Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 μM), ATR and CAT (5-100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

The thiazole derivative reduces transmembrane currents via ionic channels formed by alpha-latrotoxin and sea anemone toxin in the bilayer lipid membranes

It was shown that the thiazole derivative 3-decyloxycarbonylmethyl-4-methyl- 5-(2-hydroxyethyl)thiazole chloride (DMHT) (0.1 mM) reversibly reduced the transmembrane current in solutions of 10 mM CaCl2 and 100 mM KCl via ionic channels produced by alpha-latrotoxin from black widow spider (alpha-LT) and sea anemone toxin (RTX) in the bilayer lipid membranes (BLM). Introduction of DMHT from the cis-side of BLM inhibited transmembrane current by 31.6 +/- 3% and by 61.8 +/- 3% from the trans-side of BLM for alpha-LT channels. Application of DMHT to the cis-side BLM decreased the inward current through the RTX channels by 50 +/- 5%. Addition of Cd(2+) (0.1 mM) to the cis- or trans-side of a membrane after the DMHT induced depression of transmembrane current across the alpha-LT channels caused its further decrease by 85 +/- 5% that coincides completely with the intensity of Cd(2+)-inhibition in the control experiments without DMHT. These data suggest that DMHT may exert its inhibitory action on alpha-LT channels without considerable influence on the ionogenic groups inside the channel cavity. The comparative analysis of effective radii measured for alpha-LT and RTX channels on the cis- (0.9 nm and 0.55 nm, respectively) and the trans-side of BLM (< 0.467 nm for alpha-LT) allowed to propose the blocking action of DMHT for alpha-LT and RTX channels to result from direct penetration into the channel, achieved due to similar hydrodynamic size of blocking molecules and the size of toxin pores.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B₁ (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl₂ and KCl solutions via ionic channels produced by latrotoxins (α-latrotoxin (α-LT) and α-latroinsectotoxin (α-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl₂ inhibited transmembrane current by 31.6 ± 3% and by 61.8 ± 3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl₂ to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52 ± 4% and 50 ± 5%, respectively. Addition of Cd²⁺ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca²⁺-current across the α-LT channels caused its further decrease by 85 ± 5% that coincides favorably with the intensity of Cd²⁺ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca²⁺-selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28 ± 0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

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

Single ATP-sensitive potassium channels (K _ATP 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. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M 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 K _ATP channel can bind more divalent Mg²⁺-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 Mg²⁺-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 K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Mitochondria Present in Excised Patches From Pancreatic B-cells May Form Microcompartments With ATP-Dependent Potassium Channels

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K_ATP) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or -ketoisocaproic acid (10 mM) reduced the open probability of K_ATP channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K_ATP channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K_ATP channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K_ATP channels thus forming functional microcompartments.     

Correlations between changes in membrane capacitance induced by changes in ionic environment and the conductance of channels incorporated into bilayer lipid membranes

The action of metal polycations and pH on ionic channels produced in bilayer lipid membranes (BLM) by three different toxins was studied by measuring membrane capacitance and channel conductance. Here, we show that critical concentrations of Cd²⁺, La³⁺ or Tb³⁺ induce complex changes in membrane capacitance. The time course of capacitance changes is similar to the time course of channel blocking by these ions at low concentration. No changes in BLM capacitance or conductance were observed in the range of pH 5.8–9.0. A pH shift from 7.4 to 3–4 or 11–12 induced large changes in BLM capacitance and channel conductance. For all studied channel-forming proteins, the initial capacitance increase preceded the conductance decrease caused by addition of polycations or by a change in pH. A close relationship between membrane lipid packing and ion channel protein is suggested.     

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.     

Protein interactions with lipid bilayers: The channels of kidney plasma membrane proteolipids

Summary Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., <0.6 g/ml), the single-channel conductance is approximately 10 pS in 100mm KCl and 3 pS in 100mm NaCl. In the presence of protein concentrations above 1 g/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100mm KCl and 30 pS in 100mm NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2–3) of the proteolipid concentration present in one of the aqueous phases. In 100mm NaCl, the conductance increases at protein concentrations above 5 g/ml, whereas in 100mm KCl in increases at protein concentrations above 0.6 g/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channels incorporate in the presence of KCl than in the presence of NaCl.The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B1 (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(beta-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl2 and KCl solutions via ionic channels produced by latrotoxins (alpha-latrotoxin (alpha-LT) and alpha-latroinsectotoxin (alpha-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl2 inhibited transmembrane current by 31.6+/-3% and by 61.8+/-3% from the trans-side of BLM for alpha-LT channels. Application of DMHT in the solution of 10 mM CaCl2 to the cis-side of BLM decreased the current through the alpha-LIT and RTX channels by 52+/-4% and 50+/-5%, respectively. Addition of Cd2+ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca2+-current across the alpha-LT channels caused its further decrease by 85+/-5% that coincides favorably with the intensity of Cd2+ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on alpha-LT channels without considerable influence on the ionogenic groups of Ca2+-selective site inside the channel cavity. The binding kinetics of DMHT with the alpha-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of alpha-LT channel on either side of the BLM. The comparative analysis of effective radii measured for alpha-LT, alpha-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for alpha-LT channel on the trans-side (0.28+/-0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic cells where targets ATP-sensitive K⁺ (K_ATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K_ATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K_ATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K_ATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.     

5-Hydroxydecanoate and coenzyme A are inhibitors of native sarcolemmal KATP channels in inside-out patches

Background

5-Hydroxydecanoate (5-HD) inhibits preconditioning, and it is assumed to be a selective inhibitor of mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channels. However, 5-HD is a substrate for mitochondrial outer membrane acyl-CoA synthetase, which catalyzes the reaction: 5?HD + CoA + ATP → 5-HD-CoA (5-hydroxydecanoyl-CoA) + AMP + pyrophosphate. We aimed to determine whether the reactants or principal product of this reaction modulate sarcolemmal K_ATP (sarcK_ATP) channel activity.

Methods

Single sarcK_ATP channel currents were measured in inside-out patches excised from rat ventricular myocytes. In addition, sarcK_ATP channel activity was recorded in whole-cell configuration or in giant inside-out patches excised from oocytes expressing Kir6.2/SUR2A.

Results

5-HD inhibited (IC₅₀ ∼ 30 μM) K_ATP channel activity, albeit only in the presence of (non-inhibitory) concentrations of ATP. Similarly, when the inhibitory effect of 0.2 mM ATP was reversed by 1 μM oleoyl-CoA, subsequent application of 5-HD blocked channel activity, but no effect was seen in the absence of ATP. Furthermore, we found that 1 μM coenzyme A (CoA) inhibited sarcK_ATP channels. Using giant inside-out patches, which are weakly sensitive to “contaminating” CoA, we found that Kir6.2/SUR2A channels were insensitive to 5-HD-CoA. In intact myocytes, 5-HD failed to reverse sarcK_ATP channel activation by either metabolic inhibition or rilmakalim.

General significance

SarcK_ATP channels are inhibited by 5-HD (provided that ATP is present) and CoA but insensitive to 5-HD-CoA. 5-HD is equally potent at “directly” inhibiting sarcK_ATP and mitoK_ATP channels. However, in intact cells, 5-HD fails to inhibit sarcK_ATP channels, suggesting that mitochondria are the preconditioning-relevant targets of 5-HD.     

Potassium-chloride promiscuous channels in mitochondrial membranes

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.     

Effects of ADP upon the ATP-sensitive K+ channel in rat ventricular myocytes

Summary The effects of ADP upon the gating of ATP-sensitive K⁺ channels from rat ventricular myocytes have been investigated by patch-clamp single-channel current recording experiments. ADP was applied to the internal surface of excised insideout membrane patches and depending upon the experimental protocol and the concentration it was found that ADP could either inhibit or stimulate openings of ATP-sensitive K⁺ channels. In the absence of inactivation, ATP-sensitive K⁺ channels were inhibited by ADP in a dose-dependent manner. Partially inactivated channels, on the other hand, were stimulated by low (10 to 250 M) and inhibited by high (>250 M) concentrations of ADP. ATP-sensitive K⁺ channels which were being inhibited by ATP (<1 mM) could be opened by the simultaneous application of ADP (50 M to 1 mM). ADP had no effect upon channels inhibited by mM concentrations of ATP. The situation was further complicated when it was found that inhibition evoked by ADP was strongly attenuated by the presence of Mg²⁺ ions whilst channel stimulation, whether of partially inactivated channels or channels inhibited by ATP, required the presence of Mg²⁺ ions. The analog of ADP, ADPS, always evoked inhibition of ATP-sensitive K⁺ channels which was not affected by the presence or absence of Mg²⁺ ions.     

Novel actions of ryanodine and analogues—Perturbers of potassium channels

The effects of ryanodine, 9,21-didehydroryanodine and 9,21-didehydroryanodol on two types of K⁺ channel (a maxi, Ca²⁺-activated, 170 pS channel (BK channel) and an inward rectifier, stretch-sensitive channel of 35 pS conductance (IK channel) found in the plasma membrane of locust skeletal muscle have been investigated. 10^–9M-10^–5M ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V_rev) of the currents of both channels, i.e. from 60 mV in the absence of the alkaloid to 15 mV for 10^–5M ryanodine, measured under physiologically normal K⁺ and Na⁺ gradients. In both cases the change in the ionic selectivity was Ca²⁺-independent. 9,21-didehydroryanodine and 9,21-didehyroryanodol also reduced V_rev, but only to 35 mV during application of 10^–5M of these compounds. Additionally, 9,21-didehydroryanodine reversibly diminished the conductances of the two K⁺ channels. To test the hypothesis that ryanoids increase Na⁺ permeability by enlarging the K⁺ channels, the channels were probed with quaternary ammonium ions during ryanoid application. When applied to the cytoplasmic face of inside-out patches exised from locust muscle membrane, TEA blocked the K⁺ channels in a voltage-dependent fashion. The dissociation constant (K_d(0)) for TEA block of the IK channel was reduced from 44 mM to 1 mM by 10^–7 M ryanodine, but the voltage-dependence of the block was unaffected. Qualitatively similar data were obtained for the BK channel. Ryanodine had no effect on the K_d for cytoplasmically-applied TMA. However, the voltage-dependence for TMA block was increased for both K⁺ channels, from 0.47 to 0.8 with 10^–6M ryanodine. The effects of ryanodine on TEA and TMA block support the hypothesis that ryanodine enlarges the K⁺ channels so as to facilitate permeation of partially hydrated Na⁺ ions.     

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280100 mM KCl, five distinct K⁺-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K⁺ channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K⁺ channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent inward rectification in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (_o, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (_c, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb⁺ K⁺ > Cs⁺ > Na⁺ > Li⁺ > tetraethylammonium (TEA⁺). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA⁺ (10 mM), Ba²⁺ (3 mM), Ca²⁺ (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 M) were without effect when applied to the extracellular (cis) surface.Abbreviations and Symbols GHK Goldman-Hodgkin-Katz - I/V current/voltage - PEG polyethyleneglycol - P_o probability o f the channel being open - TEA⁺ tetraethylammonium - _c apparent mean lifetime of the channel-closed state - _o apparent mean lifetime of the channel-open state P.J.W. was supported by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Professor E.A.C. MacRobbie and M.T. by the Glaxo Junior Research Fellowship at Churchill College, Cambridge. We thank Dr. D.T. Cooke (AFRC, Long Ashton Research Station, University of Bristol, UK) and Ms. J. Marshall (University of York, UK) for their advice and assistance with the aqueous-polymer two-phase partitioning of plasma membrane from rye roots, Mr. J. Banfield and Miss P. Parmar (University of Cambridge, UK) for technical assistance and Professor E.A.C. MacRobbie, Dr. G. Thiel (University of Cambridge, UK), Dr. M.R. Blatt (Wye College, University of London, UK), Dr. D. Sanders and Dr. E. Johannes (University of York, UK) for helpful discussions.     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Differential effects of 4-aminopyridine on acetylcholine release triggered by K+ depolarization,veratridine, or A23187 in rat cerebral cortical synaptosomes

The effect of 4-aminopyridine on [³H]acetylcholine release was studied in rat cerebral cortical synaptosomes in the presence of a several secretagogues that have different mechanisms of action. As found previously, 4-aminopyridine increased [³H]acetylcholine release in a concentration-dependent manner (5–10 mM); a high concentration (10 mM) also elevated [³H]choline efflux. However, the 35 mM K⁺ induced release of [³H]acetylcholine was attenuated by 4-aminopyridine at concentrations (less than 5 mM) that had no effect on transmitter release. At no concentration of 4-aminopyridine was the release of transmitter additive with 35 mM K⁺ induced release. Veratridine-induced release was neither attenuated nor additive with low concentrations of 4-aminopyridine, even when a sub-maximal concentration of the sodium ionophore was used (10 M). In contrast, A23187-induced release was additive with that caused by 4-aminopyridine. These results suggest that: 1) 4-aminopyridine blocks potassium channels involved in regulating membrane potential in isolated cholinergic terminals; and 2) changes in the activity of these 4-aminopyridine sensitive K⁺ channels are not important in the nerve terminal's response to depolarization caused by sodium influx.     

Identification of K+, Cl−, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures

Summary K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cell suspension cultures have been investigated using the patch-clamp technique. In symmetrical 100mM K⁺, K⁺ channels opened at positive vacuolar membrane potentials (cytoplasmic side as reference) had different conductances of 57 pS and 24 pS. K⁺ channel opened at negative vacuolar membrane potentials had a conductance of 43 pS. The K⁺ channels showed a significant discrimination against Na⁺ and Cl^–. The Cl^– channel opened at positive vacuolar membrane potentials for cytoplasmic Cl^– influx had a high conductance of 110pS in symmetrical 100mM Cl^–. When K⁺ and Cl^– channels were excluded from opening, no traces were found of Ca²⁺ channel activity for vacuolar Ca²⁺ release induced by inositol 1,4,5-trisphosphate or other events. However, we found a 19pS Ca²⁺ channel which allowed influx of cytoplasmic Ca²⁺ into the vacuole when the Ca²⁺ concentration on the cytoplasmic side was high. When Ca²⁺ was substituted by Ba²⁺, the conductance of the 19 pS channel became 30 pS and the channel showed a selectivity sequence of Ba²⁺Sr²⁺Ca²⁺Mg²⁺=10.60.60.21. The reversal potentials of the channel shifted with the change in Ca²⁺ concentration on the vacuolar side. The channel could be efficiently blocked from the cytoplasmic side by Cd²⁺, but was insensitive to La³⁺, Gd³⁺, Ni²⁺, verapamil, and nifedipine. The related ion channels in freshly isolated vacuoles from red beet root cells were also recorded. The coexistence of the K⁺, Cl^–, and Ca²⁺ channels in the vacuolar membrane of tobacco cells might imply a precise classification and cooperation of the channels in the physiological process of plant cells.