Biomedical subjects

N B Standen

Publications and source records attributed to N B Standen.

At least 73 records · Page 4Linked to original sources

PubMed1991

Block of calcium-activated potassium channels in mammalian arterial myocytes by tetraethylammonium ions.

The effects of tetraethylammonium ions (TEA+) and tetrapentylammonium ions (TPeA+) on Ca2(+) -activated K+ (KCa) channels were studied in membrane patches from mesenteric arterial myocytes. External TEA+ produced a flickery block. The concentration dependence for reduction in mean unitary current was consistent with 1:1 binding, with dissociation constants (Kd) in rat and rabbit of 196 and 159 microM at 0 mV, and the block was weakly voltage dependent. Rate constants for blocking and unblocking were 380 mM-1.ms-1 and 73 ms-1, respectively. In patches containing several channels TEA+ reduced average current to the same extent as mean unitary current, implying that TEA+ block is independent of the channel state. Block was unaffected by raising external K+ to 120 mM. External TPeA+ blocked with slower kinetics and lower affinity than TEA+ (Kd, 1.49 mM). The sulfonylurea glibenclamide (10-100 microM), the hyperpolarizing vasodilator cromakalim (5 microM), and internal ATP (1 mM) were without effect on channel activity. We conclude that TEA+ is a relatively effective blocker of single KCa channels of arterial smooth muscle and should block macroscopic currents equally well, whereas external TPeA+ is about eight times less effective.

Adenosine Triphosphate↗

PubMed1991

Regulation of single calcium channels in cerebral arteries by voltage, serotonin, and dihydropyridines.

Unitary currents through Ca channels were measured from cell-attached patches on smooth muscle cells isolated from rabbit cerebral (basilar) arteries. Barium (80 and 10 mM) and calcium (80 and 10 mM) were used as the charge carriers. The dihydropyridine Ca channel agonist BAY R 5417 was used to increase open-state probability (Popen), with 500 nM BAY R 5417 increasing Popen 10-fold at 0 mV. Barium currents through single Ca channels were greater than calcium currents at any voltage, with single-channel conductances negative to -20 mV of 24.6 pS (80 mM barium), 15.1 pS (80 mM calcium), 17.2 pS (10 mM barium), and 5.8 pS (10 mM calcium). The single-channel Popen increased 2.7-fold per 5- to 7-mV membrane depolarization (negative to 0 mV) and was half maximal at +0.4 mV (80 mM calcium) and +13.5 mV (80 mM barium). Ca channels with calcium but not with barium as the charge carrier exhibited pronounced inactivation positive to -20 mV (half time, 112 ms at 0 mV). The dihydropyridine nimodipine (2 nM) inhibited average currents through Ca channels. The cerebral artery constrictor serotonin increased Popen of single Ca channels by as much as 200-fold without an effect on single-channel conductance. A second distinct amplitude of unitary currents was often observed, corresponding to a channel conductance of about one-half the more commonly observed level. The small-conductance-level channel was voltage dependent, did not inactivate over 0.5-s test pulses (with barium), and could be activated by serotonin.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1991

Role of potassium channels in the vascular response to endogenous and pharmacological vasodilators.

Many endogenous and pharmacological vasodilators hyperpolarize vascular smooth muscle and this response appears to be due to an increased conductance to potassium ions. The hyperpolarization may contribute to the mechanism of dilation by causing voltage-dependent calcium channels to close. Recent evidence indicates that the response to hyperpolarizing vasodilators is mediated through activation of ATP-sensitive potassium (KATP) channels. Single KATP channels on isolated vascular smooth muscle cells are activated by cromakalim and calcitonin gene-related peptide (CGRP). This response is inhibited by glibenclamide. Cromakalim, CGRP and other vasodilators hyperpolarize and relax arteries in vitro and these responses are reversed by glibenclamide. The hypotensive effects of these agents in vivo are antagonized by glibenclamide. We propose that activation of KATP channels and the associated membrane hyperpolarization represents an important general mechanism of vasodilation.

Animals↗

PubMed1990

Arterial dilations in response to calcitonin gene-related peptide involve activation of K+ channels.

Calcitonin gene-related peptide (CGRP) is a 37-amino-acid peptide produced by alternative processing of messenger RNA from the calcitonin gene. CGRP is one of the most potent vasodilators known. It occurs in and is released from perivascular nerves and has been detected in the blood stream, suggesting that it is important in the control of blood flow. The mechanism by which it dilates arteries is not known. Here, we report that arterial dilations in response to CGRP are partially reversed by blockers of the ATP-sensitive potassium channel (K(ATP)), glibenclamide and barium. We also show that CGRP hyperpolarizes arterial smooth muscle and that blockers of K(ATP) channels reverse this hyperpolarization. Finally, we show that CGRP opens single K+ channels in patches on single smooth muscle cells from the same arteries. We propose that activation of K(ATP) channels underlies a substantial part of the relaxation produced by CGRP.

Adenosine Triphosphate↗

PubMed1990

Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.

Resistance arteries exist in a maintained contracted state from which they can dilate or constrict depending on need. In many cases, these arteries constrict to membrane depolarization and dilate to membrane hyperpolarization and Ca-channel blockers. We discuss recent information on the regulation of arterial smooth muscle voltage-dependent Ca channels by membrane potential and vasoconstrictors and on the regulation of membrane potential and K channels by vasodilators. We show that voltage-dependent Ca channels in the steady state can be open and very sensitive to membrane potential changes in a range that occurs in resistance arteries with tone. Many synthetic and endogenous vasodilators act, at least in part, through membrane hyperpolarization caused by opening K channels. We discuss evidence that these vasodilators act on a common target, the ATP-sensitive K (KATP) channel that is inhibited by sulfonylurea drugs. We propose the following hypotheses that presently explain these findings: 1) arterial smooth muscle tone is regulated by membrane potential primarily through the voltage dependence of Ca channels; 2) many vasoconstrictors act, in part, by opening voltage-dependent Ca channels through membrane depolarization and activation by second messengers; and 3) many vasodilators work, in part, through membrane hyperpolarization caused by KATP channel activation.

Animals↗

PubMed1989

Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle.

Vasodilators are used clinically for the treatment of hypertension and heart failure. The effects of some vasodilators seem to be mediated by membrane hyperpolarization. The molecular basis of this hyperpolarization has been investigated by examining the properties of single K+ channels in arterial smooth muscle cells. The presence of adenosine triphosphate (ATP)-sensitive K+ channels in these cells was demonstrated at the single channel level. These channels were opened by the hyperpolarizing vasodilator cromakalim and inhibited by the ATP-sensitive K+ channel blocker glibenclamide. Furthermore, in arterial rings the vasorelaxing actions of the drugs diazoxide, cromakalim, and pinacidil and the hyperpolarizing actions of vasoactive intestinal polypeptide and acetylcholine were blocked by inhibitors of the ATP-sensitive K+ channels, suggesting that all these agents may act through a common pathway in smooth muscle by opening ATP-sensitive K+ channels.

Acetylcholine↗

PubMed1989

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Rubidium ions and the gating of delayed rectifier potassium channels of frog skeletal muscle.

1. Unitary currents were measured through delayed rectifier potassium channels of frog skeletal muscle, under conditions where either potassium or rubidium ions carried current. 2. Unitary currents were reduced in amplitude when Rb+ was the charge carrier, indicating that Rb+ permeated the channel less readily than did K+. On the other hand permeability ratios (PRb/PK) measured from the change in reversal potential upon ionic substitution were 0.92 for the external and 0.67 for the internal mouth of the channel. 3. Ensemble-averaged currents activated under depolarization along a similarly S-shaped time course whether K+ or Rb+ carried current, though slightly more slowly in Rb+. However, under repolarization to a negative level, tail currents were prolonged about tenfold in Rb+. 4. The duration of channel opening was substantially prolonged in Rb+. The distribution of open times was fitted by a single exponential whether K+ or Rb+ was the charge carrier, indicating a single open state. But the mean open time, averaged over all voltages investigated, was 2.65 times greater in Rb+. 5. The prolongation in Rb+ of tail currents under repolarization was associated with increases in the number of openings per burst and in the number of bursts during each tail. 6. The implications of these results for channel gating are discussed. It is argued that an early step in channel activation is more voltage dependent than later steps.

Action Potentials↗

PubMed1989

Multiple blocking mechanisms of ATP-sensitive potassium channels of frog skeletal muscle by tetraethylammonium ions.

1. Patch-clamp methods were used to study the action of tetraethylammonium ions (TEA+) and other quaternary ammonium ions on adenosine-5'-triphosphate (ATP)-sensitive K+ channels in sarcolemmal vesicles from frog skeletal muscle. The blocking ions were applied either to the external or the internal surface of the membrane patch. 2. External TEA+ caused a very fast block, so that the amplitude of single-channel currents was reduced. Open-channel variance was decreased. The block was 1:1, with a dissociation constant (Kd) of 6-7 mM. We could detect no voltage dependence of Kd. 3. External TEA+ prolonged open times in a manner consistent with the channel being unable to close when blocked by TEA+. 4. TEA+ also blocked when applied to the internal side of the membrane. This block showed two components with different kinetics and different affinities. The slow block chopped up openings into much briefer events and had a Kd of about 1.4 mM at -3 mV. The fast block reduced the amplitude of unitary currents and was of lower affinity, with Kd around 26 mM. 5. The slow block by internal TEA+ was markedly voltage dependent, the Kd decreasing e-fold for a 37 mV depolarization. Both the association and dissociation rates were dependent on voltage. In contrast, the fast block by internal TEA+ appeared virtually independent of voltage. 6. The effects of internally applied tetramethylammonium (TMA+) and tetrapentylammonium (TPA+) ions were also investigated. Internal TMA+ produced a flickery block while the block by internal TPA+ was similar to that caused by TEA+, although TPA+ was about 10 times more effective. 7. Our results suggest that the channel has three binding sites for TEA+, one of which is accessible from the outside of the membrane. Only one of the internal sites is located so as to experience a substantial fraction of the membrane voltage field.

Adenosine Triphosphate↗

PubMed1988

Noradrenaline contracts arteries by activating voltage-dependent calcium channels.

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.

Animals↗

PubMed1988

The voltage-dependent block of ATP-sensitive potassium channels of frog skeletal muscle by caesium and barium ions.

1. Patch clamp techniques were used to study the action of external Cs+ and Ba2+ on adenosine 5'-triphosphate (ATP)-dependent K+ channels in sarcolemmal vesicles from frog skeletal muscle. Both ions block channels in a voltage-dependent fashion, block increasing with hyperpolarization. 2. The Cs+ block is flickery, mean unitary current being reduced and open-level noise increased. The concentration dependence is consistent with 1:1 binding, with a dissociation constant (Kd) of 4.1 mM at -62 mV. The Kd increases e-fold for a 20 mV depolarization. 3. The kinetics of Cs+ block were analysed by amplitude distribution analysis, and by measurement of the excess open-level variance. Both methods gave similar rate constants for blocking and unblocking; about 20 mM-1 ms-1 and 75 ms-1 at -62 mV. 4. All the voltage dependence of the Cs+ block appears to lie in the blocking reaction; unblocking is independent of voltage. 5. Ba2+ blocks with slower kinetics, so that blocking events can be resolved in single-channel records. Ba2+ reduces mean open time and causes long closings. 6. The blocking rate constant for Ba2+ was measured from the open times. It was about 1.7 mM-1 ms-1 at -62 mV and increased e-fold for a 40 mV hyperpolarization. The unblocking rate, measured from closed times, yielded a Kd of about 0.1 mM at -62 mV, in agreement with that measured from the reduction in open-state probability. 7. Our results suggest that Cs+ and Ba2+ block at sites within the channel, and provide evidence that the channel is a multi-ion pore.

Adenosine Triphosphate↗

PubMed1987

Studies of the unitary properties of adenosine-5'-triphosphate-regulated potassium channels of frog skeletal muscle.

1. Patch-clamp techniques were used to study adenosine-5'-triphosphate (ATP)-dependent K+ channels in sarcolemmal vesicles from frog skeletal muscle. In addition to its ATP dependence, opening of these channels was voltage dependent, the open-state probability (P open) increasing with depolarization. 2. The reversal potential of unitary currents changed with external K+ concentration, [K+]o, as expected if the Na-K permeability ration (pNa/pK) equals 0.015. Unitary conductance increased with increasing [K+]o from 14.8 +/- 0.5 pS (n = 5) in 2.5 mM-K+ to 42.3 +/- 1.0 pS (n = 8) in 60 mM-K+. This increase was less than that expected from independence. 3. Replacement of 60 mM-external K+ by 60 mM-external Rb+ shifted the reversal potential of unitary currents by -6.7 mV, suggesting that Rb+ enters channels nearly as easily as does K+ (Rb-K permeability ration, pRb/pK = 0.76). Unitary currents were much smaller in Rb+, consistent with Rb+ binding within the channel. 4. The ATP-regulated K+ channel was blocked by both internal and external tetraethylammonium ions (TEA+). 2 mM-TEA+, applied to the cytoplasmic face of membrane patches, interrupted channel openings. Higher concentrations reduced unitary current amplitude, suggesting an increase in the rapidity of TEA+ block. 5. The reduction in P open by ATP was consistent with 1:1 binding and a dissociation constant of 0.135 mM. ATP appeared not to be hydrolysed to close channels. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) were less effective than ATP, but retained channel closing properties. Substitution of adenine with other purines or with pyrimidine bases substantially reduced activity, as did substitution of ribose by 2'-deoxyribose or by ribose 2',3'-dialdehyde. 6. Sarcoplasmic Ca2+ did not influence P open. 7. Myotubes, grown from thigh muscles of new-born rats, appeared to lack ATP-dependent K+ channels. Adult frog muscle appeared to lack high-conductance Ca2+-dependent K+ channels, at least in the surface membrane. Such channels were found in myotube membranes. 8. Open- and closed-time histograms were constructed and were consistent with at least two open and at least three closed states. Channel openings were grouped in bursts. Open times, burst lengths and the number of openings per burst were reduced by ATP. 9. The effects of [K+]o on unitary conductance and of K+ replacement with Rb+ are discussed in terms of a simple Eyring rate theory formulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1987

The action of external tetraethylammonium ions on unitary delayed rectifier potassium channels of frog skeletal muscle.

1. We have used single-channel recording to investigate the block by extracellular tetraethylammonium ions (TEA+) of delayed rectifier potassium channels of frog skeletal sarcolemma. 2. TEA+ blocks by reducing the apparent amplitude of unitary currents, without detectable increase in open-level current variance. 3. The block by TEA+ appeared to be 1:1, the fractional current being halved at 5.8 mM and -3 mV. The dissociation constant for the block was voltage dependent, increasing e-fold for a 138 mV depolarization. 4. Activation of delayed rectifier potassium currents is not altered by TEA+. 5. Open times, which in the presence of TEA+ represents bursts of open and blocked events, are not increased by TEA+, indicating that blocked channels are able to close normally.

Action Potentials↗

PubMed1985

Properties of single potassium channels in vesicles formed from the sarcolemma of frog skeletal muscle.

The patch-clamp method was used to study unitary delayed rectifier K+ channels in large vesicles formed from the membrane of frog skeletal muscle. Channels were activated by depolarizing pulses. Single-channel conductance was about 15 pS in physiological [K+]o and was doubled by raising [K+]o to 120 mM. TEA+ caused an apparent reduction in single-channel current, which we attribute to a rapid block. When depolarizations were repeated at brief intervals, records with and without channel openings were ordered non-randomly, providing evidence for a slow process which was probably inactivation. In multichannel patches the relation between variance and mean current, binomial analysis, and the distribution of times for single and double openings were all consistent with channels behaving independently. Open times were distributed exponentially. Mean open time, tau o, increased with depolarization so that 1/tau o was an exponential function of voltage. First latency histograms peaked at times later than zero and could not be fitted by a scheme having only two closed states. Channel openings occurred in bursts and closed time histograms could be fitted by the sum of three exponentials. Our results imply a scheme with at least three closed states, an open and an inactivated state.

Action Potentials↗

PubMed1984

A new preparation for recording single-channel currents from skeletal muscle.

Spherical vesicles of sarcolemma (media diameter, 78 microns) were prepared from frog skeletal muscle by prolonged exposure to enzymes (collagenase, then protease) and to isotonic KCl solutions, which promote swelling of muscle fibres. Patch clamp recording was used to measure unitary K and Na currents from this preparation. Na and K channels had conductances around 15 pS and their kinetic properties appeared unaltered by vesicle formation.

Animals↗

PubMed1983

The effects of injection of calcium ions and calcium chelators on calcium channel inactivation in Helix neurones.

1. The effects of intracellular injection of Ca, EGTA and EGTA/Ca buffers on inward currents flowing through the Ca channel in Helix aspersa neurones were studied under voltage clamp. 2. Inward currents were reversibly reduced by Ca injection. The extent of the reduction was dependent on the size and duration of the injection. Recovery from injection was exponential with a time constant around 18 s at 18-20 degrees C. 3. In our salines, which contained tetraethylammonium chloride and 4-aminopyridine, no outward current was activated by Ca injection at the holding potential. A given Ca injection reduced the inward current by the same fraction in 25 mM- and 2 . 5 mM-Sr and also at different test potentials. We conclude that Ca injection does not activate an outward current. 4. Mean resting ionized internal Ca concentration, [Ca]i, measured with a Ca-sensitive electrode was 1.9 X 10(-7) M. Our injections increased this by less than 10(-5) M, as expected if most of the injected Ca is bound. Constant-field or Eyring rate theory considerations suggest that this rise in [Ca]i would not significantly affect the inward current through open Ca channels and we conclude, therefore, that Ca injection causes Ca channel inactivation. 5. The effect of Ca injection was blocked by prior injection of EGTA. Extracellular application of carbonyl cyanide m-chlorophenylhydrazone increased the effect of Ca injection. 6. Ca injection does not inactivate Ca channels by lowering pHi since large H+ injections only caused small irreversible decreases in inward current. 7. EGTA injection increased peak Ca current (ICa) by around 30% and decreased the rate and extent of inactivation. Some inactivation remained, however, even after maximal EGTA injections. 8. Injection of EGTA/Ca buffers with free [Ca] less than 1.8 X 10(-7) M increased peak ICa, while buffers with free [Ca] greater than 8.9 X 10(-7) M decreased ICa. 9. Our results support the suggestion that Ca ions cause Ca inactivation by binding to a site which is accessible from the inside of the cell, and also suggest that there is some steady-state inactivation present at physiological [Ca]i. A simple model is presented which describes the decline of Ca current in terms of Ca binding to a site accessible from the cytoplasm.

Animals↗

PubMed1982

A binding-site model for calcium channel inactivation that depends on calcium entry.

We describe a model for the mechanism by which Ca channel inactivation might depend on calcium entry. Ca is assumed to bind to a site at the internal membrane surface to cause inactivation of Ca channels. We assume that Ca that enters through the membrane accumulates in a submembrane compartment and also make simplifying assumptions about Ca buffering and removal. Our model predicts the results of single- and double-pulse voltage-clamp experiments well. The predicted turn-off of Ca current is non-exponential. The model also predicts that procedures that slow inactivation will increase peak Ca current and suggests that both two-phase turn-off of currents and failure of normalized current to recover to 1.0 in two-pulse experiments may be explained without assuming a voltage-dependent component of inactivation or two populations of Ca channels.

Animals↗

PubMed1982

The effects of stimulation rate on calcium-dependent action potentials recorded from chick embryo heart cell aggregates.

1. Action potentials were recorded from aggregates of heart cells prepared from 3- to 7-day chick embryos. At 3 days the maximum rate of rise (+ Vmax) was insensitive to TTX; at 7 days it was considerably reduced by TTX. 2. In the presence of TTX the action potential overshoot was dependent on [Ca]0; the results may be fitted using constant field theory and assuming that the membrane is over a hundred times more permeable to Ca than to Na or K. 3. An increase in stimulation rate in the range 0.2-2 Hz led to an increase in both overshoot and + Vmax. This effect was not seen after addition of 20 mM-tetraethylammonium ions, nor when Sr was substituted for Ca in the external medium. We suggest that these rate-dependent changes may result from partial inactivation of an outward K current.

Action Potentials↗