Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposomes could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential. Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.     

Newly identified steady-state potassium channels in rat hippocampal neurons.

We report two new types of potassium channels in cultured hippocampal neurons of rat. Both channels occurred in the soma membrane of these cells at very low density. They were active in steady-state conditions, within a wide voltage range that included the resting membrane potential. Their open probability was enhanced by membrane depolarization, but not influenced by Ca ions. In symmetrical 150 mM KCl the channels showed a slope conductance of ca. 40 and 80 pS, respectively. Current-voltage relations of both K channels show a negative slope at high positive voltages.     

Effects of channel blocking on information transmission and energy efficiency in squid giant axons

Action potentials are the information carriers of neural systems. The generation of action potentials involves the cooperative opening and closing of sodium and potassium channels. This process is metabolically expensive because the ions flowing through open channels need to be restored to maintain concentration gradients of these ions. Toxins like tetraethylammonium can block working ion channels, thus affecting the function and energy cost of neurons. In this paper, by computer simulation of the Hodgkin-Huxley neuron model, we studied the effects of channel blocking with toxins on the information transmission and energy efficiency in squid giant axons. We found that gradually blocking sodium channels will sequentially maximize the information transmission and energy efficiency of the axons, whereas moderate blocking of potassium channels will have little impact on the information transmission and will decrease the energy efficiency. Heavy blocking of potassium channels will cause self-sustained oscillation of membrane potentials. Simultaneously blocking sodium and potassium channels with the same ratio increases both information transmission and energy efficiency. Our results are in line with previous studies suggesting that information processing capacity and energy efficiency can be maximized by regulating the number of active ion channels, and this indicates a viable avenue for future experimentation.     

Excitation-contraction coupling in crab muscle fibers with swollen T tubules

Single crab (Callinectes danae) fibers were equilibrated with isotonic, high KCl solutions and were subsequently returned to the control saline. This caused marked swelling of the T tubules. Fibers treated with 100 mM KCl had a 2.5-mV residual depolarization, a 50% decrease in effective membrane resistance (Reff) and a 75% reduction in membrane time constant (tau m). These fibers exhibited large increases in membrane conductance upon depolarization and were inexcitable; membrane depolarization with current pulses elicited no contraction. The effects of the KCl treatment on membrane properties were not reproduced by treatment with high potassium gluconate solutions, which did not cause tubular swelling. Tetrabutylammonium (10 mM) or Ba ions (10-20 mM), but not tetraethylammonium (40-100 mM), Sr ions (15-70 mM), or procaine (1-8 mM) reversed the effects of the KCl treatment on Reff, tau m, membrane excitability, and excitation-contraction coupling. The time course of the Ba effects was consistent with the suggestion that the KCl treatment increases the K conductance of the tubular membranes, which in turn prevents the activation of voltage-dependent Ca channels located in the membranes of the T system. This results in inhibition of the Ca-dependent electrogenesis and consequently, the absence of contraction upon depolarization of the plasma membrane.     

Interaction of permeant and blocking ions in cloned inward-rectifier K+ channels.

Blocking cloned inward-rectifier potassium (Kir) channels from the cytoplasmic side was analyzed with a rapid application system exchanging the intracellular solution on giant inside-out patches from Xenopus oocytes in <2 ms. Dependence of the pore-block on interaction of the blocking molecule with permeant and impermeant ions on either side of the membrane was investigated in Kir1.1 (ROMK1) channels blocked by ammonium derivatives and in Kir4.1 (BIR10) channels blocked by spermine. The blocking reaction in both systems showed first-order kinetics and allowed separate determination of on- and off-rates. The off-rates of block were strongly dependent on the concentration of internal and external bulk ions, but almost independent of the ion species at the cytoplasmic side of the membrane. With K+ as the only cation on both sides of the membrane, off-rates exhibited strong coupling to the K+ reversal potential (E(K)) and increased and decreased with reduction in intra and extracellular K+ concentration, respectively. The on-rates showed significant dependence on concentration and species of internal bulk ions. This control of rate-constants by interaction of permeant and impermeant internal and external ions governs the steady-state current-voltage relation (I-V) of Kir channels and determines their physiological function under various conditions.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Effects of potassium channel modulators on myotropic responses of aortic rings of pregnant rats

The contribution of potassium channels [ATP-sensitive potassium (K(ATP)) and high-conductance calcium-activated potassium (BK(Ca)) channels] in the resistance of aortic rings of term pregnant rats to phenylephrine (Phe), arginine vasopressin (AVP), and KCl was investigated. Concentration-response curves to tetraethylammonium (TEA), a nonselective K(+) channel inhibitor, were obtained in the absence or presence of KCl. TEA induced by itself concentration-dependent responses only in aortic rings of nonpregnant rats. These responses to TEA could be modulated in both groups of rings by preincubation with different concentrations of KCl. Concentration-response curves to Phe, AVP, and KCl were obtained in the absence or presence of cromakalim or NS-1619 (K(ATP) and BK(Ca) openers, respectively) and glibenclamide or iberiotoxin (K(ATP) and BK(Ca) inhibitors, respectively). Cromakalim significantly inhibited the responses to the three agonists in a concentration-dependent manner in both groups of rats. Alternatively, in the pregnant group of rats, glibenclamide increased the sensitivity to all three agonists. NS-1619 also inhibited the response to all agonists. With AVP and KCl, its effect was greater in aortic rings of pregnant than nonpregnant rats. Finally, iberiotoxin increased the sensitivity to all three agents. This effect was more important in aortic rings of nonpregnant rats and was accompanied by an increase of the maximal response to Phe and AVP. These results suggest that potassium channels are implicated in the control of basal membrane potential and in the blunted responses to these agents during pregnancy.     

Voltage-dependent cationic channels formed by a cytolytic toxin produced by Gardnerella vaginalis.

A cytolytic toxin produced by G. vaginalis was incorporated in artificial membranes and giant liposomes. The toxin formed ionic channels when incorporated in lipid bilayers. The electrical properties of such channels were studied. Current records revealed a unitary conductance of 126 pS (in symmetrical 150 mM KCl). The open state probability of the cytolysin formed channels was a function of the applied membrane potential. The permeability ratio of cations to anions was estimated to be 6.5.     

The inner quaternary ammonium ion receptor in potassium channels of the node of Ranvier

Quaternary ammonium ions were applied to the inside of single myelinated nerve fibers by diffusion from a cut end. The resulting block of potassium channels in the node of Ranvier was studied under voltage-clamp conditions. The results agree in almost all respects with similar studies by Armstrong of squid giant axons. With tetraethylammonium ion (TEA), pentyltriethylammonium ion (C₅), or nonyltriethylammonium ion (C₉) inside the node, potassium current during a depolarization begins to rise at the normal rate, reaches a peak, and then falls again. This unusual inactivation is more complete with C₉ than with TEA. Larger depolarizations give more block. Thus the block of potassium channels grows with time and voltage during a depolarization. The block reverses with repolarization, but for C₉ full reversal takes seconds at -75 mv. The reversal is faster in 120 mM KCl Ringer''s and slower during a hyperpolarization to -125 mv. All of these effects contrast with the time and voltage-independent block of potassium, channels seen with external quaternary ammonium ions on the node of Ranvier. External TEA, C₅, and C₉ block without inactivation. The external quaternary ammonium ion receptor appears to be distinct from the inner one. Apparently the inner quaternary ammonium ion receptor can be reached only when the activation gate for potassium channels is open. We suggest that the inner receptor lies within the channel and that the channel is a pore with its activation gate near the axoplasmic end.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.     

Vegetative insecticidal protein (Vip1Ac) of Bacillus thuringiensis HD201: evidence for oligomer and channel formation

The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.     

Drug receptors on single enteric neurons

There are many substances contained within enteric nerves which excite or inhibit other nerves when these substances are applied to single neurons. The actions of these substances and of drugs which mimic these actions is to open or close membrane ion channels. The effects on membrane potential are dependent on the nature of the ions which pass through the channel and whether the channel is opened or closed. In the enteric nervous system, drugs can act at one of three broad classes of receptors: [1] those which are part of an ion channel complex and which open either cation channels or chloride channels, both of which result in membrane depolarization [2] those which open potassium channels resulting in hyperpolarization or [3] those which close potassium channels resulting in depolarization. Receptors which open potassium channels are coupled to the channel via a G-protein while receptors which close potassium channels are coupled to the channel, in some cases, via a cyclic AMP-dependent system while in other cases another second messenger system is involved.     

Properties of the porin of Haemophilus influenzae type b in planar lipid bilayer membranes

The major outer membrane protein (40 kDa) of the bacterium Haemophilus influenzae type b is a porin which forms transmembrane permeability channels. It has an exclusion limit for oligosaccharides of about 1.4 kDa. When this protein was added to the aqueous phase which was bathing a planar lipid bilayer, it caused the conductance of the membrane to increase by several orders of magnitude. At low protein concentrations (2-10 pM), the conductance of the membrane increased in a stepwise fashion with an average single-channel conductance of 1.1 nS in 1 M KCl. Single-channel experiments were performed with a variety of different salts. The conductance of single channels was proportional to the specific conductance of the aqueous solution which was bathing the membrane. Current through the pores was proportional to the applied voltage, indicating that these pores are not voltage-controlled. The 40 kDa porin was very slightly cation-selective: the pores were about 1.6-times more permeable to potassium ions than to chloride ions. These properties of the 40 kDa porin are those of large water-filled channels and are characteristic of most bacterial porins. The single-channel conductance of the porin is, however, much smaller than might be expected from its exclusion limit. A model is proposed which could explain the differences in apparent pore size.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Mechanisms of spontaneous relaxation of smooth muscle (guinea pig taenia coli) depolarized in a hyperkalemic medium]

Experiments were performed on isolated strips of guinea pig taenia coli by the double sucrose-gap method. The artificial node was depolarized with potassium solution (from 120 to 167.7 mM KCl). When the bathing solution contained 0.4 mM Ca and the temperature was equal to 25 degrees C then potassium contracture was followed by fast relaxation. The muscular tone changed slightly during rectangular pulse of hyperpolarizing current, after switching off the current muscle generated a transient contractile response. The amplitude of such off-responses increased in some range with increasing in strength and duration of conditioning current. Treatment of muscle with compound D-600 resulted in a reduction of muscular tone and elimination of off-responses. The addition of Na ions to potassium solution (substitution of 47.7 mM KCl with the same quantity of NaCl) reduced muscular tone and enhanced the relaxation after off-responses. In sodium-free potassium solution each off-response was followed by increasing muscular tone but when the bathing solution contained Na ions this increase of the tone was not observed. The data obtained strongly suggest that the spontaneous relaxation of smooth muscle which was contracted in K-solution resulted from: 1) inactivation of calcium channels of surface membrane, 2) sequastration of Ca ions by intracellular storange sites, 3) extrusion of Ca in extracellular space (in part by means of Na-Ca exchange diffusion).     

Further studies on the partial double Donnan. Is isosmotic KCl solution isotonic with cells of respiratory trees of the holothurian Isostichopus badionotus Selenka?

As potassium, chloride and water traverse cell membranes, the cells of stenohaline marine invertebrates should swell if exposed to sea water mixed with an isosmotic KCl solution as they do when exposed to sea water diluted with water. To test this hypothesis respiratory tree fragments of the holothurian Isostichopus badionotus were exposed to five isosmotic media prepared by mixing artificial sodium sea water with isosmotic (611 mmol/l) KCl solution to obtain 100, 83, 71, 60 and 50% sea water, with and without 2 mmol/l ouabain. For comparison, respiratory tree fragments were incubated in sea water diluted to the same concentrations with distilled water, with and without ouabain. Cell water contents and potassium and sodium concentrations were unaffected by KCl-dilution or ouabain in isosmotic KCl-sea water mixtures. In tissues exposed to H(2)O-diluted sea water, cell water increased osmometrically and potassium, sodium and chloride concentrations decreased with dilution; ouabain caused a decrease in potasium and an increase in sodium but no effect on chloride concentrations. The isotonicity of the isosmotic KCl solution cannot be adscribed to impermeability of the cell membrane to KCl as both ions easily traverse the cell membrane. Rather, operationally immobilized extracellular sodium ions, which electrostatically hold back anions and consequently water, together with the lack of a cellward electrochemical gradient for potassium, resulting from membrane depolarization caused by high external potassium concentration, would explain the isotonicity of isosmotic KCl solution. The high external potassium concentration also antagonizes the inhibitory effect of ouabain on the Na(+)/K(+) ATPase responsible for sodium and potassium active transport.     

Inactivation of monazomycin-induced voltage-dependent conductance in thin lipid membranes. I. Inactivation produced by long chain quaternary ammonium ions

The voltage-dependent conductance induced in thin lipid membranes by monazomycin undergoes inactivation upon the introduction of quaternary ammonium ions (QA) having a long alkyl chain (e.g. dodecyltrimethylammonium [C12]) to the side containing monazomycin. That is, in response to a step of voltage the conductance rises to a peak and then falls to a much lower steady-state value. We demonstrate that the basis of this phenomenon is the ability of QA to pass through the stimulated membrane and bind to the opposite surface. As a consequence, the surface potential on that side becomes more positive, thus reducing the voltage across the membrane proper and turning off the monazomycin-induced conductance. Because the flux of QA through the membrane increases linearly with conductance, we believe that these ions pass through the monazomycin channels. QA permeability increases with alkyl chain length; remarkably, in spite of its much larger size, C12 is about 150 times more permeant than K+. It appears, therefore, that there is a hydrophobic region of the cahnnel that favors the alkyl chain; we propose that this region is formed by the hydrophobic faces of the monazomycin channels in lipid bilayers to QA inactivation of potassium channels in the squid giant azon, and suggest that there may be a common structural feature for the two channels. It is possible that some of the inactivation phenomena in excitable cells may arise from local field changes not measurable by the recording electrodes.     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

Streaming potential measurements in Ca2+-activated K+ channels from skeletal and smooth muscle. Coupling of ion and water fluxes.

Streaming potentials arising across large-conductance Ca2+-activated K+ channels incorporated into planar lipid bilayers were measured. Ca2+-activated channels obtained either from skeletal muscle or from smooth muscle membranes were used. Streaming potentials were extracted from the current-voltage relationship for the open channel obtained in the presence of an osmotic gradient. The osmotic gradient was established by adding glucose to one side of the membrane. At 300 mM KCl, the average streaming potential was 0.72 mV/osmol per kg for t-tubule channels and 0.83 mV/osmol per kg for smooth muscle channels. Streaming potential values depend on KCl concentration, they decrease as KCl concentration increases, and the value obtained by extrapolation to zero KCl concentration is 0.85 mV/osmol per kg. Assuming that water and ions cannot pass each other, at least in a region of the channel, the streaming potential values obtained indicate that this region contains a minimum of two and a maximum of four water molecules. It is concluded that the channel has a narrow region with a length of 0.6-1.2 nm.     

Effect of calcium ions on outward currents in the somatic membrane of mollusk giant neurons

Potassium currents through the somatic membrane of giant neurons ofHelix pomatia in normal (10 mM Ca) Ringer's solution and low-calcium (1 mM Ca) solution were studied by the voltage clamp method. With a decrease in the Ca concentration to 1 mM peak potassium conductance versus membrane, potential curves and inactivation curves were shifted along the voltage axis in the negative direction by about 10 mV. Inactivation of the delayed potassium current was slowed in low Ca solution. The effect of a decrease in external calcium concentration on volt-ampere and inactivation characteristics increased with a rise in external pH. These effects of a low Ca concentration on potassium mechanisms of the giant neuron somatic membrane can be attributed to changes in the negative surface potential in the region of the potassium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Biology, Hungarian Academy of Sciences, Tihany. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 400–409, July–August, 1976.