Voltage-dependent channels permeable to K+ and Na+ in the membrane ofAcer pseudoplatanus vacuoles

Summary The patch-clamp technique in whole-cell configuration was used to study the electrical properties of the tonoplast in isolated vacuoles fromAcer pseudoplatanus cultured cells. In symmetrical KCl or K₂ malate solutions, voltage- and time-dependent inward currents were elicited by hyperpolarizing the tonoplast (inside negative), while in the positive range of potential the conductance was very small. The specific conductance of the tonoplast at –100 mV, in 100mm symmetrical KCl was about 160 S/cm². The reversal potentials (E _rev) of the current, measured in symmetrical or asymmetrical ion concentrations (cation, anion or both) were very close to the values of the K⁺ equilibrium potential. Experiments performed in symmetrical or asymmetrical NaCl indicate that Na⁺ too can flow through the channels. NeitherE _rev nor amplitude and kinetics of the current changed by replacing NaCl with KCl in the external solution. These results indicate the presence of hyperpolarization-activated channels in tonoplasts, which are permeable to K⁺ as well as to Na⁺. Anions such as Cl^– or malate seem to contribute little to the channel current.     

haemolysin of Escherichia coli: Comparison of pore-forming properties between chromosome and plasmid-encoded haemolysins

Abstract Lipid bilayer experiments were performed with chromosome-encoded haemolysin of Escherichia coli . The addition of the toxin to the aqueous phase bathing lipid bilayer membranes of asolectin resulted in the formation of transient ion-permeable channels with two states at small transmembrane voltages. One is prestate (single-channel conductance 40 pS in 0.15 M KCl) of the open state, which had a single-channel conductance of 420 pS in 0.15 M KCl and a mean lifetime of 30 s. Membranes formed of pure lipids were rather inactive targets for this haemolysin. Experiments with different salts suggested that the haemolysin channel was highly cation-selective at neutral pH. The mobility sequence of the cations in the channel was similar if not identical to their mobility sequence in the aqueous phase. The single-channel data were consistent with a wide, water-filled channel with an estimated minimal diameter of about 1 nm. The pore-forming properties of chromosome-encoded haemolysin were compared with those of plasmid-encoded haemolysin. Both toxins share common features, oligomerize probably to form pores in lipid bilayer membranes. Both types of haemolysin channels have similar properties but different lifetimes.     

Single point mutations of the sodium channel drastically reduce the pore permeability without preventing its gating

1. Two mutants of the sodium channel II have been expressed inXenopus oocytes and have been investigated using the patch-clamp technique. In mutant E387Q the glutamic acid at position 387 has been replaced by glutamine, and in mutant D384N the aspartic acid at position 384 has been replaced by asparagine.2. Mutant E387Q, previously shown to be resistant to block by tetrodotoxin (Noda et al. 1989), has a single-channel conductance of 4 pS, that can be easily measured only using noise analysis. At variance with the wild-type, the openchannel current-voltage relationship of mutant E387Q is linear over a wide voltage range even under asymmetrical ionic conditions.3. Mutant D384N has a very low permeability for any of the following ions: Cl^–, Na⁺, K⁺, Li⁺, Rb⁺, Ca²⁺, Mg²⁺, NH₄ ⁺ , TMA⁺, TEA⁺. However, asymmetric charge movements similar to the gating currents of the Na⁺-selective wild-type are still observed.4. These results suggest that residues E387 and D384 interact directly with the pathway of the ions permeating the open channel.Abbreviations TTX tetrodotoxin; Na⁺, sodium; K⁺, potassium; - NFR normal frog Ringer - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis(-amino-ethyl ether) N,N,N',N'-tetra acetic acid - TEA tetraethylammonium - TMA tetramethylammonium;I _g , gating current; , single-channel conductance     

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 V₁₀O _inf28 ^sup6– . ATP concentration-response curves have Hill coefficients near three in 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 M 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 M 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. Offprint requests to: B. Neumcke     

Phloretin affects the fast potassium channels in frog nerve fibres

The effect of phloretin (20-100 M), a dipolar organic compound, on the voltage clamp currents of the frog node of Ranvier has been investigated. The Na currents are simply reduced in size but not otherwise affected. Phloretin has no effect on the slow 4-aminopyridine-resistant K channels. However, the voltage dependence and time course of the fast K conductance (g _K) is markedly altered. The g _K(E) curve, determined by measuring fast tail currents at different pulse potentials, normally exhibits a bend at –50 mV indicating the existence of two types of fats K channels. Phloretin shifts the g _K (E) curve to more positive potentials, reduces its slope and its maximum and abolishes the distinction between the two tpyes of fast K channels. The effect becomes more pronounced with time. Phloretin also markedly slows the opening of the fast K channels, but has much less effect on the closing. Opening can be accelerated again by a long depolarizing prepulse which presumably removes part of the phloretin block. It is concluded that phloretin selectively affects the fast K channels of the nodal membrane. The results are compared with similar observations on the squid giant axon. Offprint requests to: H. Meves     

Identification and characterization of a pregnane steroid recognition site that is functionally coupled to an expressed GABAA receptor

Conclusion Based on the pharmacological and biochemical evidence to date, especially that derived from the recombinantly expressed receptor studies, the suggestion that a novel GBRC-linked steroid recognition site exists becomes a cogent argument. The high affinity of the steroid site for certain naturally occurring metabolites of progesterone and glucocorticoids favors a physiologic role for these steroids in the regulation of brain excitability. Clearly, investigations of such a regulatory role is warranted. If present, it provides an important example of endocrine control of a major inhibitory neurotransmitter in the CNS. Moreover, as we gain a greater understanding of the molecular organization of the GBRC, the putative steroid site provides a novel target for the rational design of therapeutic agents for the treatment of anxiety, epilepsy, and insomnia.Special issue dedicated to Dr. Eugene Roberts.     

Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

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 proteoliposome 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.     

The species composition,occurrence and temporal stability of submerged aquatic macrophyte patches along the main channel border of pool 5A,Upper Mississippi River

Species composition, relative abundance, distribution and physical habitat associations of submerged aquatic macrophytes in the main channel border (MCB) habitat of Pool 5A, Upper Mississippi River (UMR) were investigated during the summers of 1980 and 1983. The submerged aquatic macrophytes in Pool .5A MCB were a small and stable component of the river ecosystem. Submerged plants occurred primarily in small, monospecific clumps. Clumps in close proximity to each other formed plant patches. Plant patches were stable in location and number between 1980 and 1983; 82.5% of the patches first observed in 1980 were present in 1983. Submerged macrophytes covered about 10–12 ha of the 201 ha MCB in Pool 5A. Submerged plants were most common in the lower two-thirds of the pool. Ten species of aquatic macrophytes occurred on rock channel-training structures and eleven occurred on non-rock substrates in the MCB. The most common submerged plants, in order of abundance, were Vallisneria americana Michx., Heteranthra dubia Jacq., Potamogeton pectinatus L., Ceratophyllum demersum L. and Potamogeton americanus C. & S.