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B K Krueger

Publications and source records attributed to B K Krueger.

49 records · Page 3Linked to original sources
PubMed1984

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers.

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.

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PubMed1980

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins.

Regulation of Na+ channels by neurotoxins has been studied in pinched-off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.

Animals↗
PubMed1979

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain.

Binding of 3H-saxitoxin to Na+ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1 nM; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30 nM. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K+ concentrations, or by activation of the Na+ channels with batrachotoxin plus a purified polypeptide toxin from the scorpion Leiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70--80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60--70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2 nM. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0 degrees of 0.1 min-1 for membrane bound and 0.2 min-1 for detergent-solubilized binding sites. The measured association rate constant was 6 X 10(8) M-1 min-1 at 0 degrees for membrane-bound saxitoxin binding sites.

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PubMed1977

Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes.

Agents known to inphorylation of specific endogenous proteins in intact synaptosomes from rat brain. Synaptosome preparations, preincubated in vitro with 32Pi, incorporated 32P into a variety of specific proteins. Veratridine and high (60 mM) K+, which increase Ca2+ transport across membranes, through a mechanism involving membrane depolarization, as well as the calcium ionophore A23187, each markedly stimulated the incorporation of 32P into two specific proteins (80,000 and 86,000 daltons) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. All three agents failed to stimulate protein phosphorylation in calcium-free medium containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Moreover, the Ca2+-dependent protein phosphorylation could be reversed by the addition of sufficient EGTA to chelate all free extracellular Ca2+. Veratridine, high K+, and A23187 also stimulated 45Ca2+ accumulation by synaptosomes. Tetrodotoxin blocked the stimulation both of protein phosphorylation and of 45Ca2+ accumulation by veratridine but not by high K+ or A23187. Cyclic nucleotides and several putative neurotransmitters were without effect on protein phosphorylation in these intact synaptosome preparations. The absence of any endogenous protein phosphorylation in osmotically shocked synaptosome preparations incubated with 32Pi, and the inability of added [gamma-32P]ATP to serve as a substrate for veratridine-stimulated protein phosphorylation in intact preparations, indicated that the Ca2+-dependent protein phosphorylation occurred within intact subcellular organelles. Fractionation of a crude synaptosome preparation on a discontinuous Ficoll/sucrose flotation gradient indicated that these organelles were synaptosomes rather than mitochondria. The data suggest that conditions which cause an accumulation of calcium by synaptosomes lead to a calcium-dependent increase in phosphorylation of specific endogenous proteins. These phosphoproteins may be involved in the regulation of certain calcium-dependent nerve terminal functions such as neurotransmitter synthesis and release.

Animals↗
PubMed1975

Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation.

A protein whose endogenous phosphorylation and dephosphorylation are affected by cAMP has been found in the soluble and particulate fractions of all vertebrate tissues studied. This phosphoprotein, which contained a substantial proportion of the radioactive phosphate observed on SDS-polyacrylamide gels, was estimated to have an apparent molecular weight of 49,000. In the presence of Zn++, cAMP inhibited the endogenous phosphorylation of this protein (protein 49) in the cytosol and microsomal fractions. In the presence of Mg++, cAMP stimulated the phosphorylation of protein 49 in the cytosol fractions, but had only slight effects in the microsomal fractions. The dephosphorylation of protein 49 by an endogenous protein phosphatase was markedly stimulated by cAMP in the cytosol and microsomal fractions of all tissues studied. The binding of 8-azido-cAMP (a photoaffinity analog of cAMP, which reacts specifically with cAMP-binding sites) to subcellular fractions was also studied. This binding was principally to a protein of molecular weight 49,000. These and other data suggest that a cAMP-binding protein with a molecular weight of 49,000 capable of undergoing cAMP-dependent phosphorylation and dephosphorylation, occurs in a variety of tissues.

Animals↗
PubMed1974

Adenosine 3',5'-monophosphate content in rat caudate nucleus: demonstration of dopaminergic and adrenergic receptors.

Dopamine, apomorphine, isoproterenol, and norepinephrine each increased the concentration of adenosine 3',5'-monophosphate in slice of rat caudate nucleus. The concentrations of dopamine, apomorphine isoproterenol, and norepinephrine causing half-maximal increases were 60, 150, 0.03 and 30 micromoles per liter, respectively. The effect of dopamine was blocked by fluphenazine, a dopamine receptor antagonist, but not by propranolol, a beta-andrenergic receptor antagonist. Conversely, the effect of isoproterenol was blocked by propranolol but not by fluphenazine. The results suggest that in rat caudate nucleus there are two distinct catecholamine receptors capable of causing increased concentrations of adenosine 3',5'-monophosphate, one having the characteristic of dopamine receptor, and the other having the characteristics of beta-adrenergic receptor.

Animals↗
PubMedDate not supplied

Single sodium channels from rat brain incorporated into planar lipid bilayer membranes.

A voltage- and time-dependent conductance for sodium ions is responsible for the generation of impulses in most nerve and muscle cells. Changes in the sodium conductance are produced by the opening and closing of many discrete transmembrane channels. We present here the first report of electrical recordings from voltage-dependent sodium channels incorporated into planar lipid bilayers. In bilayers with many channels, batrachotoxin (BTX) induced a steady-state sodium current that was blocked by saxitoxin (STX) at nanomolar concentrations. All channels appeared in the bilayer with their STX blocking sites facing the side of vesicle addition, allowing us to define that as the extracellular side. Current fluctuations due to the opening and closing of single BTX-activated sodium channels were voltage-dependent (unit conductance, 30 pS in 0.5 M NaCl): the channels closed at large hyperpolarizing potentials. Slower fluctuations of the same amplitude, due to the blocking and unblocking of individual channels, were seen after addition of STX. Block of the sodium channels by STX was voltage-dependent, with hyperpolarizing potentials favouring block. The voltage-dependent gating, ionic selectivity and neurotoxin sensitivity suggest that these are the channels that normally underlie the sodium conductance change during the nerve impulse.

Animals↗
PubMedDate not supplied

Voltage-dependent calcium channels from brain incorporated into planar lipid bilayers.

Many important physiological processes, including neurotransmitter release and muscle contraction, are regulated by the concentration of Ca2+ ions in the cell. Levels of cytoplasmic Ca2+ can be elevated by the entry of Ca2+ ions through voltage-dependent channels which are selective for Ca2+, Ba2+ and Sr2+ ions. We have measured currents through single, voltage-dependent calcium channels from rat brain that have been incorporated into planar lipid bilayers. Channel gating was voltage-dependent: membrane depolarization increased the channel open times and decreased the closed times. The channels were selective for divalent cations over monovalent ions. The well-known calcium channel blockers, lanthanum and cadmium, produced a concentration-dependent reduction of the apparent single-channel conductance. Contrary to expectations, the nature of the divalent cation carrying current through the channel affected not only the single-channel conductance, but also the channel open times, with mean open times being shortest for barium.

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