Patch Depolarization Research Articles

Bendroflumethiazide a potent opener of human BK channel : a new perspective for an old drug in the treatment of periodic paralysis

Carbonic‐anhydrase inhibitors and related drugs are used in the neuromuscular diseases but their effects on human Ca2+activated‐ K+‐(BK) channels are not known. The effects of acetazolamide(ACTZ), dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX) and hydrochlorthiazide(HCT) on the human BK channel alpha‐subunit(hslo) expressed in HEK293 cells were investigated using patch‐clamp technique. The data were compared with that of NS1619, quercetin(QUERC) and resveratrol(RESV). We showed that the hslo whole‐cell current was potentiated by all drugs in the range of concentration tested(10−11–10−3M) with different potency and efficacy, with the exception of HCT. The ranking of the openers at −60 mV(Vm) was BFT(EC50=1.1±3×10− 9M)>; ACTZ(EC50=1.21±2×10−7M) >;DCP(EC50=4.3±1×10−7M) ≥RESV(EC50=5.5±1×10−7M)≥ ETX(EC50=7.1±0.1×10−7M)>; NS1619(EC50=5.5±1×10−5M)>; MTZ(EC50=1.17±1×10−4M)≥ QUERC(EC50=1.5±0.1×10−4M). We found that BFT and ACTZ were the most potent drugs at −60 mV(Vm). Patch depolarization potentiated the drugs action. The observed ACTZ and DCP actions on hslo channel at −60 mV(Vm) at sub‐micromolar concentrations explain their use in hypokalemic‐PP. BFT may be used in the hyperkalemic‐PP. This work is supported by Telethon Grants.

Exocytosis at the Ribbon Synapse of Retinal Bipolar Cells Studied in Patches of Presynaptic Membrane

The distribution of exocytic sites and ion channels in the synaptic terminal of retinal bipolar cells was investigated by measuring capacitance and conductance changes in cell-attached patches of presynaptic membrane. Patch depolarization evoked capacitance and conductance increases that were inhibited by blocking Ca(2+) influx or loading the terminal with EGTA. The increase in capacitance declined as the depolarization approached the reversal potential for Ca(2+), indicating that it was a result of Ca(2+)-dependent exocytosis. The conductance increase was caused by K(Ca) channels that were also activated by Ca(2+) influx. Two observations indicated that sites of exocytosis and endocytosis colocalized with clusters of Ca(2+) channels and K(Ca) channels; the initial rate of exocytosis was correlated with the activation of K(Ca) channels, and exocytosis did not occur in the 41% of patches lacking this conductance. Electron microscopy demonstrated that there were approximately 16 vesicles docked to the plasma membrane at each active zone marked by a ribbon, but vesicles were also attached to the rest of the membrane at a density of 1.5/microm(2). The density of ribbons was 0.10 +/- 0.02/microm(2), predicting that approximately 43% of cell-attached patches would lack an active zone. The density of Ca(2+) channel clusters assayed by capacitance and conductance responses was therefore similar to the density of ribbons. These results are consistent with the idea that Ca(2+) channel clusters were colocalized with ribbons but do not exclude the possibility that calcium channels also occurred at other sites. The wide distribution of vesicles docked to the plasma membrane suggests that exocytosis might also be triggered by the spread of Ca(2+) from Ca(2+) channel clusters.

Nitric oxide and thiol reagent modulation of Ca2+-activated K+ (BKCa) channels in myocytes of the guinea-pig taenia caeci.

The modulation of large conductance Ca2+-activated K+ (BKCa) channels by the nitric oxide (NO) donors S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) and agents which oxidize or reduce reactive thiol groups were compared in excised inside-out membrane patches of the guinea-pig taenia caeci. When the cytosolic side of excised patches was bathed in a physiological salt solution (PSS) containing 130 mM K+ and 15 nM Ca2+, few BKCa channel openings were recorded at potentials negative to 0 mV. However, the current amplitude and open probability (NPo) of these BKCa channels increased with patch depolarization. A plot of ln(NPo) against the membrane potential (V) fitted with a straight line revealed a voltage at half-maximal activation (V0.5) of 9.4 mV and a slope (K) indicating an e-fold increase in NPo with 12.9 mV depolarization. As the cytosolic Ca2+ was raised to 150 nM, V0.5 shifted 11.5 mV in the negative direction, with little change in K (13.1 mV). NOCys (10 microM) and SNP (100 microM) transiently increased NPo 16- and 3. 7-fold, respectively, after a delay of 2-5 min. This increase in NPo was associated with an increase in the number of BKCa channel openings evoked at positive potentials by ramped depolarizations (between -60 and +60 mV). Moreover, this NOCys-induced increase in NPo was still evident in the presence of 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), the specific blocker of soluble guanylyl cyclase. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 microM) and reduced glutathione (GSH; 1 mM) also significantly increased NPo (at 0 mV) 7- to 9-fold, as well as increasing the number of BKCa channel openings evoked during ramped depolarizations. Sulfhydryl oxidizing agents thimerosal (10 microM) and 4,4'-dithiodipyridine (4,4DTDP; 10 microM) and the thiol-specific alkylating agent N-ethylmaleimide (NEM; 1 mM) significantly decreased NPo (at 0 mV) to 40-50% of control values after 5-10 min. Ramped depolarizations to +100 mV evoked relatively few BKCa channel openings. The effects of thimerosal on NPo were readily reversed by DTT, while the effects of NOCys were prevented by NEM. It was concluded that both redox modulation and nitrothiosylation of cysteine groups on the cytosolic surface of the alpha subunit of the BKCa channel protein can alter channel gating.

Voltage-gated potassium channels activated during action potentials in layer V neocortical pyramidal neurons.

To investigate voltage-gated potassium channels underlying action potentials (APs), we simultaneously recorded neuronal APs and single K(+) channel activities, using dual patch-clamp recordings (1 whole cell and 1 cell-attached patch) in single-layer V neocortical pyramidal neurons of rat brain slices. A fast voltage-gated K(+) channel with a conductance of 37 pS (K(f)) opened briefly during AP repolarization. Activation of K(f) channels also was triggered by patch depolarization and did not require Ca(2+) influx. Activation threshold was about -20 mV and inactivation was voltage dependent. Mean duration of channel activities after single APs was 6.1 +/- 0.6 ms (mean +/- SD) at resting membrane potential (-64 mV), 6.7 +/- 0.7 ms at -54 mV, and 62 +/- 15 ms at -24 mV. The activation and inactivation properties suggest that K(f) channels function mainly in AP repolarization but not in regulation of firing. K(f) channels were sensitive to a low concentration of tetraethylammonium (TEA, 1 mM) but not to charybdotoxin (ChTX, 100 nM). Activities of A-type channels (K(A)) also were observed during AP repolarization. K(A) channels were activated by depolarization with a threshold near -45 mV, suggesting that K(A) channels function in both repolarization and timing of APs. Inactivation was voltage dependent with decay time constants of 32 +/- 6 ms at -64 mV (rest), 112 +/- 28 ms at -54 mV, and 367 +/- 34 ms at -24 mV. K(A) channels were localized in clusters and were characterized by steady-state inactivation, multiple subconductance states (36 and 19 pS), and inhibition by 5 mM 4-aminopyridine (4-AP) but not by 1 mM TEA. A delayed rectifier K(+) channel (K(dr)) with a unique conductance of 17 pS was recorded from cell-attached patches with TEA/4-AP-filled pipettes. K(dr) channels were activated by depolarization with a threshold near -25 mV and showed delayed long-lasting activation. K(dr) channels were not activated by single action potentials. Large conductance Ca(2+)-activated K(+) (BK) channels were not triggered by neuronal action potentials in normal slices and only opened as neuronal responses deteriorated (e.g., smaller or absent spikes) and in a spike-independent manner. This study provides direct evidence for different roles of various K(+) channels during action potentials in layer V neocortical pyramidal neurons. K(f) and K(A) channels contribute to AP repolarization, while K(A) channels also regulate repetitive firing. K(dr) channels also may function in regulating repetitive firing, whereas BK channels appear to be activated only in pathological conditions.

Ca2+-inhibited non-inactivating K+ channels in cultured rat hippocampal pyramidal neurones.

1. Whole-cell perforated-patch recording from cultured CA1-CA3 pyramidal neurones from neonatal rat hippocampus (20-22 C; [K+]o = 2.5 mM) revealed two previously recorded non-inactivating (sustained) K+ outward currents: a voltage-independent 'leak' current (Ileak) operating at all negative potentials, and, at potentials >= -60 mV, a time- and voltage-dependent 'M-current' (IK(M)). Both were inhibited by 1 mM Ba2+ or 10 microM oxotremorine-M (Oxo-M). In ruptured-patch recording using Ca2+-free pipette solution, Ileak was strongly enhanced, and was inhibited by 1 mM Ba2+ but unaffected by 0.5 mM 4-aminopyridine (4-AP), 1 mM tetraethylammonium (TEA) or 1-10 nM margatoxin. 2. Single channels underlying these currents were sought in cell-attached patch recordings. A single class of channels of conductance approximately 7 pS showing sustained activity at resting potential and above was identified. These normally had a very low open probability (Po < 0. 1), which, however, showed a dramatic and reversible increase (to about 0.9 at approximately 0 mV) following the removal of Ca2+ from the bath. Under these (Ca2+-free) conditions, single-channel Po showed both voltage-dependent and voltage-independent components on patch depolarization from resting potential. The mean activation curve was fitted by a modified Boltzmann equation. When tested, all channels were reversibly inhibited by addition of 10 microM Oxo-M to the bath solution. 3. The channels maintained their high Po in patches excised in inside-out mode into a Ca2+-free internal solution and were strongly inhibited by application of Ca2+ to the inner face of the membrane (IC50 = 122 nM); this inhibition was observed in the absence of MgATP, and therefore was direct and unrelated to channel phosphorylation/dephosphorylation. 4. Channels in patches excised in outside-out mode were blocked by 1 mM Ba2+ but were unaffected by 4-AP or TEA. 5. Channels in cell-attached patches were inhibited after single spikes, yielding inward ensemble currents lasting several hundred milliseconds. This was prevented in Ca2+-free solution, implying that it was due to Ca2+ entry. 6. The properties of these channels (block by internal Ca2+ and external Oxo-M and Ba2+, and the presence of both voltage-dependent and voltage-independent components in their Po/V relationship) show points of resemblance to those expected for channels associated with both Ileak and IK(M) components of the sustained macroscopic currents. For this reason we designate them Ksust ('sustained current') channels. Inhibition of these channels by Ca2+ entry during an action potential may account for some forms of Ca2+-dependent after-depolarization. Their high sensitivity to internal Ca2+ may provide a new, positive feedback mechanism for cell excitation operating at low (near-resting) [Ca2+]i.

The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization.

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Delta myo5Delta cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A-induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37 degrees C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Delta myo5Delta cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Delta myo5Delta cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.

Ca2+ sparks triggered by patch depolarization in rat heart cells.

The goal of this study was to examine the relationship between Ca2+ entry through L-type Ca2+ channels and local [Ca2+]i transients (Ca2+ sparks) in single rat cardiac ventricular cells. L-type Ca2+ channels were activated by depolarization of cell-attached membrane patches, and [Ca2+]i was measured simultaneously as fluo 3 fluorescence using laser scanning confocal microscopy. Patch depolarization with Ca2+ as the charge carrier (10 or 110 mmol.L(-1)) significantly increased the probability of the occurrence of Ca2+ sparks (Ca2+ spark rate) only in the volume of cytoplasm located immediately beneath the membrane patch (basal Ca2+ spark rate, 119 Ca2+ sparks.cell(-1).s(-1); patch depolarization Ca2+ spark rate, 610 Ca2+ sparks.cell(-1).s(-1); P<.005). With Ba2+ in the pipette solution (10 mmol.L(-1)), patch depolarization was not associated with an increased Ca2+ spark rate at the position of the pipette or at any other sites distant from the pipette. Therefore, Ca2+ entry and not voltage per se was a necessary event for the occurrence of Ca2+ sparks. Under identical experimental conditions, patch depolarization experiments opened single L-type Ca2+ channels with a single-channel conductance of 19 pS with Ba2+ as the charge carrier. Although single-channel openings could not be resolved when Ca2+ was the charge carrier, ensemble averages yielded an inward current of up to 0.75 pA. The results suggest that voltage-activated Ca2+ entry through one or a small number of L type Ca2+ channels triggers the release of Ca2+ only from the sarcoplasmic reticulum in direct proximity to those L-type Ca2+ channels. The relatively low probability of triggering Ca2+ sparks may have resulted from some alteration of excitation-contraction coupling associated with the technique of the cell-attached patch clamp.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Endothelin Activates Large-Conductance K+ Channels in Rat Lactotrophs: Reversal by Long-Term Exposure to Dopamine Agonist

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nm ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nm ET-1 on these channels was markedly attenuated in the presence of the selective ETA receptor antagonist BQ-610 (200 nm), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mm KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 μm Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BKCa) channels were blocked by 20 mm tetraethylammonium but were insensitive to the K+ channel blockers apamin (1μ m), charybdotoxin (200 nm), or iberiotoxin (200 nm). Acute application of 10 μm dopamine and 20 nm ET-1 caused activation of BKCa channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (±)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nm), bath application of 20 nm ET-1 resulted in inhibition of spontaneously active BKCa channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BKCa channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ETA receptor to BKCa channels.

Regulation of M-type Potassium Channels in Mammalian Sympathetic Neurons: Action of Intracellular Calcium on Single Channel Currents

Currents through single M-type potassium channels were recorded in membrane patches excised from rat superior cervical sympathetic neurons. Application of Ca2+ to the internal face of inside-out patches produce two forms of M-channel inhibition: a slow, all-or-nothing suppression of activity; and a fast block associated with a concentration-dependent shortening of open times compatible with open-channel block. Both forms of block were enhanced by patch depolarization. Neither was replicated or affected by Mg2+, and both could be recorded in the absence of intracellular ATP, implying that they did not involve phosphorylation. Since the block was reversible in the absence of ATP and since alkaline phosphatase did not reduce channel activity, block was unlikely to have resulted from dephosphorylation. In cell-attached patch recordings, M-channel activity increased during exposure of the cell to Ca2+-free solution and was rapidly reduced on applying 2 mM Ca2+ to the extra-patch solution. This suggests that M-channel activity in these neurons may be tonically regulated by variations in resting intracellular [Ca2+]. Copyright © 1996 Elsevier Science Ltd

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+].

Cation and anion unitary ion channel currents in cultured bovine microglia

The use of excised patches has led to the identification and characterization of two channels not previously reported in microglia. A calcium-dependent K + channel K(Ca) was activated in inside-out patches obtained from cultured bovine microglia and had a unitary conductance of 240 pS with symmetrical 140 mM K + across patches. Mean open times of K(Ca) were exponentially dependent on patch potential and were increased with patch depolarization. Channel open probability (Popen) was increased with patch depolarization with either 5 mM or 140 mM internal K + and was attributable to potential dependent enhancement of both opening frequency and mean open time. Whole cell currents showed the presence of a slowly activating K + conductance, blocked by external TEA (at 2 mM) and likely the macroscopic correlate of the unitary K(Ca); half-maximal activation of the current occurred at +25 mV. An anion channel (unitary conductance of 325 pS with symmetrical Cl − across patches) was activated in inside-out patches with depolarizing and hyperpolarizing potential steps from 0 mV. Channel activation was not dependent on internal Ca 2+. The anion currents inactivated during maintained potential steps with the time constant for inactivation faster with increased patch depolarization. The properties of the K(Ca) and anion channels in microglia membrane may have relevance to cell function in response to neuronal damage in the CNS.

Properties of inward and outward potassium currents in cultured mouse motoneurons

Inward rectifier potassium currents and calcium-dependent potassium currents have been studied in cultured embryonic mouse motoneurons. Sustained unitary inward rectifier potassium currents were recorded from cell-attached patches and the channel conductance was dependent on external K+ concentration with a value of 25 pS when external K+ was 140 mM. The channel open probability exhibited a sigmoidal dependence on potential with the largest values (near 0.7) at depolarizing patch potentials. Inactivating inward rectifier potassium currents were also recorded in some cell-attached patches following voltage steps to hyperpolarizing potentials with the rate of inactivation faster with larger hyperpolarizing steps. Whole-cell inward rectifier potassium currents increased from an initial level to a steady-state level with hyperpolarizing steps to -120 mV from a holding potential of -60 mV; with larger hyperpolarizing commands the peak currents decayed to the steady-state. The steady-state current-voltage relation exhibited a region of negative slope resistance. External Cs+ (0.5-1 mM) reduced the amplitudes of macroscopic currents and diminished the open times of unitary currents consistent with block of open rectifying channels with an estimated KD for channel block of 1 mM. A large conductance calcium-dependent potassium channel was isolated in inside-out patches with a conductance of 240 pS with symmetrical 140 mM K+ across the patches and a conductance of 110 pS when the external K+ was reduced to 5 mM. With symmetrical K+ the channel open probability exhibited a sigmoid dependence on potential with the largest values, in excess of 0.8, associated with patch depolarization. The dependence of open probability on potential was dependent on the concentrations of internal Ca2+ and external K+. Properties of inward rectifier and calcium-dependent K+ channels, such as the voltage dependence of open probability, are involved in the establishment of cellular excitability in motoneurons. Future studies will be useful to investigate whether channel properties of motoneurons are altered after cell treatment with neurotoxic agents including oxygen radicals or excitotoxic amino acids.

Membrane currents controlled by physical forces in cultured mesangial cells

Mechanically-activated ion channels (MACs) of cultured rat mesangial cells were stimulated by applying suction to patch pipets or by exposing cells to hypoosmotic media. MAC density was estimated as 1.5 +/- 0.4 per mu 2. In the absence of any stimulus, MAC open probabilities (N * P) were < 0.0001 increasing as a function of stretch or extracellular hypoosmolarity. Single channel mean open time during stretch increased with patch depolarization whereas hyperpolarization of the membrane delayed MAC inactivation. Ionic conductance of MACs, based on average slope conductances at hyperpolarized potentials, was 76 pS in high external K+ (N = 5) and 40 pS in high external Na+ (N = 8). PK+/PNa+ was estimated to be 4.7. MACs did not permeate Cl-, at least outwardly. Whole cell currents in response to voltage steps applied to resting cells in control conditions were approximately ohmic between -120 mV and 40 mV and were linearly and reversibly dependent on extracellular osmolarity. Our results demonstrate that: (1) MACs can be activated by both negative hydrostatic pressures applied to the pipet and by osmotic gradients; (2) MAC kinetic behavior is sensitive to membrane potential; (3) MACs may participate in cellular responses to physical forces.

Ca2(+)‐activated K+ current involvement in neuronal function revealed by in situ single‐channel analysis in Helix neurones.

1. The properties of single calcium-activated potassium channels (or C-channels) were studied in cell-attached patches using the patch-clamp technique. Experiments were performed on identified Ca2(+)-dependent U cells in juvenile specimens (1-2 months old) of Helix aspersa. 2. The criteria used to identify C-channels were based on comparison between macroscopic C-currents and currents reconstructed from unitary recordings. Both currents had a slow activation rate at large positive potentials which turned into fast activation after large Ca2+ entries. Both currents were blocked by intracellularly injected EGTA. 3. The unitary conductance in normal (5 mM) or reduced (0.5 mM) [K+]o ranged from 24 to 65 pS (mean +/- S.D., 48 +/- 13; n = 64). With 85-110 mM [K+]o, which is approximately equal to the internal [K+], the conductance was 64 pS and the reversal potential was approximately 0 mV. 4. C-channels in U cells were distributed in clusters of three to ten channels (mean 5.05 channels in seventy-five patches). Calcium channels were present in patches containing clustered C-channels. C-channels within clusters behaved independently. 5. With patch electrode containing 8 mM-calcium, C-channels opened transiently upon patch depolarization. Reopenings in quiescent depolarized patches were induced by whole-cell spikes triggered by current pulses applied to an intracellular electrode. Apparent inactivation of C-channels in depolarized patches was in fact due to a decrease in [Ca2+]i resulting from inactivation of Ca2+ channels. 6. Calcium-free saline solutions in the patch electrodes prevented C-channels from opening upon patch depolarization. Entry of calcium through the surrounding membrane induced delayed openings in the patch. Peak opening probability Po occurred 330 +/- 30 ms after a brief Ca2+ entry with a lag period of 50-80 ms. With patch electrodes filled with Ca2(+)-containing saline solutions and under conditions which maximized C-channel opening, peak Po was reached in 20-50 ms. The same value was observed for the whole-cell C-current. 7. The peak Po at a given patch potential and in response to a whole-cell spike was not altered by a previous long-lasting patch depolarization, or by producing several successive Ca2+ entries. Thus, C-channels did not appear to be inactivated by depolarization or increase in [Ca2+]i. 8. C-channels were found to be relatively highly voltage dependent, with an e-fold increase in Po per 14.9 mV increase in potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivating and non-inactivating outward current channels in cell-attached patches of Helix neurons

Two species of inactivating outward current channels and a non-inactivatingvoltage-dependent current were seen in cell-attached patches of Helix neurons. Large, slowly inactivating channels had a slope conductance of 44 pS as measured with patch pipets containing the normal extracellular ion concentrations, including 4 mM potassium. Latency to maximal opening was 50–220 ms, and the inactivation time constant averaged 350 ms. Channel opening was decreased by preceding depolarization. The channels were selective for potassium and inhibited by 50 mM TEA. Small, quickly inactivating channels were 14 pS and had kinetics and voltage dependence similar to I A. Patch depolarization also activated a non-inactivating voltage-dependent outward current having channel conductance and/or kinetics such that individual channel openings and closings could not be distinguished. Such current was also seen in the presence of 50 mM TEA, but not in the presence of Co 2+, characteristics which are similar to outward hydrogen ion currents, described by others in Helix neurons.

An apparatus for precise control of pipetting blood samples

Membrane currents controlled by physical forces in cultured mesangial cells. Mechanically-activated ion channels (MACs) of cultured rat mesangial cells were stimulated by applying suction to patch pipets or by exposing cells to hypoosmotic media. MAC density was estimated as 1.5 ± 0.4 per µ2. In the absence of any stimulus, MAC open probabilities (N * P) were < 0.0001 increasing as a function of stretch or extracellular hypoosmolarity. Single channel mean open time during stretch increased with patch depolarization whereas hyperpolarization of the membrane delayed MAC inactivation. Ionic conductance of MACs, based on average slope conductances at hyperpolarized potentials, was 76 pS in high external K+ (N = 5) and 40 pS in high external Na+ (N = 8). PK+/PNa+ was estimated to be 4.7. MACs did not permeate Cl-, at least outwardly. Whole cell currents in response to voltage steps applied to resting cells in control conditions were approximately ohmic between -120 mV and 40 mV and were linearly and reversibly dependent on extracellular osmolarity. Our results demonstrate that: (1) MACs can be activated by both negative hydrostatic pressures applied to the pipet and by osmotic gradients; (2) MAC kinetic behavior is sensitive to membrane potential; (3) MACs may participate in cellular responses to physical forces.