Channel Activity In Inside-out Patches Research Articles

HAEM Modulation of Arterial Smooth Muscle Cell Large-Conductance Ca2+-Activated K+ (BK) Channel Activity

Several hemolytic diseases are associated with vasospasm. Haem release during hemolysis can build up to toxic levels. Under normal conditions haem is degraded by haem oxygenase to carbon monoxide (CO), bilverdin and Fe2+. The mechanisms causing vasospasm are poorly understood. Arterial smooth muscle cells (SMCs) express BK channels which produce spontaneous transient outward currents (STOCs) when activated by Ca2+ sparks. STOCs hyperpolarize SMCs resulting in cell relaxation and vasodilation. Previous studies show that intracellular haem inhibits whereas CO enhances BK channel activity in inside-out patches. We hypothesize that haem interaction with BK channels might contribute to the vasospasm. Therefore, the current study aims to investigate the mechanism of action of haem by examining the effects of extracellular haem and CO on whole-cell BK currents. SMCs were isolated from the mesenteric artery of male Wistar rats. Depolarizing pulse-induced BK currents and STOCs were recorded using whole-cell patch techniques. CO was applied using CO-releasing molecule 3 (CORM-3) which was prepared before each use. Extracellular haem (5 µM) increased pulse-induced BK currents and STOC amplitudes by 2.3 ± 0.5 (p≤0.01, n=9) and 1.28 ± 0.09 (p≤0.05, n=5) times respectively. Extracellular CORM-3 (30 µM) had no significant effect on pulse induced BK currents (n=8) but doubled STOC frequency (2.06 ± 0.61, p≤0.05, n=6) without affecting STOC amplitude (n=6). In the presence of the HO-inhibitor, zinc protoporphyrin-IX, haem increased STOC amplitude (1.5 ± 0.04, p≤0.001, n=4) and frequency (2.4 ± 0.5, p≤0.05, n=4). In summary, our results suggest that the stimulatory effect of haem on STOCs has a component that is independent of its degradation product, CO. Furthermore, other intracellular factors could be contributing to the positive haem effects. Information gained from further studies could improve our understanding of the changes in vascular tone that occur during haemolytic diseases.

A new interaction between proximal and distal C-terminus of Cav1.2 channels

Cardiac Cav1.2 channels, coupling membrane stimulation to intracellular Ca2+ signaling, are regulated by multiple cytoplasmic factors, such as calmodulin (CaM), phosphorylation, Ca2+, ATP and intramolecular fragments of the channel. The interaction between distal and proximal C-terminal regulatory domains (DCRD and PCRD) of Cav1.2 channel is suggested to inhibit the channel activity, while PKA-mediated phosphorylation facilitates Cav1.2 channel by releasing such an interaction. Here, we report that the interaction between the distal C-terminus (CT3) and the proximal C-terminus (CT1) are inhibited by CaM in a Ca2+-dependent manner. Furthermore, CT3D (a short CT3 with DCRD truncated) interacts with CT1B (a short CT1 with EF-hand and PCRD truncated), revealing a new interaction between distal and proximal C-terminus. Ca2+/CaM inhibited the binding of CT3D to CT1B more strongly than the binding between CT3 and CT1, implying that the interaction of DCRD/PCRD (in CT3/CT1) might cooperate with the binding of CT3D to CT1B. We name the new CT1B-binding region of CT3D as CaM-competitive domain (CCD). The electrophysiological experiments show that CT3D inhibits while CT1B facilitates Cav1.2 channel activity in inside-out patches in guinea-pig ventricular myocytes. These results suggest that distal C-terminus inhibits Cav1.2 channel through modulation of the CaM-binding property of the channels.

Direct activation of Ca2+ and voltage-gated potassium channels of large conductance by anandamide in endothelial cells does not support the presence of endothelial atypical cannabinoid receptor

Endocannabinoid anandamide induces endothelium-dependent relaxation commonly attributed to stimulation of the G-protein coupled endothelial anandamide receptor. The study addressed the receptor-independent effect of anandamide on large conductance Ca2+-dependent K+ channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10µM anandamide did not significantly influence the resting membrane potential. In a Ca2+-free solution the cells were depolarized by ~10mV. Further administration of 10µM anandamide hyperpolarized the cells by ~8mV. In voltage-clamp mode, anandamide elicited the outwardly rectifying whole-cell current sensitive to paxilline but insensitive to GDPβS, a G-protein inhibitor. Administration of 70µM Mn2+, an agent used to promote integrin clustering, reversibly stimulated whole-cell current, but failed to further facilitate the anandamide-stimulated current. In an inside-out configuration, anandamide (0.1–30µM) facilitated single BKCa channel activity in a concentration-dependent manner within a physiological Ca2+ range and a wide range of voltages, mainly by reducing mean closed time. The effect is essentially eliminated following chelation of Ca2+ from the cytosolic face and pre-exposure to cholesterol-reducing agent methyl-β-cyclodextrin. O-1918 (3µM), a cannabidiol analog used as a selective antagonist of endothelial anandamide receptor, reduced BKCa channel activity in inside-out patches. These results do not support the existence of endothelial cannabinoid receptor and indicate that anandamide acts as a direct BKCa opener. The action does not require cell integrity or integrins and is caused by direct modification of BKCa channel activity.

Role of protein phosphatases in the run down of guinea pig cardiac Cav1.2 Ca2+ channels.

This study aimed to investigate protein phosphatases involved in the run down of Cav1.2 Ca(2+) channels. Single ventricular myocytes obtained from adult guinea pig hearts were used to record Ca(2+) channel currents with the patch-clamp technique. Calmodulin (CaM) and ATP were used to restore channel activity in inside-out patches. Inhibitors of protein phosphatases were applied to investigate the role of phosphatases. The specific protein phosphatase type 1 (PP1) inhibitor (PP1 inhibitor-2) and protein phosphatase type 2A (PP2A) inhibitor (fostriecin) abolished the slow run down of Cav1.2 Ca(2+) channels, which was evident as the time-dependent attenuation of the reversing effect of CaM/ATP on the run down. However, protein phosphatase type 2B (PP2B, calcineurin) inhibitor cyclosporine A together with cyclophilin A had no effect on the channel run down. Furthermore, PP1 inhibitor-2 mainly prolonged the open time constants of the channel, specifically, the slow open time. Fostriecin primarily shortened the slow close time constants. Our data suggest that PP1 and PP2A were involved in the slow phase of Cav1.2 Ca(2+) channel run down. In addition, they exerted different effects on the open-close kinetics of the channel. All above support the view that PP1 and PP2A may dephosphorylate distinct phosphorylation sites on the Cav1.2 Ca(2+) channel.

Direct Inhibitory Effect of Fluoxetine on TREK-2 Channel

Earlier studies have reported that channel activity of TREK-1, a member of two-pore domain K+ (K2P) channel family, is inhibited by antidepressants, such as fluoxetine, norfluoxetine, and paroxetine. However, TREK-2, which is present in the same family, has been less studied than TREK-1. Well-known properties of TREK-1 are also exhibited by TREK-2. This study was performed to identify whether and how TREK-2 channel is inhibited by fluoxetine treatment. In HEK-293A cells transfected with rat TREK-2 or mutants (deletion of the amino or carboxyl terminal domain: TREK-2 ΔN, TREK-2 ΔC, and chimeras) DNA. Fluoxetine significantly inhibited both TREK-1 and TREK-2 activities in a dose-dependent manner. Under whole-cell mode and excised mode, fluoxetine showed inhibitory effect on TREK-2 current, suggesting that fluoxetine may modulate TREK-2 either directly or indirectly with different mechanism. However, fluoxetine produced high effects on TREK-2 channel activity in inside-out patches containing TREK-2 compared to whole-cell and outside-out patches. To identify the regions responsible for fluoxetine effect, we studied the role of the cytoplasmic regions of TREK-2. TREK-2 ΔN had no effect on fluoxetine-induced TREK-2 inhibition. TREK-2 ΔC abolished the inhibition in response to fluoxetine. The regions that allow inhibition by fluoxetine were localized to a charged region near the proximal C-terminus (KKTKEE). The deletion of KKTKEE abolished sensitivity to fluoxetine. From these results, we suggest that fluoxetine may modulate TREK-2 directly through the C-terminus (in particular KKTKEE), which is found to be critical for their channel sensitivity to fluoxetine.

TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca(2+) buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca(2+) buffering conditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca(2+) (0 [Ca(2+)](o)) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 nm Ang II and blocked the inhibitory action of [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca(2+) influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.

Biphasic effects of H2O2 on BKCa channels

The inhibitory or activating effect of H2O2 on large conductance calcium and voltage-dependent potassium (BKCa) channels has been reported. However, the mechanism by which this occurs is unclear. In this paper, BKCa channels encoded by mouse Slo were expressed in HEK 293 cells and BKCa channel activity was measured by electrophysiology. The results showed that H2O2 inhibited BKCa channel activity in inside-out patches but enhanced BKCa channel activity in cell-attached patches. The inhibition by H2O2 in inside-out patches may be due to oxidative modification of cysteine residues in BKCa channels or other membrane proteins that regulate BKCa channel function. PI3K/AKT signaling modulates the H2O2-induced BKCa channel activation in cell-attached patches. BKCa channels and PI3K signaling pathway were involved in H2O2-induced vasodilation and H2O2-induced vasodilation by PI3K pathway was mainly due to modulation of BKCa channel activity.

TRPC3 properties of a native constitutively active Ca2+‐permeable cation channel in rabbit ear artery myocytes

Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 microm, 25 nm, 1.5 microm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Nitric Oxide Stimulates a Large-Conductance Ca2+-Activated K+ Channel in Human Skin Fibroblasts through Protein Kinase G Pathway

In order to investigate the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel and determine the effects of nitric oxide (NO) on the channel in human skin fibroblasts, we performed electrophysiological patch clamp recordings on 5th-passage cells of human genital skin cultures. The whole-cell outward K⁺ current was increased with depolarization, and proved to be sensitive to NS1619 (a selective BK_Ca channel activator) and iberiotoxin (a specific BK_Cachannel inhibitor). The single-channel currents showed 226 pS of mean conductance in symmetrical K⁺. Sodium nitroprusside (SNP; an NO donor) significantly increased the K⁺ current amplitude in the whole-cell mode, and open probability of the channel (NPo) in the cell-attached mode, but not in the inside-out mode. S-nitroso-N-acetylpenicillamine (an NO donor) and 8-Br-cGMP (a membrane-permeant cGMP analogue) also increased the BK_Cachannel activity. The stimulatory effect of SNP on BK_Ca channels was inhibited by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor), or KT5823 [a specific protein kinase G (PKG) inhibitor]. Cytoplasmic PKG also increased the channel activity in inside-out patches. In conclusion, the present data indicate that BK_Ca channels constitute a significant fraction of K⁺ current in human skin fibroblasts, and that NO increases NPo of BK_Ca channels, which are mediated via the cGMP/PKG pathway, without direct effects on the channel.

Intracellular spermine decreases open probability of N-methyl- d-aspartate receptor channels

Spermine and related polyamines have been shown to be endogenous regulators of several ion channel types including ionotropic glutamate receptors. The effect of spermine on N-methyl- d-aspartate (NMDA) receptors in cultured rat hippocampal neurons was studied using single-channel and whole-cell patch clamp recordings. Intracellular spermine resulted in the dose-dependent inhibition of NMDA-induced responses. Spermine reversibly inhibited the single NMDA receptor channel activity in inside-out patches suggesting a membrane-delimited mechanism of action. Open probability of NMDA receptor channels was decreased in a dose-dependent manner. Mechanism of spermine-induced inhibition of NMDA receptor was different from that of intracellular Ca 2+-induced NMDA receptor inactivation. Both pharmacological studies and single channel analysis indicate that in contrast to the effect of extracellular spermine the intracellular spermine effect is not dependent on the NMDA receptor subunit composition. We propose that intracellular spermine has a direct inhibitory effect on NMDA receptors that is different from calcium-induced NMDA receptor inactivation and spermine-induced voltage-dependent inhibition of AMPA/kainate receptors. Spermine-induced tonic change in the open probability of NMDA receptor channels may play a role in mechanisms underlying short-term changes in the synaptic efficacy.

Biophysical properties and metabolic regulation of a TASK-like potassium channel in rat carotid body type 1 cells.

The single channel properties of TASK-like oxygen-sensitive potassium channels were studied in rat carotid body type 1 cells. We observed channels with rapid bursting kinetics, active at resting membrane potentials. These channels were highly potassium selective with a slope conductance of 14-16 pS, values similar to those reported for TASK-1. In the absence of extracellular divalent cations, however, single channel conductance increased to 28 pS in a manner similar to that reported for TASK-3. After patch excision, channel activity ran down rapidly. Channel activity in inside-out patches was markedly increased by 2 and 5 mM ATP and by 2 mM ADP but not by 100 microM ADP or 1 mM AMP. In cell-attached patches, both cyanide and 2,4-dinitrophenol strongly inhibited channel activity. We conclude that 1) whilst the properties of this channel are consistent with it being a TASK-like potassium channel they do not precisely conform to those of either TASK-1 or TASK-3, 2) channel activity is highly dependent on cytosolic factors including ATP, and 3) changes in energy metabolism may play a role in regulating the activity of these background K+ channels.

Tedisamil blocks BK-type Ca 2+-dependent K + channels and modulates action potentials in rat hippocampal neurons

Tedisamil, a bradycardic compound in heart, also acts on K + channels in neurons. We determined the actions of tedisamil on action potentials in CA1 pyramidal neurons in hippocampal slices and on BK-type Ca 2+-activated K + channel activity in inside-out patches excised from hippocampal neurons. In slices, tedisamil (5 μM) attenuated the fast afterhyperpolarization (AHP) and prolonged the repolarization phase of the action potential. Additionally, the compound induced burst-firing activity and enhanced the slow AHP that follows a train of action potentials. The single channel data showed tedisamil actions to be consistent with open channel blockade of the BK-type of K + channel. Together, the results are consistent with the possibility that prolongation of the action potential by tedisamil is mediated by a tetraethylammonium-like effect of the agent to block BK-type Ca 2+-activated K + channels. The study also points to a number of effects that may contribute to the known nervous system toxicity induced by tedisamil.

Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells.

The effect of intracellular Ca(2+) on the activity of the inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K(+) channel rapidly declined with an application of ionomycin (1 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.

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.

Alteration of the membrane lipid environment by L-palmitoylcarnitine modulates K(ATP) channels in guinea-pig ventricular myocytes.

Sarcolemmal adenosine 5'-triphosphate-sensitive K+ channels (K(ATP)) are dramatically up-regulated by a membrane phospholipid, phosphatidyl-inositol-4,5-bisphosphate (PIP2). During ischaemia, L-palmitoylcarnitine (L-PC), a fatty acid metabolite, accumulates in the sarcolemma and deranges the membrane lipid environment. We therefore investigated whether alteration of the membrane lipid environment by L-PC modulates the K(ATP) channel activity in inside-out patches from guinea-pig ventricular myocytes. L-PC (1 microM) inhibited KATP channel activity, without affecting the single channel conductance, through interaction with Kir6.2. L-PC simultaneously enhanced the ATP sensitivity of the channel [concentration for half-maximal inhibition (IC50) fell from 62.0+/-2.7 to 30.3+/-5.5 microM]. In contrast, PIP2 attenuated the ATP sensitivity (IC50 343.6+/-54.4 microM) and restored Ca2+-induced inactivation of KATP channels (94.1+/-13.7% of the control current immediately before the Ca2+-induced inactivation). Pretreatment of the patch membrane with 1 microM L-PC, however, reduced the magnitude of the PIP2-induced recovery to 22.7+/-6.3% of the control (P<0.01 vs. 94.1+/-13.7% in the absence of L-PC). Conversely, after the PIP2-induced recovery, L-PC's inhibitory action was attenuated, but L-PC partly reversed the PIP2-mediated decrease in the ATP sensitivity (IC50 fell from 310+/-19.2 to 93.1+/-9.8 microM). Thus, interaction between L-PC and PIP2 in the plasma membrane appears to regulate K(ATP) channels.

Oxygen‐sensing persistent sodium channels in rat hippocampus

1. Persistent sodium channel activity was recorded before and during hypoxia from cell-attached and inside-out patches obtained from cultured hippocampal neurons at a pipette potential (Vp) of +30 mV. Average mean current (IU) of these channels was very low under normoxic conditions and was similar in cell-attached and excised inside-out patches (-0.018 +/- 0.010 and -0.025 +/- 0.008 pA, respectively, n = 24). 2. Hypoxia increased the activity of persistent sodium channels in 10 cell-attached patches (IU increased from -0. 026 +/- 0.016 pA in control to -0.156 +/- 0.034 pA during hypoxia, n = 4, P = 0.013). The increased persistent sodium channel activity was most prominent at a VP between +70 and +30 mV (membrane potential, Vm = -70 to -30 mV) and could be blocked by lidocaine, TTX or R56865 (n = 5). Sodium cyanide (NaCN, 5 mM; 0.5-5 min) increased persistent sodium channel activity in cell-attached patches (n = 3) in a similar manner. 3. Hypoxia also increased sodium channel activity in inside-out patches from hippocampal neurons. Within 2-4 min of exposure to hypoxia, I had increased 9-fold to -0. 18 +/- 0.04 pA (n = 21, P = 0.001). Sodium channel activity increased further with longer exposures to hypoxia. 4. The hypoxia-induced sodium channel activity in inside-out patches could be inhibited by exposure to 10-100 microM lidocaine applied via the bath solution (I = -0.03 +/- 0.01 pA, n = 8) or by perfusion of the pipette tip with 1 microM TTX (I = -0.01 +/- 0.01 pA, n = 3). 5. The reducing agent dithiothreitol (DTT, 2-5 mM) rapidly abolished the increase in sodium channel activity caused by hypoxia in excised patches (I = -0.01 +/- 0.01 pA, n = 4). Similarly, reduced glutathione (GSH, 5-20 mM) also reversed the hypoxia-induced increase in sodium channel activity (IU = -0.02 +/- 0.02 pA, n = 5). 6. These results suggest that persistent sodium channels in neurons can sense O2 levels in excised patches of plasma membrane. Hypoxia triggers an increase in sodium channel activity. The redox reaction involved in increasing the sodium channel activity probably occurs in an auxiliary regulatory protein, co-localized in the plasma membrane.

An oxygen-, acid- and anaesthetic-sensitive TASK-like background potassium channel in rat arterial chemoreceptor cells.

The biophysical and pharmacological properties of an oxygen-sensitive background K+ current in rat carotid body type-I cells were investigated and compared with those of recently cloned two pore domain K+ channels. Under symmetrical K+ conditions the oxygen-sensitive whole cell K+ current had a linear dependence on voltage indicating a lack of intrinsic voltage sensitivity. Single channel recordings identified a K+ channel, open at resting membrane potentials, that was inhibited by hypoxia. This channel had a single channel conductance of 14 pS, flickery kinetics and showed little voltage sensitivity except at extreme positive potentials. Oxygen-sensitive current was inhibited by 10 mM barium (57% inhibition), 200 microM zinc (53% inhibition), 200 microM bupivacaine (55% inhibition) and 1 mM quinidine (105 % inhibition). The general anaesthetic halothane (1.5%) increased the oxygen-sensitive K+ current (by 176%). Halothane (3 mM) also stimulated single channel activity in inside-out patches (by 240%). Chloroform had no effect on background K+ channel activity. Acidosis (pH 6.4) inhibited the oxygen-sensitive background K+ current (by 56%) and depolarised type-I cells. The pharmacological and biophysical properties of the background K+ channel are, therefore, analogous to those of the cloned channel TASK-1. Using in situ hybridisation TASK-1 mRNA was found to be expressed in type-I cells. We conclude that the oxygen- and acid-sensitive background K+ channel of carotid body type-I cells is likely to be an endogenous TASK-1-like channel.

Activation of inwardly rectifying K+ channel in OK proximal tubule cells involves cGMP-dependent phosphorylation process.

The inwardly rectifying K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cell is activated by cyclic AMP-dependent protein kinase (PKA). In this study, we further examined the involvement of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent process in modulation of this K+ channel by using the patch-clamp technique. In cell-attached patches, channel activity was increased by the application of either N2, 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (DBcGMP, 100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 100 microM), and it was inhibited by KT5823 (10 microM), a membrane-permeable specific inhibitor of cGMP-dependent protein kinase (PKG). The effect of DBcGMP on channel activity was abolished by the pretreatment of cells with KT5823 (10 microM), but it was observed in the presence of KT5720 (200 nM), a specific inhibitor of PKA. Furthermore, atrial natriuretic peptide (ANP, 10 nM) increased channel activity, which was also prevented by the application of KT5823 (10 microM). In inside-out patches, ATP (3 mM) was required to maintain channel activity, which was inhibited by KT5823 (10 microM), but it was not increased by cGMP (100 microM) alone. The channel activity was increased by the coapplication of PKG (500 U/ml) and cGMP (100 microM). These results suggest that cGMP activates the inwardly rectifying K+ channel in OKP cells through PKG-mediated phosphorylation processes independent of PKA-mediated processes, and that ANP is an agonist which stimulates PKG-mediated processes in the proximal tubule cell. Furthermore, it is suggested that the ATP-dependent channel activity in inside-out patches is maintained at least in part by PKG, which is the membrane-bound catalytic domain.

Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

Protein kinase C stimulates the small-conductance K+ channel in the basolateral membrane of the CCD.

We have used the patch-clamp technique to study the regulation of the activity of the basolateral small-conductance K+ channel (SK) in the cortical collecting duct (CCD) of the rat kidney. Addition of 50-75 nM calphostin C, an agent which specifically inhibits protein kinase C (PKC), reduced channel activity by 90% in cell-attached patches. In contrast, addition of 1 microM phorbol 12-myristate 13-acetate, a stimulator of PKC, led to addition of "new" K+ channel currents in 9 of 20 patches in the basolateral membrane of the CCD, and the mean increase in NP0, a product of channel number (N) and open probability (Pzero), was 0.90 in these 9 patches. However, application of 1 nM exogenous PKC had no significant effect on channel activity in inside-out patches, suggesting that the PKC effect on the activity of the SK observed in cell-attached patches was not a result of a membrane-delimited action, such as a direct phosphorylation of the SK or closely associated proteins. The effect of calphostin C on the SK can be reversed by addition of either 10 microM S-nitroso-N-acetylpenicillamine, a donor of nitric oxide, or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate. In addition, the inhibitory effect of calphostin C on the SK was completely abolished by pretreatment of the cells with 1 microM okadaic acid, an inhibitor of protein phosphatase. However, 100 microM N omega-nitro-L-arginine methyl ester, an agent that inhibits nitric oxide synthases (NOS), blocked the SK in cell-attached patches in the presence of okadaic acid, suggesting that the effect of okadaic acid on calphostin C-induced inhibition of the SK was a step before formation of nitric oxide. We conclude that PKC is involved in the stimulation of the SK and that the effect of PKC on the SK may be mediated by regulation of NOS activity in the CCD of the rat kidney.