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N B Standen

Publications and source records attributed to N B Standen.

At least 55 records · Page 3Linked to original sources
PubMed1996

A rate theory model for Mg2+ block of ATP-dependent potassium channels of rat skeletal muscle.

1. We have studied the block by intracellular Mg2+ (0.08-4mM) of ATP-dependent potassium channels (KATP channels) from rat skeletal muscle using inside-out excised sarcolemmal patches. The block is voltage dependent, is relieved by extracellular potassium and has rapid kinetics, allowing the use of amplitude distribution analysis to estimate on and off rates. 2. To gain insight into the pore properties necessary to produce such a block, we have used an energy barrier model based on Eyring rate theory. The model has two energy wells and three barriers for K+ within the pore, while intracellular Mg2+ has access only to the inner well. We fitted the model to unitary current-voltage relations in different [Mg2+], to on and off rates, and to dissociation constants for Mg2+ block. 3. The voltage dependence of block was almost entirely due to the rate constant for unblocking. This implies that the inner energy barrier is asymmetrical, so that Mg2+ entry senses little of the voltage field, but Mg2+ exit senses about 20% of the voltage field. Best fits were obtained by placing the barrier and binding site 0.01 and 0.22, respectively, of the electrical distance through the pore from the inside. 4. The relief of block by [K+]o resulted from an increase in the unblocking rate for Mg2+, implying ionic repulsion between ions in the pore.

Adenosine Triphosphate↗
PubMed1996

Effect of adenosine and intracellular GTP on KATP channels of mammalian skeletal muscle.

We investigated the action of adenosine and GTP on KATP channels, using inside-out patch clamp recordings from dissociated single fibers of rat flexor digitorum brevis (FDB) skeletal muscle. In excised patches, KATP channels could be activated by a combination of an extracellular adenosine agonist and intracellular Mg2+-ATP and GTP or GTP-gamma-S. The activation required hydrolyzable ATP and could be partially reversed with Mg2+, suggesting that it may involve a G-protein dependent phosphorylation of KATP channels. We found that KATP channels of the rat FDB could not be activated by Mg2+-ATP alone or by Mg2+-ATP in the presence of extracellular adenosine. Patches whose channel activity had been 'rundown' by Ca2+ could not be recovered by adenosine, GTP or Mg2+-ATP. KATP channels activated by adenosine receptor agonists had a similar ATP sensitivity to those under control conditions; but adenosine appears to be able to switch these KATP channels from an inactive to an active mode.

Adenosine↗
PubMed1996

Evidence against the association of the sulphonylurea receptor with endogenous Kir family members other than KATP in coronary vascular smooth muscle.

We used whole-cell patch clamp to record inward rectifier (KIR) and ATP-sensitive (KATP) K+ currents from pig coronary arterial myocytes. KIR currents were blocked by Ba2+ ions with a KD around 3 microM, but were unaffected by 10 microM glibenclamide, and only reduced 16% by 100 microM of the sulphonlyurea (n=4). In contrast, pinacidil-activated KATP currents were over 1000 times more sensitive to glibenclamide, being inhibited with a KD close to 100 nM (n=5). Our findings suggest that the sulphonylurea receptor (SUR) in these cells associates with the appropriate subunits of the Kir family to form KATP channels, but does not show promiscuous association with subunits that form the strong inward rectifier KIR.

ATP-Binding Cassette Transporters↗
PubMed1996

Effects of the BKCa channel activator, NS1619, on rat cerebral artery smooth muscle.

1. We have investigated the actions of NS1619, a putative activator of large conductance calcium-activated potassium channels (BKCa) by use of the patch-clamp technique on smooth muscle cells enzymatically isolated from the rat basilar artery. 2. Using whole cell current-clamp to measure membrane potential, addition of 30 microM NS1619 produced cellular hyperpolarization, moving the membrane potential towards the calculated equilibrium potential for potassium. This hyperpolarization was rapidly reversed by IbTX (100 nM), a selective inhibitor of BKCa. 3. In whole cell recordings made from cells voltage-clamped at 0 mV using the perforated-patch technique, addition of NS1619 (10-30 microM) activated an outward current, which reversed following washout of NS1619. 4. This outward current was unaffected by application of either glibenclamide (5 microM), an inhibitor of ATP-sensitive potassium channels, or apamin (100 nM), an inhibitor of small-conductance calcium-activated potassium channels. However, this current was almost completely abolished by iberiotoxin (IbTX; 50-100nM). 5. Depolarizing voltage steps activated small outward currents from cells held at -15 mV. Application of NS1619 (10-30 microM) increased the size of these currents, producing a shift to the left of the current-voltage (I-V) relationship. These currents were largely inhibited by IbTX (100 nM). 6. Measurements of the unitary amplitude of the single channels activated by NS1619 which could be resolved in whole cell recordings yielded a value of 5.6 +/- 0.14 pA at 0 mV. 7. NS1619 (10-30 microM) directly activated single channels contained in excised inside-out and outside-out membrane patches. In both configurations NS1619 (10-30 microM) rapidly increased the open probability of a large conductance calcium-dependent channel. The activation produced by NS1619 was calcium-dependent and inhibited by external IbTX (100 nM). The unitary current amplitude was unaffected by NS1619. 8. By use of conventional whole cell recording methods and conditions that suppressed BKCa openings, outward potassium currents were activated by depolarizing potentials positive to -35 mV from a holding potential of -65 mV. NS1619 (10-30 microM) inhibited this current in a concentration-dependent manner. This inhibition was reversed following washout of NS1619, recovering to 60-90% of control values within 2 min. 9. Ba2+ currents, measured by conventional whole cell recording, were activated by depolarizing voltage steps from negative holding potentials. NS1619 (1-30 microM) inhibited the evoked current in a concentration-dependent manner, yielding an IC50 value of 7 microM with a Hill coefficient approaching unity. This inhibition was reversible, with the currents recovering to 65-100% of control values after washout of NS1619 for 2 min. 10. NS1619 (0.3-100 microM) induced concentration-dependent relaxation of basilar artery segments contracted with histamine/5-HT (IC50 = 12.5 +/- 2.0 microM; n = 4). This relaxation curve was shifted to the right, but not abolished, when the tissue was treated with a blocker of BKCa channels (IbTX; 100nM). Additionally, NS1619 produced concentration-dependent relaxation of basilar artery contracted with a depolarizing, isotonic salt solution containing 80 mM K+. 11. Thus NS1619 produces hyperpolarization of basilar artery myocytes through direct activation of BKCa and also directly inhibits Ca2+ currents and voltage-activated K+ channels. We discuss the implications of these results for its vasorelaxant actions.

Animals↗
PubMed1995

Activation of ATP-dependent K+ channels by hypoxia in smooth muscle cells isolated from the pig coronary artery.

1. The perforated patch technique with amphotericin B was used to record whole-cell currents activated by hypoxia in smooth muscle cells, isolated enzymatically from pig coronary arteries. 2. Superfusion with hypoxic solution (O2 partial pressure, 25-40 mmHg) activated an inward current at -60 mV in 143 mM extracellular K+. The reversal potential of the current induced by hypoxia shifted with extracellular [K+] as expected for a K+ current, while its current-voltage relation was consistent with the channels showing little voltage dependence. 3. The hypoxia-induced current was inhibited by glibenclamide (10 microM), but was unaffected by charybdotoxin (50 nM). 4. In whole-cell recordings at -60 mV in 143 mM K+ solution, openings of single channels passing a current close to -2 pA could sometimes be detected in normoxic solution. Openings became more frequent during the onset of the response to hypoxia, when several levels could be detected. Channels with a similar conductance were activated by hypoxia in cell-attached patches. 5. Our results suggest that hypoxia activates ATP-dependent K+ channels. We discuss possible mechanisms by which this activation may occur.

Adenosine Triphosphate↗
PubMed1994

The effect of intracellular anions on ATP-dependent potassium channels of rat skeletal muscle.

1. We have used excised inside-out patches to study the effects of anions bathing the cytoplasmic surface of the membrane on ATP-dependent K+ channels of rat flexor digitorum brevis muscle. Channels were closed by ATP applied to the cytoplasmic face of the patch with a concentration for half-closure (Ki) of 14 microM, were highly selective for K+ and had unitary conductances of 62 pS in symmetrical 155 mM K+ and 27 pS in 5 mM [K+]o. 2. In 139 mM Cl- internal solution channel activity declined rapidly after excision of the patch. Inclusion of 40 mM potassium gluconate (substituted for KCl) in the solution both restored channel activity and greatly slowed its subsequent run-down. 3. The action of gluconate was concentration dependent. The effect did not involve a change in ATP binding, since the Ki for ATP was not significantly changed by gluconate, and was specific for the cytoplasmic face of the patch. 4. The anions pyruvate, lactate and acetate were all able to restore channel activity after run-down, though less well than gluconate, while sulphate and methylsulphate were without effect. 5. Analysis of single channel kinetics showed that gluconate did not affect mean open lifetime, but led to a decrease in the number and duration of long closings. 6. Anions are most likely to act by stabilizing the structure of the channel protein. Changes in the intracellular concentration of certain anions may play a role in regulating channel activity.

Adenosine Triphosphate↗
PubMed1994

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+.

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole-cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e-fold as the driving force on K+(V-EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current-voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.

Animals↗
PubMed1994

Properties of the ATP-sensitive K+ current activated by levcromakalim in isolated pulmonary arterial myocytes.

Tension and patch clamp recording techniques were used to investigate the relaxation of rabbit pulmonary artery and the properties of the K+ current activated by levcromakalim in isolated myocytes. Under whole-cell voltage clamp, holding at -60 mV in symmetrical 139 mM K+, levcromakalim (10 microM) induced a noisy inward current of -116 +/- 19 pA (n = 13) which developed over 1 to 2 min. This current could be blocked by either glibenclamide (10 microM) or phencyclidine (5-50 microM) and was unaffected when extracellular Ca2+ was removed. Both these drugs inhibited the levcromakalim-induced relaxation of muscle strips precontracted with 20 mM [K+]o. Application of voltage ramps in symmetrical 139 mM K+ confirmed that the levcromakalim-induced current was carried by K+ ions and was weakly voltage dependent over the potential range from -100 to +40 mV. The unitary current amplitude and density of the channels underlying the levcromakalim-activated whole-cell K+ current was estimated from the noise in the current record. We estimate that levcromakalim caused activation of around 300 channels per cell, with a single channel current of 1.1 pA, corresponding to a slope conductance of about 19 pS. Furthermore, cells dialyzed with an ATP-free pipette solution developed a large noisy inward current at -60 mV, which could subsequently be blocked by flash photolysis of caged ATP. Analysis of the noise associated with this current indicated that the single channel amplitude underlying the ATP-blocked current was 1.4 pA, a value similar to that estimated for the levcromakalim-induced current.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation.

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 microM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

KATP channels in vascular smooth muscle.

ATP sensitive potassium channels (KATP channels) appear widely distributed in the vascular system. At the single channel level, channels with both small and large conductance have been described, though the former appear to be activated by potassium channel openers or ATP depletion in whole cell studies. KATP channels are inhibited by cytoplasmic ATP, and may be activated by intracellular nucleotide diphosphates. Regulation by intracellular metabolites confers a degree of sensitivity of the channel to the metabolic status of the cell, and there is evidence that KATP currents are activated during metabolic inhibition. In general, activation of KATP channels will lead to membrane hyperpolarisation and so to vasorelaxation. The functional role of the channel is being intensively studied at present. The channel may form a target for a number of endogenous vasodilators, and may be inhibited by some vasoconstrictors. It may be involved in the vasodilator response to hypoxia, and may contribute to the resting membrane potential of smooth muscle in some blood vessels.

Adenosine Triphosphate↗
PubMed1994

The role of the membrane potential of endothelial and smooth muscle cells in the regulation of coronary blood flow.

In the mammalian heart the supply of oxygen and energy-rich substrates through the coronary arterioles is continuously adapted to the variations of cardiac work. The coronary resistance arteries and the surrounding myocardium form a functional unit with multiple interactions between coronary endothelial cells, smooth muscle cells, perivascular nerves, and cardiac muscle cells. We describe the mechanisms underlying the electrical and chemical communication between the different cell types, the ionic channels contributing to the resting potential of endothelial and smooth muscle cells, and the mechanisms responsible for modulation of the resting potential. The main conclusion of our analysis is that the membrane potential of coronary endothelial and smooth muscle cells is one of the major determinants of coronary blood flow, and that modulation of the membrane potential provides a way to dilate or constrict coronary resistance arteries. It is proposed that the membrane potential of the myo-endothelial regulatory unit, i.e., of the endothelial cells and the underlying smooth muscle cells in the terminal arterioles, may function as an integrator of the numerous local and global vasodilator and constrictor signals that provide for the adaptation of coronary blood flow to the metabolic demands of the heart.

Animals↗
PubMed1993

Calcium currents elicited by voltage steps and steady voltages in myocytes isolated from the rat basilar artery.

1. Whole-cell patch clamp methods were used to record Ca2+ channel currents from isolated rat basilar arterial myocytes either in response to voltage steps or at steady holding potentials. Inward currents were rapidly and reversibly blocked by 2 mM Co2+, and this solution was routinely used to subtract leakage currents. 2. Peak currents measured in response to voltage steps were -85 +/- 23 pA (n = 7) in physiological Ca2+ (1.8 mM) and -256 +/- 39 pA (n = 19) in 10 mM Ba2+. The time course of activation and inactivation were unaffected by changing the holding potential from -88 to -48 mV, and thus we found no evidence for a component of current flowing through transient Ca2+ channels. 3. Activation curves were constructed by dividing the macroscopic current by the single Ca2+ channel current. The product of the number of functional channels and the open-state probability (NPo) measured in this way rose to around 2000. Its dependence on voltage was fitted by a Boltzmann function with half-activation at -8.6 mV in physiological Ca2+ concentrations and a steepness factor in the range 6-8 mV. 4. In physiological solution, block by external Mg2+ reduced peak current through Ca2+ channels by 17%. 5. Steady-state currents were measured by holding the cell at a fixed voltage and rapidly applying Co2+ to block the current through Ca2+ channels. Steady-state currents could be detected at voltages as negative as -58 mV, and persisted for more than 15 min, suggesting that Ca2+ channels in this artery may provide a steady-state Ca2+ influx that contributes to resting contractile tone.

Animals↗
PubMed1993

Adenosine-activated potassium current in smooth muscle cells isolated from the pig coronary artery.

1. The perforated patch technique with nystatin or amphotericin was used to record whole cell currents activated by adenosine in smooth muscle cells isolated enzymatically from pig coronary arteries. 2. Adenosine (5-40 microM) activated an outward current at a holding potential of 0 mV in 5 mM [K+]o and an inward current at -60 mV in 143 mM [K+]o. The dependence of the reversal potential for the adenosine-activated current on [K+]o suggests that it flows through K+ channels, while its current-voltage relation is consistent with the channels showing little voltage dependence. 3. The adenosine-activated current was inhibited by the sulphonylurea glibenclamide (5 microM) and by phencyclidine (5 microM). It was unaffected by charybdotoxin (50 nM) or apamin (100 nM), blockers of large and small conductance Ca(2+)-activated K+ channels respectively. 4. At -60 mV in 143 mM K+ solution, openings of single channels passing a current of just over -2 pA could sometimes be detected in the absence of adenosine. Openings became more frequent after the application of adenosine, with several levels then being detected. Openings of channels with a larger conductance were sometimes also seen in the presence of adenosine. Fluctuation analysis gave somewhat lower estimates of unitary current than did direct measurements. 5. The effect of adenosine could be mimicked by the A1 receptor agonist CCPA (2-chloro-N6-cyclopentyladenosine), while the A2 agonist CGS 21680 (2-p-(2-carboxethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride) was without effect. The response to adenosine was inhibited by the A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), but was unaffected by the A2 antagonist CGS 15943A (5-amino-9-chloro-2-(2-furanyl)-1,2,4- triazolo[1,5-C]quinazoline monomethanesulphonate). 6. Our results suggest that adenosine acts at an A1 receptor to activate K+ channels. We consider it most likely that these are ATP-dependent K+ channels. We discuss the mechanism by which K+ channel activation may lead to hyperpolarization and so vasorelaxation.

Adenosine↗
PubMed1992

Activation of ATP-dependent K+ currents in intact skeletal muscle fibres by reduced intracellular pH.

We have used three-microelectrode voltage clamp in conjunction with the ammonium prepulse method to investigate the effects of lowered intracellular pH (pHi) on resting potassium currents of frog skeletal muscle fibres. Potassium currents were recorded in 40 mM K+, Cl(-)-free solution in response either to voltage steps or ramps. An ammonium prepulse (2 h) reduced pHi to 6.45 from a control value of 7.19. The intracellular ATP concentration, measured with high-pressure liquid chromatography (HPLC), was unchanged by this procedure. Mean outward potassium currents were larger in low pHi than in control fibres, being about twice as large at +40 mV, whereas mean inward currents were very similar in control and low-pHi fibres. The sulphonylurea glibenclamide blocked single KATP channels in excised patches with a Kd of 3 microM. In intact fibres 50 microM glibenclamide had no effect on K+ currents in controls but reduced currents in low-pHi fibres. In the presence of glibenclamide, K+ currents in low-pHi fibres were not significantly different from those in control fibres. We suggest that reduced pHi in intact skeletal muscle fibres opens ATP-dependent potassium channels (KATP channels), as has been shown to occur in excised patches of membrane.

Adenosine Triphosphate↗
PubMed1992

The effect of intracellular pH on ATP-dependent potassium channels of frog skeletal muscle.

1. We have used patch-clamp methods to study the effects of pH at the cytoplasmic surface of the membrane on ATP-dependent K+ channels (KATP channels) in patches excised from frog (Rana temporaria) skeletal muscle, and to study the kinetics of ATP binding. 2. In the absence of ATP, a reduction in pH led to a slight decrease in single-channel current amplitude, an increase in the number of very brief closings, an increase in the apparent mean open time, and an increase in burst duration. After correction for missed closings, the change in mean open time was slight. Despite these changes in detailed kinetics, the channel open-state probability, Popen, changed little with changes in pH in the absence of ATP. 3. In the presence of ATP, a decrease in internal pH (pHi) reduced the degree of channel inhibition by ATP, shifting the curve relating Popen and [ATP] to higher concentrations of ATP without altering its steepness. The ATP concentration for half-inhibition of channel activity (Ki) was 17 microM at pH 7.2 and 260 microM at pH 6.3. 4. The effect of pH could be modelled by assuming that one or two protons bind to the channel and prevent ATP binding to exert its effect of causing channel closure. The predicted dissociation constants for ATP and H+ respectively were 5.4 and 0.11 microM. 5. The rate constants for binding and unbinding of ATP were estimated from the dependence of the mean open time on [ATP] and from the Ki. The apparent rate constants for ATP binding were 0.6 and 0.04 mM-1 ms-1 at pH 7.2 and 6.3 respectively, while the rate constant for unbinding was 0.01 ms-1. In terms of our model the calculated true rate constant for ATP binding was 1.85 mM-1 ms-1. ATP binding also led to a reduction in burst duration. 6. The effect of pH described here differs from findings in cardiac muscle and pancreatic B-cells. The results are discussed in relation to the possible function of KATP channels in skeletal muscle during exercise.

Adenosine Triphosphate↗
PubMed1991

The flickery block of ATP-dependent potassium channels of skeletal muscle by internal 4-aminopyridine.

We have examined the effects of 4-aminopyridine (4-AP) on single ATP-dependent potassium channels in patches excised from frog skeletal muscle. 4-AP applied to the internal face of the membrane caused a flickery block. We could not detect any flickery block when 10 mM 4-AP was applied to the external surface of the membrane. The reduction in mean unitary current by internal 4-AP was consistent with 1:1 binding with a Kd of 3.3 mM at 0 mV. The block was voltage-dependent, increasing with depolarization with an effective valency of 0.57. Rate constants for blocking and unblocking by 4-AP were obtained by fitting beta functions to the distribution of current amplitudes. Both rate constants were voltage-dependent. At 0 mV they were 17 mM-1 ms-1 and 61 ms-1. Simulation of the block using these rate constants produced a flickery block very similar to that observed experimentally.

4-Aminopyridine↗
PubMed1991

ATP-dependent potassium channels of muscle cells: their properties, regulation, and possible functions.

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a low Popen at physiological [ATP]i. The unitary conductance is 15-20 pS at physiological [K+]o, and the channels are highly selective for K+. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. KATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pHi. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and close states. The main effect of ATP is to increase occupancy of long-lived close states. The channels may have a role in the control of excitability and probably act as a route for K+ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.

Adenosine Triphosphate↗