Channels In Cell-attached Patches Research Articles

Increase in cytosolic Ca2+ produced by hypoxia and other depolarizing stimuli activates a non‐selective cation channel in chemoreceptor cells of rat carotid body

The current model of O2 sensing by carotid body chemoreceptor (glomus) cells is that hypoxia inhibits the outward K(+) current and causes cell depolarization, Ca(2+) influx via voltage-dependent Ca(2+) channels and a rise in intracellular [Ca(2+)] ([Ca(2+)]i). Here we show that hypoxia (<5% O2), in addition to inhibiting the two-pore domain K(+) channels TASK-1/3 (TASK), indirectly activates an ∼20pS channel in isolated glomus cells. The 20pS channel was permeable to K(+), Na(+) and Cs(+) but not to Cl(-) or Ca(2+). The 20pS channel was not sensitive to voltage. Inhibition of TASK by external acid, depolarization of glomus cells with high external KCl (20mm) or opening of the Ca(2+) channel with FPL64176 activated the 20pS channel when 1mm Ca(2+) was present in the external solution. Ca(2+) (10μm) applied to the cytosolic side of inside-out patches activated the 20pS channel. The threshold [Ca(2+)]i for activation of the 20pS channel in cell-attached patches was ∼200nm. The reversal potential of the 20pS channel was estimated to be -28mV. Our results reveal a sequential mechanism in which hypoxia (<5% O2) first inhibits the K(+) conductance and then activates a Na(+)-permeable, non-selective cation channel via depolarization-induced rise in [Ca(2+)]i. Our results suggest that inhibition of K(+) efflux and stimulation of Na(+) influx both contribute to the depolarization of glomus cells during moderate to severe hypoxia.

Single KATP Channel Opening in Response to Action Potential Firing in Mouse Dentate Granule Neurons

ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

CYP-omega-hydroxylation-dependent metabolites of arachidonic acid inhibit the basolateral 10 pS chloride channel in the rat thick ascending limb

Metabolites of arachidonic acid influence sodium chloride (NaCl) transport in the thick ascending limb. Because a 10 pS Cl channel is the major type of chloride channel in the basolateral membrane of this nephron segment, we explored the effect of arachidonic acid on this channel in cell-attached patches. Addition of 5 micromol arachidonic acid significantly decreased channel activity (a product of channel number and open probability) while linoleic acid had no effect. To determine if this was mediated by acachidonic acid per se or by its metabolites, we measured channel activity in the presence of the cyclooxygenase inhibitor indomethacin, the selective lipoxygenase inhibitor nordihydroguaiaretic acid, and the cytochrome P-450 (CYP)-omega-hydroxylation inhibitor 17-octadecynoic acid. Neither cyclooxygenase nor lipoxygenase inhibition had an effect on basal chloride channel activity; further they failed to abolish the inhibitory effect of arachidonate on the 10 pS channel. However, inhibition of CYP-omega-hydroxylation completely abolished the effect of arachidonic acid. The similarity of the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid suggests that the effect of arachidonic acid was mediated by CYP-omega-hydroxylation-dependent metabolites. We conclude that arachidonic acid inhibits the 10 pS chloride channel in the basolateral membrane of the medullary thick ascending limb, an effect mediated by the CYP-omega-hydroxylation-dependent metabolite 20-HETE.

Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin

High-conductance apical K+ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K+ losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+ enrichment. We found that PKA activation using 50 μmol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 μmol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 μmol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 μmol/l Na+ orthovanadate and 10 μmol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

Activation of BK Channels in GH3 Cells by ac-PLA2-Dependent G-Protein Signaling Pathway

BK-channels in GH3 cells are activated by arachidonic acid produced by c-PLA2. beta-adrenergic agonists also activate BK channels and were presumed to do so via production of cAMP. We, however, show for the first time in GH3 cells that a beta-adrenergic agonist activates a pertussis-toxin-sensitive G protein that activates c-PLA2. The arachidonic acid produced by c-PLA2 then activates BK channels. We examined BK channels in cell-attached patches and in excised patches from untreated GH3 cells and from GH3 cells exposed to c-PLA2 antisense oligonucleotides. For the cell-attached patch experiments, physiologic pipette and bath solutions were used. For the excised patches, 150 mM KCl was used in both the pipette and bath solutions, and the cytosolic surface contained 1 microM free Ca2+ (buffered with 5 mM K2EGTA). Treatment of GH3 cells with the G protein activator, fluoroaluminate, (AlF4-) produced an increase in the Po of BK channels of 177 +/- 41% (mean +/- SD) in cell-attached patches. Because G proteins are membrane associated, we also added an activator of G proteins, 100 microM GTP-gamma-S, to the cytosolic surface of excised patches. This treatment leads to an increase in Po of 50 +/- 9%. Similar treatment of excised patches with GDP-beta-S had no effect on Po. Isoproterenol (1 microM), an activator of beta-adrenergic receptors and, consequently, some G proteins, increased BK channel activity 229 +/- 37% in cell-attached patches from cultured GH3 cells. Western blot analysis showed that GH3 cells have beta-adrenergic receptor protein and that isoproterenol acts through these receptors because the beta-adrenergic receptor antagonist, propanolol, blocks the action of isoproterenol. To test whether G protein activation of BK channels involves c-PLA2, we studied the effects of GTP-gamma-S on excised patches and isoproterenol on cell attached patches from GH3 cells previously treated with c-PLA2 antisense oligonucleotides or pharmacological inhibitors of c-PLA2. Neither isoproterenol nor GTP-gamma-S had any effect on Po in these patches. Similarly, neither isoproterenol nor GTP-gamma-S had any effect on Po in cultured GH3 cells pretreated with pertussis toxin. Isoproterenol also significantly increased the rate of arachidonic production in GH3 cells. These results show that some receptor-linked, pertussis-toxin-sensitive G protein in GH3 cells can activate c-PLA2 to increase the amount of arachidonic acid present and ultimately increase BK-channel activity.

Electrophysiological properties of BK channels in Xenopus motor nerve terminals.

Single channel properties of Ca(2+)-activated K(+) (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone-muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca(2+) affinity of the channel (K(d)= 7.7 microM at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca(2+)](i) from 1 microM to 1 mM. The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at > or =+20 mV. The activation rate increased approximately 10-fold in response to a [Ca(2+)](i) increase from 1 to 100 microM, but increased only approximately 2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca(2+)](i) of 100 microM or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca(2+) affinity and rapid Ca(2+) binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca(2+)] at the inner membrane surface.

Distinct electrophysiological alterations in dentate gyrus versus CA1 glial cells from epileptic humans with temporal lobe sclerosis

Previous studies have characterized the electrophysiological properties of astrocytes in the CA1 region of hippocampi resected from patients with intractable temporal lobe epilepsy (TLE). However, the properties of hilar astrocytes from such patients have not been studied although astrocytes display regional heterogeneity and a non-uniform response to injury. Thus, we performed patch-clamp recordings of putative astrocytes in hilar and CA1 regions of surgically removed epileptic hippocampi with and without sclerosis (mesial TLE, MTLE patients, and paradoxical TLE, PTLE patients, respectively), and non-epileptic, non-sclerotic hippocampi (tumor patients). Our data show that the current profile of hilar astrocytes undergoes significant changes in MTLE but not in PTLE or tumor hippocampi. In particular, inwardly rectifying K + (K IR) and outwardly rectifying K + currents were reduced, inward Na + currents and membrane resistances were increased in putative astrocytes from MLTE cases compared to PTLE and tumor cases. Because the conductance of K IR channels in cell-attached patches (∼34 pS) from MTLE tissue was not altered, a reduction in the number of K IR channels likely accounts for the decrease in whole-cell K IR conductance. Presumed astrocytes in the CA1 region from each patient group displayed intercellular coupling and a passive current profile; these characteristics were never observed in hilar glial cells. No apparent changes in the current profile of coupled CA1 glial cells could be detected between MTLE, PTLE and tumor tissues. Additionally, CA1 glial cells expressed a high density of 34 pS K IR channels. These data suggest that K + buffering via K IR channels may be functionally compromised in hilar astrocytes of epileptic and sclerotic (MTLE) human hippocampi. By contrast, CA1 astrocytes retained their intercellular coupling and K IR channel expression necessary for K + buffering.

Regulation of A-type potassium channels in murine colonic myocytes by phosphatase activity.

A rapidly inactivating K(+) current (A-type current) participates in the regulation of colonic muscle excitability. We found 19-pS K(+) channels in cell-attached patches of murine colonic myocytes that activated and inactivated with kinetics similar to the A-type current. The A-type current in colonic myocytes is regulated by Ca(2+)/calmodulin-dependent protein kinase II. Therefore, we studied regulation of the 19-pS K(+) channels by Ca(2+)-dependent phosphorylation/dephosphorylation. The rates of inactivation of ensemble-averaged currents resulting from 19-pS K(+) channels were increased by the calmodulin antagonist W-7. Inhibitors of calcineurin, cyclosporin A and FK-506, slowed the inactivation of the 19-pS K(+) channels. Okadaic acid, an inhibitor of the calcineurin/inhibitor-1/protein phosphatase 1 cascade, also slowed inactivation of the 19-pS K(+) channels. Polymerase chain reaction detected transcripts encoding calcineurin A in isolated colonic smooth muscle cells, and immunohistochemical studies demonstrated specific expression of calcineurin A-like immunoreactivity in colonic muscle tissues and in colonic myocytes. These data, when considered with previous findings, suggest that Ca(2+)-dependent phosphorylation/dephosphorylation regulates the A-type current in murine colonic smooth muscle cells.

Somatic Colocalization of Rat SK1 and D class (Cav 1.2) L-type Calcium Channels in Rat CA1 Hippocampal Pyramidal Neurons

In hippocampal neurons, the firing of a train of action potentials is terminated by generation of the slow afterhyperpolarization (AHP). Recordings from hippocampal slices have shown that the slow AHP likely results from the activation of small-conductance calcium-activated potassium (SK) channels by calcium (Ca(2+)) entry through L-type Ca(2+) channels. However, the relative localization of these two channel subtypes is not known. The cloning and characterization of three subtypes of SK channel has suggested that SK1 may underlie generation of the slow AHP. Using a novel antibody directed against rat SK1 (rSK1), it has been determined that the rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In conjunction with antibodies directed against C (Ca(v)1.2) and D (Ca(v)1.3) class L-type Ca(2+) channel alpha1 subunits, it was observed that rSK1 channels were selectively colocalized with D class L-type channels. This colocalization supports the functional coupling of L-type and SK channels previously observed in cell-attached patches from hippocampal neurons. However, it appears contrary to the slow rise and decay of the slow AHP. Induction of delayed facilitation of L-type Ca(2+) channels in cell-attached patches from hippocampal neurons evoked delayed opening of coupled SK channels. Generation of ensemble currents produced waveforms identical to the ionic current underlying the slow AHP (I(sAHP)). Therefore, these data indicate that the slow AHP is somatic in origin, resulting from delayed facilitation of D class L-type Ca(2+) channels colocalized with rSK1 channels.

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD.

We investigated the effect of inhibiting Na+-K+-ATPase on the basolateral 18-pS K+ channel in the cortical collecting duct (CCD) of the rat kidney. Inhibiting Na+-K+-ATPase with strophanthidin decreased the activity of the 18-pS K+ channel and increased the intracellular Ca2+ to 420 nM. Removal of extracellular Ca2+ abolished the effect of strophanthidin. When intracellular Ca2+ was raised with 5 microM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the 18-pS K+ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism of Ca2+-induced inhibition, the effect of 400 nM Ca2+ on channel activity was studied in the presence of calphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62, inhibitors of calmodulin-dependent kinase II. Addition of calphostin C or KN-93 or KN-62 failed to block the inhibitory effect of high concentrations of Ca2+ . This suggested that the inhibitory effect of high concentrations of Ca2+ was not mediated by protein kinase C or calmodulin-dependent kinase II pathways. To examine the possibility that the inhibitory effect of high concentrations of Ca2+ was mediated by the interaction of nitric oxide with superoxide, we investigated the effect of 400 nM Ca2+ on channel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or N(omega)-nitro-L-arginine methyl ester. Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonic acid or N(omega)-nitro-L-arginine methyl ester completely abolished the inhibitory effect of 400 nM Ca2+ on channel activity. Moreover, application of 4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effect of strophanthidin. We conclude that the effect of inhibiting Na+-K+-ATPase is mediated by intracellular Ca2+ and the inhibitory effect of high concentrations of Ca2+ is the result of interaction of nitric oxide with superoxide.

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells.

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.

Bicuculline, pentobarbital and diazepam modulate spontaneous GABAA channels in rat hippocampal neurons

Spontaneously opening, chloride-selective channels that showed outward rectification were recorded in ripped-off patches from rat cultured hippocampal neurons and in cell-attached patches from rat hippocampal CA1 pyramidal neurons in slices. In both preparations, channels had multiple conductance states and the most common single-channel conductance varied. In the outside-out patches it ranged from 12 to 70 pS (Vp=40 mV) whereas in the cell-attached patches it ranged from 56 to 85 pS (-Vp=80 mV). Application of GABA to a patch showing spontaneous channel activity evoked a rapid, synchronous activation of channels. During prolonged exposure to either 5 or 100 microM GABA, the open probability of channels decreased. Application of GABA appeared to have no immediate effect on single-channel conductance. Exposure of the patches to 100 microM bicuculline caused a gradual decrease on the single-channel conductance of the spontaneous channels. The time for complete inhibition to take place was slower in the outside-out than in the cell-attached patches. Application of 100 microM pentobarbital or 1 microM diazepam caused 2 - 4 fold increase in the maximum channel conductance of low conductance (<40 pS) spontaneously active channels. The observation of spontaneously opening GABA(A) channels in cell-attached patches on neurons in slices suggests that they may have a role in neurons in vivo and could be an important site of action for some drugs such as benzodiazepines, barbiturates and general anaesthetics.

Purinergic activation of BK channels in clonal kidney cells (Vero cells).

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP >> UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.

Diazepam, γ-aminobutyric acid, and progesterone open K + channels in myocytes from coronary arteries

Benzodiazepines enhance coronary blood flow and lower blood pressure, but the cellular basis of this action remains unclear. The present study now demonstrates a direct effect of diazepam, γ-aminobutyric acid (GABA), and progesterone on the large conductance, Ca 2+- and voltage-activated K + channel (BK Ca) in single myocytes isolated from porcine coronary arteries. These GABA receptor agonists significantly increased whole-cell (perforated patch) K + currents and stimulated the activity of single BK Ca channels in cell-attached patches dramatically. This effect is not mediated via cyclic AMP or cyclic GMP, but involves stimulation of Ca 2+ influx in response to activation of a bicuculline-sensitive GABA A-like receptor. We propose that localized, subsarcolemmal increases in Ca 2+ levels open BK Ca channels, thereby promoting K + efflux, membrane repolarization, and coronary relaxation. This transduction pathway can now account, at least in part, for the direct vasodilatory effects of diazepam, progesterone, and GABA.

Large conductance Ca(2+)-activated K(+) channels in human meningioma cells.

Cells from ten human meningiomas were electrophysiologically characterized in both living tissue slices and primary cultures. In whole cells, depolarization to voltages higher than +80 mV evoked a large K(+) outward current, which could be blocked by iberiotoxin (100 nm) and TEA (half blocking concentration IC(50) = 5.3 mm). Raising the internal Ca(2+) from 10 nm to 2 mm shifted the voltage of half-maximum activation (V(1/2)) of the K(+) current from +106 to +4 mV. Respective inside-out patch recordings showed a voltage- and Ca(2+)-activated (BK(Ca)) K(+) channel with a conductance of 296 pS (130 mm K(+) at both sides of the patch). V(1/2) of single-channel currents was +6, -12, -46, and -68 mV in the presence of 1, 10, 100, and 1000 microm Ca(2+), respectively, at the internal face of the patch. In cell-attached patches the open probability (P(o)) of BK(Ca) channels was nearly zero at potentials below +80 mV, matching the activation threshold for whole-cell K(+) currents with 10 nm Ca(2+) in the pipette. Application of 20 microm cytochalasin D increased P(o) of BK(Ca) channels in cell-attached patches within minutes. These data suggest that the activation of BK(Ca) channels in meningioma cells does not only depend on voltage and internal Ca(2+) but is also controlled by the cytoskeleton.

Fatty acid-activated K+ channels in autonomic neurons: activation by an endogenous source of free fatty acids.

Application of arachidonic acid evoked robust activation of large-conductance K+ channels in cell-attached and excised inside-out patches from acutely isolated chick ciliary ganglion neurons. A similar effect was produced by 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analogue of arachidonic acid. The unitary conductance of fatty acid-activated channels was 35-40 pS at +20 mV with physiological gradients of K+ and 165 pS at +20 mV with an extracellular K+ concentration of 37.5 mM and an intracellular K+ concentration of 150 mM. Gating of these channels in cell-attached patches was potentiated by membrane stretch. Channel gating evoked by both lipids was concentration-dependent, with detectable activation apparent at 4 microM in the majority of patches and maximal activation occurring between 32 and 64 microM. Gating was relatively voltage-independent. Large-conductance K+ channels were also activated in inside-out patches by the monounsaturated fatty acid 11-cis-eicosenoic acid but not by the fully saturated fatty acid arachidic acid. Application of 100 microM H2O2, an agent that activates cytosolic phospholipase A2, also caused activation of large-conductance K+ channels in intact neurons. The stimulatory effects of H2O2 were blocked by pretreatment with 20 microM 4-bromophenacyl bromide, an irreversible inhibitor of phospholipase A2. Therefore, mobilization of endogenous fatty acids can cause activation of large-conductance K+ channels in autonomic neurons.

Estrogen modulates a large conductance chloride channel in cultured porcine aortic endothelial cells.

Estrogen is known to exert a protective effect on cardiovascular disease, but the mechanism for this effect is unclear. It has, however, been reported that estrogen and antiestrogen modify ionic currents and membrane potential in various cells. The aim of this study was to clarify whether the chloride channel of aortic endothelial cells was, in fact, modified by estrogen and antiestrogen with inside-out patch and cell-attached patch recording methods. Tamoxifen activated a large-conductance (368 +/- 23 pS, n = 6, in symmetric 150 mM Cl- solution) chloride channel of endothelial cells grown in the presence of 1 microg/ml colchicine. The channels were activated mainly between +/-40 mV, but were inactivated at more extreme potentials. The open probability of channels in cell-attached patches increased from <0.01 to 0.37 +/- 0.08 (n = 8) when cells were treated with 15 microM tamoxifen. This effect can be blocked by 17beta-estradiol, but not by progesterone. The results showed that tamoxifen increased chloride channel activity in the presence of colchicine in cultured endothelial cells, and this action was suppressed by 17beta-estradiol but not by progesterone. This rapid effect by estrogens suggests that these hormones exert nongenomic, short-term activity and do not appear to affect the nuclear estrogen receptor. With these effects, estrogen and antiestrogen bind to the endothelial cells plasma membrane site and subsequently may activate an intracellular second messenger pathway.

Apamin-sensitive, small-conductance, calcium-activated potassium channels mediate cholinergic inhibition of chick auditory hair cells.

Acetylcholine released from efferent neurons in the cochlea causes inhibition of mechanosensory hair cells due to the activation of calcium-dependent potassium channels. Hair cells are known to have large-conductance, "BK"-type potassium channels associated with the afferent synapse, but these channels have different properties than those activated by acetylcholine. Whole-cell (tight-seal) and cell-attached patch-clamp recordings were made from short (outer) hair cells isolated from the chicken basilar papilla (cochlea equivalent). The peptides apamin and charybdotoxin were used to distinguish the calcium-activated potassium channels involved in the acetylcholine response from the BK-type channels associated with the afferent synapse. Differential toxin blockade of these potassium currents provides definitive evidence that ACh activates apamin-sensitive, "SK"-type potassium channels, but does not activate carybdotoxin-sensitive BK channels. This conclusion is supported by tentative identification of small-conductance, calcium-sensitive but voltage-insensitive potassium channels in cell-attached patches. The distinction between these channel types is important for understanding the segregation of opposing afferent and efferent synaptic activity in the hair cell, both of which depend on calcium influx. These different calcium-activated potassium channels serve as sensitive indicators for functionally significant calcium influx in the hair cell.

Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K(V)LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.