Differential activation of ATP-sensitive potassium channels during energy depletion in CA1 pyramidal cells and interneurones of rat hippocampus.

In the hippocampus, pyramidal cells are more vulnerable than granule cells and interneurones to energy depletion during hypoxia and ischaemia. The aim of the present study was to explore whether this difference is related to the lower expression of adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channels in pyramidal cells compared to other hippocampal neurones. Hippocampal slices were prepared from 10- to 13-day-old rats, and CAI pyramidal cells and interneurones of the stratum radiatum were visually and electrophysiologically identified. Energy depletion was produced by removing glucose from the bath or by inhibiting mitochondrial metabolism using rotenone. In the perforated-patch configuration, both protocols elicited outward currents in only a minority of the pyramidal cells but in most of the interneurones. The currents started to develop 9-57 min after glucose deprivation and 4-16 min after rotenone application and reversed near the K+ equilibrium potential. Bath-applied diazoxide (0.3 mM), an opener of K(ATP) channels, could activate additional currents. The sulphonylureas tolbutamide (0.5 mM) or glibenclamide (20 microM), two blockers of K(ATP) channels, totally inhibited the currents induced by energy depletion and activated by diazoxide. The results demonstrate the differential activation of K(ATP) channels during energy depletion in pyramidal cells and interneurones, and suggest that channel activation is neuroprotective against the deleterious effects of energy depletion.

Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus.

1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.

Endogenous trypsin receptors in Xenopus oocytes: linkage to internal calcium stores.

The effects of the protease trypsin, externally applied to full-grown oocytes of Xenopus laevis, were studied using electrophysiology and fluorometry. The following results were obtained: trypsin in concentrations of 0.1 microgram/ml to 1 mg/ml liberated Ca2+ from internal stores and evoked large transient currents of up to 5 microA in bath solutions containing 1 mM or no Ca2+. The response desensitized for 50 minutes and recovered at longer times. Transient currents could also be elicited by tryptic impurities in commercially available collagenase used for defolliculation of oocytes. Application of chymotrypsin (0.01 or 1 mg/ml) or of thrombin (3.4 ng/ml or 0.34 mg/ml) neither evoked currents nor desensitized trypsin responses. Incubation with 1 microgram/ml Pertussis toxin for 20 to 25 hours prevented the Ca2+ release from internal stores and the activation of transient currents by trypsin. We propose that endogenous receptors in the oolemma, specific for trypsin, are linked to internal Ca2+ stores via Pertussis toxin-sensitive G proteins. Thus, receptor activation by external trypsin raises internal Ca2+ and thereby opens Ca(2+)-activated Cl channels in the oolemma.

Inhibition of ATP-sensitive K+ channels of mouse skeletal muscle by disopyramide.

Single ATP-sensitive K+ channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. The class Ia antiarrhythmic, disopyramide (5-100 microM), applied to the cytoplasmic membrane surface inhibited KATP channels at -40 and +40 mV. Channel inhibition by disopyramide started slowly and reached an almost stationary level within 1 min. Recovery from channel inhibition by disopyramide was incomplete. At pH 7.4, the disopyramide concentrations producing 50% channel inhibition were 8.1 microM at -40 mV and 7.1 microM at +40 mV. The Hill coefficients of the concentration-response curves were close to unity at both potentials. Raising the internal pH from 7.4 to 8.0 had no significant effect on the actions of disopyramide, but lowering the pH to 6.5 greatly potentiated the inhibition of KATP channels by the antiarrhythmic. Thus the open probabilities of KATP channels at -40 mV and in the presence of disopyramide (20 microM) were smaller by a factor of 18 at pH 6.5 than at pH 7.4. The results suggest that disopyramide interacts with KATP channels through the lipid phase of the membrane and that lowering the intracellular pH increases the affinity of KATP channels to disopyramide. Thus disopyramide at therapeutic concentrations (6-15 microM) affects muscular KATP channels, in particular at reduced intracellular pH values that occur under ischaemic conditions and during fatiguing exercise.

Morphological and electrophysiological properties of centrifuged stratified Xenopus oocytes.

To separate and concentrate various cytoplasmic organelles in wild type and albino Xenopus oocytes, defolliculated cells were loaded on a Ficoll-400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10,000 g for 5 min at 20 degrees C. The cells became pear-shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100-300 microns, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between -35 and -65 mV, and the membrane resistances were in the 500 k omega to 1 M omega range. Under voltage clamp conditions, the oocytes exhibited Ca(2+)-activated Cl- currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messengers in the cytosol.

Diverse effects of pinacidil on KATP channels in mouse skeletal muscle in the presence of different nucleotides.

OBJECTIVE: The potassium channel opener pinacidil relaxes smooth muscle and exerts cardioprotective effects. The aim of the study was to investigate the actions of pinacidil on ATP sensitive potassium channels (KATP channels) in mammalian skeletal muscle and to explore the interrelations of this drug with various nucleotides. METHODS: Single skeletal muscle fibres were prepared enzymatically from flexor digitorum brevis muscles of adult mice. Membrane patches of the inside-out configuration were excised in a Ca(2+)-free solution, and currents through single KATP channels were recorded at -40 mV. The cytoplasmic face of the patch was exposed to a K(+)-rich solution with MgCl2 (1 mM), and pinacidil (0.1 or 0.4 mM) and the nucleotides (0.1 mM) were added to this internal solution. RESULTS: KATP channels were not activated by pinacidil in the presence of the nonhydrolysable ATP analogue AMP-PNP, in contrast to the reported channel activation by pinacidil and ATP. KATP channels had a high activity in the control and were blocked by ADP; the subsequent addition of pinacidil did not enhance the open probability of KATP channels. Pinacidil in the presence of a mixture of AMP-PNP and ADP activated KATP channels. CONCLUSIONS: The diverse effects of pinacidil are interpreted with a model of the KATP channel containing a binding site for pinacidil and two sites for nucleotides, one activatory (A) site and one inhibitory (I) site. Occupation of the A site by ATP or ADP activates the channel, while occupation of the I site by ATP, AMP-PNP, ADP closes the channel. Pinacidil activates the channel and displaces blockers from the I site only if the A site is occupied.

Internal Ca2+ ions inactivate and modify ATP-sensitive potassium channels in adult mouse skeletal muscle.

The effects of internal Ca2+ ions on single ATP-sensitive potassium channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel activity was high when patches were excised in a Ca(2+)-free, Na(+)-rich solution and declined irreversibly within seconds in the presence of internal Ca2+ (0.1 and 2 mM). After Ca(2+)-dependent inactivation of the channels, the ATP concentration-response curve was steeper and 50% channel blockage occurred at a lower ATP concentration than before inactivation. ATP (50 microM) in a K(+)-rich solution bathing the intracellular membrane surface reduced the open-probability of KATP channels to 18% before and to 10% after exposure of the patch to internal Ca2+ (0.1 mM). The block of KATP channels by ATP (50 microM) was also enhanced by internal Ca2+ at a concentration of 13 microM. It is concluded that internal Ca2+ ions can both inactivate KATP channels and modify the active channels. KATP channels modified by Ca2+ are blocked more strongly by ATP than unmodified channels.

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP.

Single ATP-sensitive potassium channels (KATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl2 with K(+)-rich solutions bathing the internal membrane surface. Currents through single. KATP channels were recorded at -40 and +40 mV. AMP-PNP (5 to 500 microM; Li salt) reduced the open-probability po of KATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of po at -40 mV was observed at nucleotide concentrations of 29 microM in the absence and of 39 microM in the presence of Mg2+. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg2+, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl2. The efficacies of channel blockage by AMP-PNP at -40 and +40 mV were not significantly different. The results indicate that a KATP channel can bind more divalent Mg(2+)-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg(2+)-free solution on the single channel current i. At a Li+ concentration of 10 mM, i was hardly affected at -40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of KATP channels by internal Li+ ions.

Negative cooperativity may explain flat concentration-response curves of ATP-sensitive potassium channels.

Blockage of ATP-sensitive K+ channels by various drugs has been reported to exhibit a weak concentration dependence with Hill coefficients below unity. This phenomenon is interpreted by a negative cooperativity between K+ channels whereby drug binding to one channel lowers the drug affinities of neighbouring channels. Results are presented for a dimeric and a tetrameric channel model and compared with published experimental data.

The cardiotonic bipyridine AWD 122-60 inhibits adenosine triphosphate-sensitive potassium channels of mouse skeletal muscle.

The inhibition of ATP-sensitive potassium channels in mouse skeletal muscle by the cardiotonic bipyridine AWD 122-60 was investigated with the patch-clamp technique. In excised patches of the inside-out configuration, internally applied AWD 122-60 (10(-6)-10(-3) mol/l) reversibly reduced the open-probability of single ATP-sensitive potassium channels. The agent shortened the periods of channel activity but did not affect the channel conductance. At positive membrane potentials channel inhibition by AWD 122-60 was more pronounced than at negative potentials, the drug concentrations producing 50% channel inhibition were 11 mumol/l at +40 mV and 29 mumol/l at -40 mV. The Hill coefficients of the concentration-response curves were in the range between 0.5 and 0.6 for both potentials. Internal application of another cardiotonic bipyridine, milrinone (10(-4) mol/l), had no effects on ATP-sensitive potassium channels in skeletal muscle. Possible effects of the inhibition of ATP-sensitive potassium channels in heart muscle by AWD 122-60 are discussed.

Vanadate as an activator of ATP--sensitive potassium channels in mouse skeletal muscle.

The inside-out mode of the patch-clamp technique was used to study adenosine-5'-triphosphate (ATP)-sensitive K+ channels in mammalian skeletal muscle. Vanadate, applied to the cytoplasmic face of excised patches, was a potent activator of ATP-sensitive K+ channels. Divalent cations (Mg2+, Ca2+) were a prerequisite for the activating process. The maximal effect was achieved using 1 mM vanadate dissolved in Ringer, increasing the open-state probability about ninefold. The active 5 + redox form of vanadate which stimulates ATP-sensitive K+ channels is likely to be decavanadate V10O28(6-). ATP concentration-response curves have Hill coefficients near three internal Na(+)-rich Ringer and between one and two in internal KCl solutions. Half-maximal channel blockage was observed at ATP concentrations of 4 and 8 microM in Ringer and KCl solutions, respectively. Internal vanadate shifted the curves towards higher ATP concentrations without affecting their slopes. Thus 50% channel blockage occurred at 65 microM ATP in internal Ringer containing 0.5 mM vanadate. The results indicate that the affinity and stoichiometry of ATP binding to ATP-sensitive K+ channels are strongly modulated by internal cations and that the ATP sensitivity is weakened by vanadate.

Effects of potassium channel openers on single potassium channels in mouse skeletal muscle.

The patch-clamp technique was used to study the effects of the potassium channel openers cromakalim, pinacidil, RP 49356 and diazoxide on single potassium channels in mouse skeletal muscle. In excised patches in the inside-out configuration, one type of potassium channel, the ATP-sensitive potassium channel, could be activated by internally applied RP 49356 even in the absence of internal ATP. At a concentration of 0.4 and 0.8 mmol/l, RP 49356 increased the open-probability of the channels by a factor of 2.7 and 17.4 respectively. The stimulating effect of cromakalim (0.2-0.8 mmol/l) and pinacidil (0.4 mmol/l) depended on the presence of ATP (0.1 mmol/l) at the cytoplasmic side of the patch membrane. The two drugs were able to restore the open-probability of the channels blocked by internal ATP (0.1 mmol/l) to 50-90% of its value in ATP-free solution. No channel reactivation could be observed at a higher ATP concentration (1 mmol/l). Diazoxide (0.4 mmol/l) had almost no effect. None of these channel openers could stimulate the other prominent type of potassium channel in skeletal muscle, the large-conductance Ca2(+)-activated potassium channel. The results show that cromakalim, pinacidil and RP 49356 are specific openers of ATP-sensitive potassium channels in skeletal muscle. It is suggested that the drugs displace the channel blocker ATP and that RP 49356 in addition recruits inactive channels.

Interaction of monovalent cations with tetrodotoxin and saxitoxin binding at sodium channels of frog myelinated nerve.

Na currents and Na-current fluctuations were measured in myelinated frog nerve fibres to study interactions between monovalent externally applied cations and the binding of the Na-channel blockers tetrodotoxin (TTX) or saxitoxin (STX). Adding 110 mM NaCl to Ringer's solution increased the maximum peak Na conductance by a factor of 2.51 in the presence of 12 nM TTX and by a factor of 2.43 in the presence of 4 nM STX. According to the analysis of Na-current fluctuations this increase of the Na conductance is mainly caused by an increase of the number N of unblocked Na channels per node, while the conductance of a single channel saturates in the hyperosmolar solutions. The increase of N is interpreted by displacement of TTX or STX from Na channels by external Na+. Relief of TTX blockage was also observed by adding 110 mM chloride salts of Li+, hydrazine+, guanidine+ and K+ to Ringer, but not in Ringer + 110 mM tetramethylammonium chloride or 250 mM sucrose. The increase of N by the external cations is a saturating function of the permeability of the Na channel to these ions. The results are interpreted by a toxin receptor in a superficial prefilter to the Na channel, which contributes to cation discrimination at the outer channel region.

ATP-sensitive potassium channels in adult mouse skeletal muscle: different modes of blockage by internal cations, ATP and tolbutamide.

Single ATP-sensitive K channels were studied in membrane patches excised from enzymatically dissociated mouse toe muscle. The channel conductance is 74 pS in symmetrical 160 mM KCl solutions. Replacement of K+ by Na+ in the internal solution or 2 mM internal Ca2+ or Mg2+ induced a rectification of the current-voltage curve at positive potentials. No change of the current-voltage curve was observed by adding small amounts of the channel blockers ATP (20-100 microM) or tolbutamide (0.5 mM) to internal 160 mM KCl solutions. The openings of the channel occurred in bursts. Open (tau o), closed (tau c) times within bursts and pauses (tau p) between bursts were determined over a wide range of positive and negative membrane potentials. At increasing potentials tau o increases, tau c reaches a minimum near 0 mV and tau p decreases. According to the voltage dependence and the time scale of channel blockage three types of blocking agents could be distinguished: (i) small internal cations (Na+, Ca2+, Mg2+) are "fast" blockers at positive voltages; at negative voltages they decrease tau o and increase tau c. (ii) Internal ATP anions produce a voltage-dependent decline of the open-state probability and strongly decrease tau o. (iii) Tolbutamide causes a voltage-independent decrease of the open-probability and its main effect is an increase of tau p. The results suggest that the ATP-sensitive K channel has an internal gate like those of other voltage-gated cation channels and that different blockers interfere with different transitions in channel gating.

ATP-sensitive potassium channels in adult mouse skeletal muscle: characterization of the ATP-binding site.

Single K+-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160 mM KCl solutions were blocked reversibly by 10 microM internal ATP and thus identified as ATP-sensitive K+ channels. The channels were also blocked reversibly by mM concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K+ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5 mM chloramine-T, 50 mM hydrogen peroxide or 2 mM N-ethylmaleimide (NEM). Large-conductance Ca-activated K+ channels were not affected by these reagents. The presence of 1 mM internal ATP prevents the irreversible inhibition of ATP-sensitive K+ channels by NEM. The results suggest that internal Na+ ions increase the affinity of the ATP-sensitive K+ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.

Low-conductance states of K+ channels in adult mouse skeletal muscle.

Single-channel currents were recorded from Ca2+-activated or ATP-sensitive K+ channels in inside-out membrane patches excised from isolated mouse toe muscles. In addition to the closed and fully open configurations, both types of channels may exhibit several intermediate low-conductance states which are clustered near multiples of elementary conductance units. The units are 1/8 or 1/6 of the channel conductance for Ca2+-activated channels and 1/4 or 1/3 for ATP-sensitive channels. Normally, low-conductance states are rare, but they occur more frequently directly after patch excision. An increased probability of low-conductance states of ATP-sensitive K+ channels was also observed in the presence and during washout of the internal channel blocker adenine. The results suggest that Ca2+-activated and ATP-sensitive K+ channels are composed of several membrane pores with strong positive cooperativity among the elementary conductance units.

A high-conductance anion channel in adult amphibian skeletal muscle.

Membrane patches were excised from enzymatically dissociated frog toe muscle. High-conductance anion channels could be induced in previously quiet patches by 20-120 s depolarizations beyond +20 mV and then studied in the potential range from -80 to +60 mV for a long time. From reversal potentials the estimated permeability ratios PCl/PNa and PCl/Pglucuronate were near 3.5 and 4, respectively. There were probably 5 or more conductance levels (substates) for a single channel, the most common in symmetrical 110 mM NaCl being 260 and 70 pS at 10 degrees C. Gating was complex, with rapid and slow events and several gating modes, including periods of rapid flickering. Channels closed reversibly at potentials more negative than -50 mV. The channel was blocked by application to the cytoplasmic face of tannic acid, gallic acid, and zinc but not of DIDS or 9-anthracene-carboxylic acid, and it was blocked by extracellular zinc.

Conductance properties and voltage dependence of an anion channel in amphibian skeletal muscle.

Single anion-selective channels were studied in excised membrane patches of adult frog toe muscle. The conductance gamma and the probability po of the main open state were determined for various ionic compositions of the extra- and intracellular solutions. gamma = 280 pS in symmetrical 110 mM NaCl, pH 7.4 solutions at 15 degrees C. Higher gamma values were found at elevated internal or external NaCl concentrations, in 70 mM external CaCl2 and at lower extracellular pH. The po(E) curve declined steeply with hyperpolarization and was shifted towards more negative potentials at increased internal ionic strength and at higher external pH. Positive shifts were induced by extracellular Ca. The results show that the anion channel saturates at Cl concentrations greater than 110 mM, that the potential profile of an open channel is almost symmetrical and that channel gating is affected by neighboring channels. It is suggested that the anion channel has a voltage sensor (effective gating charge 4.3) and a similar collection of local fixed charges on the extra- and intracellular sides as voltage-gated cation channels.