PubMed Health⌕ Search

Biomedical subjects

J C Tanaka

Publications and source records attributed to J C Tanaka.

15 recordsLinked to original sources
PubMed1995

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers.

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl. The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Molecular interactions of 3',5'-cyclic purine analogues with the binding site of retinal rod ion channels.

Photoreceptor outer segments transduce information about incoming light levels through a class of ion channels that respond directly to changes in cytosolic 3',5'-cyclic guanosine monophosphate levels. A series of 3',5'-cyclic purine analogues with alterations at N1, C2, C6, or C8 positions was used to examine molecular interactions between the nucleotide and the channel. The maximal current activated by C2-altered analogues in excised membrane patches was less than the current activated by cGMP, and the K0.5, the concentration which activates 50% of the current in a patch, was increased. Nonpolar C8-substituted cAMP analogues activated more current than the parent cAMP with lower K0.5 values. This was in contrast to 8-amino-cAMP, which exhibited greatly reduced activity. The rank order of activity, based on K0.5 values, for C8-cAMP substituents was as follows: 8-azido- > 8-methylamino- > 8-benzylamino- > cAMP > 8-bromo- > 8-hydroxy- >> 8-amino-cAMP. 1,N6-Etheno-cAMP and N6-monobutyryl-cAMP activated a small fraction of the total possible current with high K0.5 values. Other analogues with alterations at N1 or C6 positions including N1-oxide-cAMP, 2-aminopurine riboside 3',5'-monophosphate, and N6-monosuccinyl-cAMP do not bind to the channel, suggesting that interactions with the channel in this region are essential for binding. In order to help interpret the changes in maximal current and K0.5 values compared to cGMP, molecular models of the active analogues were constructed and then docked into a molecular model of the cyclic nucleotide binding site of the retinal channel. This model, proposed by Kumar and Weber [(1992) Biochemistry 31, 4643-4649], was based on the crystal structure of cAMP bound to catabolite activator protein. Our modeling showed that the analogues were sterically accommodated within the binding site. No hydrogen bonds were predicted between the purine rings of cAMP and the pocket; however, Phe 533 on the beta 5 strand was predicted to form weak electrostatic interactions with C6 substituents on both cAMP and cGMP. The importance of contacts in this region of the binding pocket is further emphasized by the inactive analogues, all of which are altered at N1 or C6.

Ambystoma↗
PubMed1993

Divalent effects on cGMP-activated currents in excised patches from amphibian photoreceptors.

The light-sensitive current in photoreceptors is conducted by a single class of ion channels gated by the binding of multiple molecules of cytoplasmic cGMP. Both Na and Ca ions enter the outer segment through this channel and Ca behaves as a blocking ion, greatly reducing the influx of Na. Because intracellular Ca functions as the cytosolic messenger for light adaptation, and this channel is the major entry point for Ca into the outer segment, we seek a better understanding of the selectivity properties of the channel and how they affect intracellular Ca levels. In these studies, we added divalent cations to the cytoplasmic face of an excised patch at constant, symmetrical [Na]. Our results suggest a novel high-affinity divalent binding site at the internal face of the channel. At constant low levels of cGMP, the addition of 10-100 nM cytoplasmic Ca or Mg attenuated the current 5- to 10-fold. There is also a low-affinity site, midway through the transmembrane field; saturation of this site reduces the divalent-free current approximately 100-fold. The presence of a high-affinity cytoplasmic site raises the question of whether Ca regulates the photoreceptor current through a direct interaction with the channel perhaps altering the channel selectivity or kinetics.

Animals↗
PubMed1993

The effects of protons on 3',5'-cGMP-activated currents in photoreceptor patches.

Macroscopic 3',5'-guanine cyclic monophosphate (cGMP)-activated currents from photoreceptor outer segment membranes were examined as the pH on the cytoplasmic face of inside/out patches was reduced. In the absence of divalent cations, protons reduced the current in both directions without affecting the shape of the current-voltage relation consistent with a voltage-independent block. When Ca2+ was added to the bath, increasing the [H+] relieved the Ca2+ block and eliminated the Ca(2+)-induced reversal potential shifts seen at pH 7.4. These results suggest that protons alter Na+/Ca2+ permeability of the channel and relieve Ca2+ block of the sodium transport.

Animals↗
PubMed1990

Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel.

Monovalent cation selectivity has been characterized for the 3',5'-cyclic guanosine monophosphate (cGMP)-activated channel in vertebrate photoreceptor outer segment plasma membranes without divalent cations. Macroscopic currents in excised, inside-out patches were activated with saturating concentrations of cGMP (200 microM). Using a bi-ionic protocol with symmetrical 120 mM ion concentrations across the membrane, alkali metal ions and certain organic cations were substituted for sodium on the cytoplasmic face. The relative permeabilities, determined from shifts in the reversal potential (Erev), were NH4 much greater than Na greater than guanidinium greater than K greater than Li greater than Rb greater than Cs (3.34: 1.0: 0.97: 0.93: 0.92: 0.74: 0.50, respectively). Erev's were also measured as a function of [Na], [NH4], and [Cs], and the slope of the relation was -59.8, -52.1, and -49.1 mV/decade, respectively. The slopes for NH4 and Cs differ significantly from the Nernst-Planck prediction of -58.2 mV/decade expected for a single ion channel. Relative permeabilities were also determined for the alkali metal series of ions with 20 mM ionic concentrations on both sides of the membrane. The permeability sequence at 20 mM was unchanged, but the relative permeability for NH4 and Cs deviated significantly from the measurements at 120 mM with 1.46 and 0.75 ratios, respectively. The dependence of Erev on absolute concentrations and the deviation from Nernst-Planck predictions are best explained by multi-ion occupancy of the cGMP-activated channel. Selectivity was also examined by comparing the conductance ratios as a function of potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Photoreceptor channel activation by nucleotide derivatives.

Cyclic nucleotide activated sodium currents were recorded from photoreceptor outer segment membrane patches. The concentration of cGMP and structurally similar nucleotide derivatives was varied at the cytoplasmic membrane face; currents were generated at each concentration by the application of a voltage ramp. Nucleotide-activated currents were analyzed as a function of both concentration and membrane potential. For cGMP, the average K0.5 at 0 mV was 24 microM, and the activation was cooperative with an average Hill coefficient of 2.3. Of the nucleotide derivatives examined, only 8-[[(fluorescein-5-yl-carbamoyl)methyl]thio]-cGMP (8-Fl-cGMP) activated the channel at lower concentrations than cGMP with a K0.5 of 0.85 microM. The next most active derivative was 2-amino-6-mercaptopurine riboside 3',5'-monophosphate (6-SH-cGMP) which had a K0.5 of 81 microM. cIMP and cAMP had very high K0.5 values of approximately 1.2 mM and greater than 1.5 mM, respectively. All nucleotides displayed cooperativity in their response and were rapidly reversible. Maximal current for each derivative was compared to the current produced at 200 microM cGMP; only 8-Fl-cGMP produced an identical current. The partial agonists 6-SH-cGMP, cIMP, and cAMP activated currents which were approximately 90%, 80%, and 25% of the cGMP response, respectively. 5'-GMP, 2-aminopurine riboside 3',5'-monophosphate, and 2'-deoxy-cGMP produced no detectable current. The K0.5 values for cGMP activation, examined from -90 to +90 mV, displayed a weak voltage dependence of approximately 400 mV/e-fold; the index of cooperativity was independent of the applied field.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Photoreceptor channel activation: interaction between cAMP and cGMP.

cAMP activates a current in excised patches from rod outer segments. The current at saturating concentrations of cAMP is approximately 25% of the current activated with 200 microM cGMP, the terminal cytoplasmic messenger in phototransduction. The K0.5 for cAMP is greater than 1.5 mM, and the index of cooperativity is approximately 1.4. cAMP activates the same population of channels as activated by cGMP since currents in the presence of both nucleotides are less than the sum of the individual responses. When increasing concentrations of cAMP, less than its K0.5, are added to a fixed, subsaturating concentration of cGMP, cAMP significantly enhances the total current compared with the current produced by cGMP alone. These results are predicted by a three-site, linear, sequential binding scheme where either cAMP or cGMP may bind to the same site on the channel. At approximately 5 microM cGMP, which is estimated to be the steady-state dark level in vertebrate photoreceptors, cAMP between 1 and 100 microM produces a large increase in the photoreceptor current. A possible physiological role for cAMP-cGMP interaction in phototransduction is discussed.

Animals↗
PubMed1988

Patch electrode glass composition affects ion channel currents.

The influence of patch electrode glass composition on macroscopic IV relations in inside-out patches of the cGMP-activated ion channel from rod photoreceptors was examined for a soda lime glass, a Kovar sealing glass, a borosilicate glass, and several soft lead glasses. In several glasses the shape or magnitude of the currents changed as the concentration of EGTA or EDTA was increased from 200 microM to 10 mM. The changes in IV response suggest that, at low concentrations of chelator, divalent cations are released from the electrode glass and interact with the cGMP-activated channel. Possible mechanisms are discussed to explain the observations, and several comments are made concerning the choice of a glass for patching.

Animals↗
PubMed1987

Incorporation of a retinal rod cGMP-dependent conductance into planar bilayers.

The light-modulated current of vertebrate retinal rods flows through a 3',5'-cyclic GMP-dependent conductance located in the outer segment plasma membrane. We report the incorporation into planar bilayers of a conductance derived from vertebrate rod outer segment membranes specifically activated by cGMP but not by cAMP, 5'-GMP, GTP, or 5'-AMP. When the mean currents were measured as a function of increasing cGMP concentration, maximal activation occurred at concentrations less than 50 microM. Washout of cGMP rapidly reversed the effect. The apparent half-saturating concentrations were between 12 and 27 microM. Sodium, lithium, cesium, and potassium supported current in the presence of low concentrations of Ca2+, Mg2+, and 100 microM cGMP; choline did not. Removal of the divalent cations reversibly increased the currents. When calcium was the only current-carrying cation, attenuated currents were seen. These experiments support the hypothesis that calcium is a permeant blocker of the conductance. At low concentrations of cGMP in solutions also containing 0.5 mM EDTA, brief current spikes occurred with amplitudes from 0.5 to 4 pA at 50 mV. These spikes differed from the well-defined, unitary conductance steps usually associated with the opening and closing of ion channels. Occasionally we saw longer-lasting channel-like events; however, amplitude histograms did not resolve discrete conductance levels.

Adenosine Monophosphate↗
PubMed1986

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

We have examined the voltage-dependent gating of batrachotoxin-modified sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and single-channel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with half-maximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance (500 mM Na+ cis, 200 mM Na+ trans) averaged 20 pS and was independent of membrane potential. Channels usually inserted with their extracellular faces on the trans side of the bilayer; addition of tetrodotoxin to the cis side had no effect, whereas addition to the trans side blocked greater than 95% of channel openings at -77 mV. A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphatidylcholine vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K+ gradients in the presence of valinomycin. After batrachotoxin activation, Vm was altered by shifts of K+o. All of the specific 22Na+ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified T-tubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltage-dependent activation of the batrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure to this toxin. Most importantly, this voltage-dependent gating is a property of all of the purified channels capable of specific pharmacological activation.

Animals↗
PubMed1986

Developmental appearance of sodium channel subtypes in rat skeletal muscle cultures.

22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K = 0.6-5.0 X 10(-8) M) and an insensitive population (Ki = 3.3-4.9 X 10(-6) M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X 10(-7)M). A single population was also found in 6-day-old cultures (Ki = 5.3 X 10(-7) M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki = 1.3 X 10(-9) M and 9.6 X 10(-7) M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.

Animals↗
PubMed1985

Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes.

The voltage-sensitive sodium channel has been purified from rabbit T-tubular membranes and reconstituted into defined phospholipid vesicles. Membranes enriched in T-tubular elements (specific [3H]nitrendipine binding = 41 +/- 9 pmol/mg of protein, n = 7) were isolated from fast skeletal muscle. After solubilization with Nonidet P-40, the sodium channel protein was purified to greater than 95% of theoretical homogeneity based on the specific activity of [3H]saxitoxin binding. Two subunits of Mr approximately 260,000 and 38,000 were found; these bands co-distributed with the peak of [3H]saxitoxin binding on sucrose gradients. The purified protein was reconstituted into egg phosphatidylcholine vesicles and retained the ability to gate specific 22Na+ influx in response to activation by batrachotoxin or veratridine. All activated fluxes were blocked by saxitoxin and tetrodotoxin. On sucrose gradients, the distribution of protein capable of functional channel activity paralleled the distribution of specific [3H]saxitoxin binding and of the Mr 260,000 and 38,000 components. The cation selectivity for the reconstituted, batrachotoxin-activated channel was Na+ greater than K+ greater than Rb+ greater than Cs+, with flux ratios of 1:0.13:0.02:0.008. Nine of 25 monoclonal antibodies raised against the rat sarcolemmal sodium channel cross-reacted with the rabbit T-tubular sodium channel in a solid-phase radioimmunoassay. Six of these antibodies showed specific binding to immunoblot transfers of T-tubular membrane proteins. Each labeled a single band at Mr approximately 260,000 corresponding in mobility to the large subunit of the sodium channel.

Animals↗
PubMed1984

Sodium channels in vertebrate hearts. Three types of saxitoxin binding sites in heart.

The affinity of saxitoxin binding to cardiac sarcolemmal and cytosolic fractions was examined across species. In amphibia (frog) the plasma membrane site demonstrated a high affinity (Kd approx. 5 X 10(-9) M) but the majority of the total sites in the homogenate appeared to be high affinity soluble sites (Kd approx. 2 X 10(-9) M). Chicken and turtle cardiac plasma membrane fractions bound [3H]saxitoxin with 500-fold less affinity (Kd values of approx. 2 X 10(-6) M). No binding was seen in the cytosol. The affinity of cardiac sarcolemmal binding in amphibians correlates quantitatively with the K0.5 for the inhibition of sodium currents. Physiological correlation of the low affinity saxitoxin sites in chicken and turtle with toxin concentrations necessary to inhibit the sodium current remains unclear. The hypothesis that frog cytosolic saxitoxin binding sites originated from sarcolemma during homogenization is examined. The presence of three types of saxitoxin binding sites in cardiac preparations supports the existence of sodium channel subtypes.

Animals↗
PubMed1983

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma.

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.

Animals↗