine activates coronary KATP channels via signal transduction involving the D1-like dopaminergic receptor-protein kinase A-signaling pathway.

Effects of Prostaglandin E1 on Vascular ATP-Sensitive Potassium Channels

Prostaglandin E1 (PGE1) has been reported to activate ATP-sensitive potassium (KATP) channels, which induces vasorelaxation. However, direct evidence of PGE1 interactions with vascular KATP channels is limited. The present study investigated the effects and mechanisms of PGE1 on vascular KATP channels in both isometric tension and patch clamp experiments. Isometric tension experiments were performed in rat thoracic aortic rings without an endothelium. Electrophysiologic experiments were performed using patch-clamp techniques to monitor KATP channels in rat vascular smooth muscle cells. PGE1 significantly decreased the isometric tension in a concentration-dependent manner, which was partially inhibited by pretreating with a KATP channel inhibitor, glibenclamide (1 microM), or an inhibitor of protein kinase A (PKA), Rp-cAMPS (100 microM). Application of PGE1 to the bath solution during cell-attached recordings induced a significant increase in KATP channel activity, whereas PGE1 failed to activate KATP channels in the inside-out patches. The PGE1-induced KATP channel currents in cell-attached patches were abolished by pretreating with Rp-cAMPS (100 microM). The results indicate that the activation of vascular KATP channels played an important role in the PKA-dependent PGE1-induced vasorelaxation. Furthermore, an electrophysiological experiment demonstrated that PGE1 activated vascular KATP channels via PKA activation.

Nav Channel Mechanosensitivity: Activation and Inactivation Accelerate Reversibly with Stretch

Voltage-gated sodium channels (Nav) are modulated by many bilayer mechanical amphiphiles, but whether, like other voltage-gated channels (Kv, HCN, Cav), they respond to physical bilayer deformations is unknown. We expressed human heart Nav1.5 pore α-subunit in oocytes (where, unlike αNav1.4, αNav1.5 exhibits normal kinetics) and measured small macroscopic currents in cell-attached patches. Pipette pressure was used to reversibly stretch the membrane for comparison of I Na( t) before, during, and after stretch. At all voltages, and in a dose-dependent fashion, stretch accelerated the I Na( t) time course. The sign of membrane curvature was not relevant. Typical stretch stimuli reversibly accelerated both activation and inactivation by ∼1.4-fold; normalization of peak I Na( t) followed by temporal scaling (∼1.30- to 1.85-fold) resulted in full overlap of the stretch/no-stretch traces. Evidently the rate-limiting outward voltage sensor motion in the Nav1.5 activation path (as in Kv1) accelerated with stretch. Stretch-accelerated inactivation occurred even with activation saturated, so an independently stretch-modulated inactivation transition is also a possibility. Since Nav1.5 channel-stretch modulation was both reliable and reversible, and required stretch stimuli no more intense than what typically activates putative mechanotransducer channels (e.g., stretch-activated TRPC1-based currents), Nav channels join the ranks of putative mechanotransducers. It is noteworthy that at voltages near the activation threshold, moderate stretch increased the peak I Na amplitude ∼1.5-fold. It will be important to determine whether stretch-modulated Nav current contributes to cardiac arrhythmias, to mechanosensory responses in interstitial cells of Cajal, to touch receptor responses, and to neuropathic (i.e., hypermechanosensitive) and/or normal pain reception.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca 2+-activated K + channel current in cultured rat hippocampal astrocytes

Brief hypoxia differentially regulates the activities of Ca 2+-activated K + channels (K Ca) in a variety of cell types. We investigated the effects of hypoxia (<2% O 2) on K Ca channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K Ca current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K Ca currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K Ca channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K Ca current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca 2+ external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K Ca channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K Ca currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K Ca single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K Ca currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients.

We investigated the types and distribution of voltage-gated K+ channels in the soma and apical dendrite of layer 5 (L5) neocortical pyramidal neurones, of young rats (postnatal days 13-15), in acute brain slices. A slow inactivating outward K+ current and a fast inactivating outward K+ current were detected in nucleated patches. The slow K+ current was completely blocked by tetraethylammonium (TEA) with an IC50 of 5 +/- 1 mM (mean +/- s.e.m.) and was partially blocked by 4-aminopyridine (4-AP). The fast K+ current was blocked by 4-AP with an IC50 of 4.2 +/- 0.5 mM, but was not blocked by TEA. The activation kinetics of the slow K+ current were described by a second order Hodgkin-Huxley model. The slow K+ current displayed bi-exponential inactivation. A fourth order Hodgkin-Huxley model for activation and first order for inactivation described the kinetics of the fast K+ current. In somatic cell-attached recordings, three classes of single K+ channels could be differentiated based on their unitary conductance and inactivation kinetics, a fast inactivating channel having a conductance of 13 +/- 1 pS, a slow inactivating channel having a conductance of 9.5 +/- 0.5 pS, and a very slowly inactivating channel having a conductance of 16 +/- 1 pS. The inactivation time constants of the slow and of the very slow K+ channel corresponded to the two inactivation time constants of the slow K+ current observed in nucleated patches. This suggested that two distinct K+ channels mediated the slow K+ current in nucleated patches. The three subtypes of K+ channels that were observed in somatic recordings were present along the apical dendrite. The amplitude of ensemble K+ currents in cell-attached patches decreased along the apical dendrite as the distance from the soma increased, with a slope of -0.9 +/- 0.3 pA per 100 microm. The results suggest that the decrease of the voltage-gated K+ channel density from the soma along the apical dendrite of L5 pyramidal neurones helps to define a distal, low threshold region for the initiation of dendritic regenerative potentials.

Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.

Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.

Heat transduction in rat sensory neurons by calcium-dependent activation of a cation channel.

The mechanism of heat transduction in vertebrate sensory neurons was investigated in vitro by using cultured dorsal root ganglion neurons from adult rat. In response to a physiologically relevant range of stimulus temperatures (23-45 degrees C), a subpopulation of small dorsal root ganglion neurons are depolarized by a cation current (heat-activated current, Iheat) that is antagonized by extracellular cesium. Heat-induced single-channel currents in cell-attached patches are evoked at a similar range of temperatures. Iheat is a calcium-dependent current activated indirectly by heat-evoked release of calcium from intracellular stores. This suggests that the channel itself is not the transducer of thermal energy. Similar to nociceptive heat sensation in vivo, Iheat is enhanced by the hyperalgesic agent prostaglandin E2 and only partially adapts during prolonged heat stimuli. To our knowledge, these data provide the first demonstration that ion channels can mediate heat transduction in mammalian sensory neurons and provide evidence that heat causes the channels to open via an increase in the intracellular second messenger calcium.

Investigations on the mechanism of action of the antiproliferant and ion channel antagonist flufenamic acid.

The compound flufenamic acid has been previously described as an inhibitor of chloride- and non-selective cation channels. Moreover, this compound showed antiproliferative effects in the mouse fibroblast cell line LM(TK-). In this study, we investigated the effects of this compound on cell proliferation and membrane currents induced by mitogens (such as fetal calf serum, FCS) or platelet-derived growth factor (PDGF) in LM(TK-) cells. After a brief application of FCS or PDGF (5-15 s), the electrical response of the cells was biphasic: First, a transient potassium conductance was activated, which appeared 8.3 +/- 0.7 s after the onset of stimulation and lasted for 30.1 +/- 2.9 s. The corresponding single channel currents in cell-attached patches had an amplitude of 3-4 pA (at a holding potential of +60 mV). The second effect of serum or PDGF was the occurrence of a cation conductance for monovalent ions (sodium, potassium and cesium) and calcium. In contrast to the potassium current, this conductance activated later (11.8 +/- 1.6 s after onset of fetal calf serum stimulation) and remained activated for minutes. Flufenamic acid inhibited the proliferation of LM(TK-) cells reversibly and in a concentration-dependent manner. This effect can be correlated with the inhibitory effects of flufenamic acid on mitogen-induced membrane currents: The compound inhibited the non-selective cation current with an IC50 of 38 microM, whereas 135 microM were necessary for halfmaximal inhibition of the potassium current; this is very close to the concentration for halfmaximal inhibition of cell proliferation (120 microM). Hence, on the grounds of this comparison the blockade of the non-selective cation current appears to be of only minor importance for the blockade of cell proliferation.

Voltage-activated proton currents in membrane patches of rat alveolar epithelial cells.

1. Voltage-activated H(+)-selective currents were studied in cell-attached and excised, inside-out patches of membrane from rat alveolar epithelial cells in primary culture. The pH was varied from 5.5 to 7.5 in the pipette (pHo) and in the bath (pHi). 2. H+ currents in cell-attached patches exhibited activation kinetics and voltage dependence intermediate between their behaviour after excision into pH 6.5 and pH 7.5 solutions, consistent with reported pHi values for alveolar epithelial cells. 3. In inside-out patches, increasing pHo shifted the threshold voltage for activating H+ currents and the relationship between the time constant of activation of H+ currents (tau act) and voltage (V) negatively along the voltage axis by 40-50 mV (unit pH)-1. 4. Decreasing pHi shifted the activation threshold and the tau act-V relationship negatively along the voltage axis by 40-50 mV (unit pH)-1. In addition, at lower pHi the activation of H+ currents upon patch depolarization was markedly faster, with tau act increasing approximately 4- to 5-fold per unit pHi. 5. The limiting slope H+ conductance increased only by a factor of 1.7 per unit pH when pHi was lowered in the range 7.5-5.5 (at constant pHo 7.5). This result suggests that H+ permeation through voltage-activated H+ 'channels' occurs by a mechanism distinct from H+ permeation through water-filled ion channels, in which the conductance might be expected to increase in direct proportion to [H+].

Burst kinetics of single NMDA receptor currents in cell-attached patches from rat brain cortical neurons in culture.

Burst kinetics of single NMDA receptor currents in cell-attached patches from rat brain cortical neurons in culture.

Single-channel activity in cultured cortical neurons of the rat in the presence of a toxic dose of glutamate.

Rat cortical neurons grown in cell culture were exposed to 500 microM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca(2+-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K+ channel requiring intracellular calcium ([Ca2+]i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 +/- 6 pS; 'BK channel'). Another Ca(2+)-dependent channel permeable for Cl- (conductance for outward currents in cell-attached patches 72 +/- 17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K+ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca2+]i. Both phases of increasing channel activity required the presence of extracellular Ca2+ suggesting that [Ca2+]i was mainly increased by Ca2+ influx. The N-methyl-D-aspartate (NMDA) antagonists dizocilpine (MK-801, 10 microM) and DL-2-amino-5-phosphonovaleric acid (AP5; 100 microM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca2+ through NMDA receptor channels causes a strong activation of Ca(2+)-dependent K+ channels, which is likely to result in pronounced loss of intracellular K+. NMDA receptor channels seem to remain active for a long time (> 10 min) after the end of glutamate application.

GABAb receptors are coupled to a barium-insensitive outward rectifying potassium conductance in premotor respiratory neurons

1. Labeled premotor respiratory neurons from neonatal rats in culture were used to study the effects of (+/-)baclofen, a selective gamma-aminobutyric acid (GABA)b receptor agonist known to inhibit rhythmic breathing movements in mammals. Bath application of (+/-)baclofen-activated outward currents in cell-attached patches, suggesting that a second messenger system linked the (+/-)baclofen-activated conductance (GBac) to the GABAb receptor. 2.GBac channels exhibited outward rectification and were insensitive to blockade by Ba2+ and Cs+. The single-channel conductance was 100 pS in symmetrical K+ solutions and decreased as [K+]o was reduced. The reversal potential for the GBac channel shifted 45 mV/decade when [K+]o was changed indicating that it was predominantly selective for K+ ions. These properties were similar to those of the S-channel in Aplysia sensory neurons. 3. The properties of GBac channels were distinct from those associated with the GABAb mediated slow inhibitory postsynaptic potential (IPSP), indicating that GABAb receptors can be associated with more than one type of K+ channel. We propose that GBac channels modulate the repetitive firing activity of premotor respiratory neurons and may also participate in presynaptic inhibition.

Single-channel dose-response studies in single, cell-attached patches

A method for carrying out dose-response studies of ion channel currents in cell-attached patches has been devised. Patch pipettes are filled at the tip with a solution containing one concentration of ligand and then backfilled with another. The concentration of ligand at the membrane is described as a function of time by the equation for diffusion in a cone, allowing response vs. time data to be transformed into a dose-response curve. For Xenopus myocyte cholinergic receptors, examples of the use of this method are given for several concentration-dependent reactions including blockade by the local anesthetic QX-222, activation by acetylcholine, and modulation of current amplitude by sodium ions. Several methods of analyzing the nonstationary channel kinetics are presented, including a pseudo-stationary approach that uses interval likelihood maximization.