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R J French

Publications and source records attributed to R J French.

At least 19 recordsLinked to original sources
PubMed1996

Identification and properties of an ATP-sensitive K+ current in rabbit sino-atrial node pacemaker cells.

1. Single myocytes were isolated from rabbit sino-atrial (SA) node by enzymatic dissociation. Spontaneous pacemaker activity, whole-cell and single-channel currents were recorded under conditions known to modulate ATP-sensitive K+ (KATP) channels. 2. The KATP channel openers, cromakalim and pinacidil, slowed or abolished the pacemaker activity, and caused hyperpolarization of the maximum diastolic potential (MDP). Glibenclamide, a KATP channel blocker, reversed these effects. Cromakalim- and pinacidil-activated currents reversed near the potassium equilibrium potential, EK. Glibenclamide had no effect on the L-type calcium current, ICa(L), the hyperpolarization-activated inward current, If, or the delayed rectifier potassium current, IK. 3. Sodium cyanide, which inhibits mitochondrial ATP production, induced a macroscopic current that reversed near EK and was blocked by glibenclamide. 4. In excised, inside-out patches from SA node cells, single KATP channels showed a slope conductance of 52 +/- 8 pS (mean +/- S.D.) when measurements were made at negative voltages in symmetric, 140 mM K+. Channels from ventricular myocytes showed a somewhat larger slope conductance (70 +/- 5 pS). 5. Raising the intracellular ATP concentration caused a concentration-dependent reduction in the open probability of the KATP channels (IC50, 16 microM; Hill coefficient, approximately 1; at both pH 7.4 and 6.8). 6. In excised inside-out patches, cromakalim or pinacidil induced significant increases in KATP channel activity in the presence of 50 microM or 1 mM intracellular ATP. This channel activity was blocked by glibenclamide. 7. Our results suggest that sino-atrial node cells express a distinct isoform of KATP channel which may play an important role in pharmacological and pathophysiological modulation of pacemaker activity.

Action Potentials↗
PubMed1996

Interactions between a pore-blocking peptide and the voltage sensor of the sodium channel: an electrostatic approach to channel geometry.

Few experimental data illuminate the relationship between the molecular structures that mediate ion conduction through voltage-dependent ion channels and the structures responsible for sensing transmembrane voltage and controlling gating. To fill this void, we have used a strongly cationic, mutated mu-conotoxin peptide, which only partially blocks current through voltage-dependent sodium channels, to study voltage-dependent activation gating in both bound and unbound channels. When the peptide binds to the ion-conducting pore, it inhibit channel opening, necessitating stronger depolarization for channel activation. We show that this activation shift could result entirely from electrostatic inhibition of the movement of the voltage-sensing S4 charges and estimate the approximate physical distance through which the S4 charges move.

Animals↗
PubMed1996

Protein kinase C-induced changes in the stoichiometry of ATP binding activate cardiac ATP-sensitive K+ channels. A possible mechanistic link to ischemic preconditioning.

Activation of both ATP-sensitive K+ (KATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (< or = 50 mumol/L), PKC inhibits KATP channel activity. This finding is surprising, as both KATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximately 2 to approximately 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATP channels at millimolar levels of ATP. The effects of PKC on single KATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on KATP channel properties. KATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKC's action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in KATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates KATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.

Adenosine Triphosphate↗
PubMed1995

Regulation of adenosine triphosphate-sensitive potassium channels from rabbit ventricular myocytes by protein kinase C and type 2A protein phosphatase.

Myocytes from rabbit ventricle were enzymatically dissociated and the effects of protein kinase C (PKC) on the properties of single ATP-sensitive (KATP) channels were studied using excised inside-out membrane patches. Application of a purified, constitutively active form of PKC (20 nM) to the intracellular surface of inside-out patches caused a 48% +/- 4% (n = 18) reduction in the open probability of single KATP channels. In the presence of the PKC inhibitors peptide PKC(19-31) or chelerythrine chloride, PKC had no effect on KATP channel properties. Heat-inactivated PKC had no effect on channel properties. KATP channel activity returned spontaneously after removal of PKC. However, application of okadaic acid, at a concentration (5 nM) appropriate for specific inhibition of type 2A protein phosphatase (PP-2A), after removal of PKC, prevented spontaneous recovery of channel activity. Treatment with purified PP-2A during the PKC-mediated inhibition of KATP channel activity caused a partial or full restoration of activity. The Hill coefficient for ATP binding was reduced from 2.2 (control) to 1.2 in the presence of PKC. The apparent inhibition constant (Ki) for ATP was unaffected by PKC [Ki(control) = 21 microM; Ki(PKC) = 20 microM]. PKC is, therefore, capable of inhibiting cardiac KATP channel activity, and the extent to which the channels remain phosphorylated appears to be dependent on membrane-associated PP-2A activity. These enzymes may, therefore, be involved in signal transduction mechanisms which serve to regulate the activity of cardiac KATP channels.

Adenosine Triphosphate↗
PubMed1995

Sodium current inhibition by internal calcium: a combination of open-channel block and surface charge screening?

Internal application of millimolar concentrations of calcium to batrachotoxin (BTX)-activated rat skeletal muscle sodium channels, bathed symmetrically in 200 mM NaCl, causes a reduction in apparent single-channel amplitude without visibly increasing noise at a bandwidth of 50 Hz. A greater calcium-induced reduction occurred upon removal of external sodium ions. Internal calcium acted similarly in high ionic strength solutions (3M NaCl), where surface charges are effectively screened, suggesting that calcium acts, in part, by binding within the pore and occluding the conducting pathway. In low ionic strength solutions (20 mM NaCl), internal addition of N-Methyl-Glucamine (NMG) ions decreased the single channel amplitude consistent with screening of negative surface charges. An accurate description of the dose dependence of calcium inhibition, using either a simple blocking model, or rate theory calculations of ion permeation and block, also required surface charge screening. Hence, our data support the view that sodium current inhibition by internal calcium arises from a combination of both open-channel block and surface charge effects.

Animals↗
PubMed1994

Glibenclamide selectively blocks ATP-sensitive K+ channels reconstituted from skeletal muscle.

Glibenclamide, a blocker of ATP-sensitive K+ (KATP) channels, was tested on three different types of rat skeletal K+ channels incorporated into bilayers. Glibenclamide (10 microM) blocked a class of KATP channels (unitary conductance of 57 pS in symmetric 150 mM KCl), which were inhibited by ATP. High concentrations of glibenclamide (100 microM) had no effect on either voltage-gated K+ channels (37 pS), or Ca(2+)-activated K+ channels (210 pS). Our results show that glibenclamide, even at high concentrations (100 microM) that may be required for quick action in whole muscle experiments, is a selective and specific blocker of skeletal KATP channels.

Adenosine Triphosphate↗
PubMed1994

Amine blockers of the cytoplasmic mouth of sodium channels: a small structural change can abolish voltage dependence.

Many drugs block sodium channels from the cytoplasmic end (Moczydlowski, E., A. Uehara, X, Guo, and J. Heiny. 1986. Isochannels and blocking modes of voltage-dependent sodium channels. Ann. N.Y. Acad. Sci. 479:269-292.). Lidocaine, applied to either side of the membrane, induces two blocking modes, a rapid, voltage-dependent open-channel block, and a block of the inactivated channel that occurs on a 1000-fold slower timescale. Here we describe the actions of several lidocaine-related amines on batrachotoxin(BTX)-activated bovine cardiac sodium channels incorporated into planar lipid bilayers. We applied blocking amines from the intracellular side and examined the structural determinants of fast, open-channel block. Neither hydroxyl nor carbonyl groups, present in the aryl-amine link of lidocaine, were necessary, indicating that hydrogen bonding between structures in the aryl-amine link and the channel is not required. Block, however, was significantly enhanced by addition of an aromatic ring, or by the lengthening of aliphatic side chains, suggesting that a hydrophobic domain strengthens binding while the amine group blocks the pore. For most blockers, depolarizing potentials enhanced block, with the charged amine group apparently traversing 45-60% of the transmembrane voltage. By contrast, block by phenylhydrazine was essentially voltage-independent. The relatively rigid planar structure of phenylhydrazine may prevent the charged amino end from entering the electric field when the aromatic ring is bound. The relation between structural features of different blockers and their sensitivity to voltage suggests that the transmembrane voltage drops completely over less than 5 A. We raise the possibility that the proposed hydrophobic binding domain overlaps the endogenous receptor for the inactivation gate. If so, our data place limits on the distance between this receptor and the intrapore site at which charged amines bind.

Amines↗
PubMed1994

Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.

Animals↗
PubMed1994

Open-channel block by internally applied amines inhibits activation gate closure in batrachotoxin-activated sodium channels.

We have studied the action of several pore-blocking amines on voltage-dependent activation gating of batrachotoxin(BTX)-activated sodium channels, from bovine heart and rat skeletal muscle, incorporated into planar lipid bilayers. Although structurally simpler, the compounds studied show general structural features and channel-inhibiting actions that resemble those of lidocaine. When applied to the cytoplasmic end of the channel, these compounds cause a rapid, voltage-dependent, open-channel block seen as a reduction in apparent single-channel amplitude (companion paper). Internal application of phenylpropanolamine, phenylethylamine, phenylmethylamine, and diethylamine, as well as causing open-channel block, reduces the probability of channel closure, producing a shift of the steady-state activation curve toward more hyperpolarizing potentials. These gating effects were observed for both cardiac and skeletal muscle channels and were not evoked by addition of equimolar N-Methyl-D-Glucamine, suggesting a specific interaction of the blockers with the channel rather than a surface charge effect. Kinetic analysis of phenylpropanolamine action on skeletal muscle channels indicated that phenylpropanolamine reduced the closed probability via two separate mechanisms. First, mean closed durations were slightly abbreviated in its presence. Second, and more important, the frequency of the gating closures was reduced. This action was correlated with the degree, and the voltage dependence, of open-channel block, suggesting that the activation gate cannot close while the pore is occluded by the blocker. Such a mechanism might underlie the previously reported immobilization of gating charge associated with local anesthetic block of unmodified sodium channels.

Amines↗
PubMed1994

Ion permeation, divalent ion block, and chemical modification of single sodium channels. Description by single- and double-occupancy rate-theory models.

Calcium ions, applied internally, externally, or symmetrically, have been used in conjunction with rate-theory modeling to explore the energy profile of the ion-conducting pore of sodium channels. The block, by extracellular and/or intracellular calcium, of sodium ion conduction through single, batrachotoxin-activated sodium channels from rat brain was studied in planar lipid bilayers. Extracellular calcium caused a reduction of inward current that was enhanced by hyperpolarization and a weaker block of outward current. Intracellular calcium reduced both outward and inward sodium current, with the block being weakly dependent on voltage and enhanced by depolarization. These results, together with the dependence of single-channel conductance on sodium concentration, and the effects of symmetrically applied calcium, were described using single- or double-occupancy, three-barrier, two-site (3B2S), or single-occupancy, 4B3S rate-theory models. There appear to be distinct outer and inner regions of the channel, easily accessed by external or internal calcium respectively, separated by a rate-limiting barrier to calcium permeation. Most of the data could be well fit by each of the models. Reducing the ion interaction energies sufficiently to allow a small but significant probability of two-ion occupancy in the 3B2S model yielded better overall fits than for either 3B2S or 4B3S models constrained to single occupancy. The outer ion-binding site of the model may represent a section of the pore in which sodium, calcium, and guanidinium toxins, such as saxitoxin or tetrodotoxin, compete. Under physiological conditions, with millimolar calcium externally, and high potassium internally, the model channels are occupied by calcium or potassium much of the time, causing a significant reduction in single-channel conductance from the value measured with sodium as the only cation species present. Sodium conductance and degree of block by external calcium are reduced by modification of single channels with the carboxyl reagent, trimethyloxonium (TMO) (Worley et al., 1986) Journal of General Physiology. 87:327-349). Elevations of only the outermost parts of the energy profiles for sodium and calcium were sufficient to account for the reductions in conductance and in efficacy of calcium block produced by TMO modification.

Animals↗
PubMed1994

Cation fluxes and cation channels in outer segment membranes of bovine retinal rods: contamination by antibiotics applied to cattle?

Membrane vesicles were prepared from intact rod outer segments (ROSs) isolated from bovine retinas and were examined for the presence of cation-selective conductances. We performed macroscopic flux measurements in an ensemble of ROS membrane vesicles and single-channel measurements after fusion of ROS membrane vesicles with planar bilayer membranes. Two K(+)-permeable conductances were observed, the well-established cyclic GMP (cGMP) gated channel and an apparently new K+ channel with some unusual properties. Flux and single-channel data showed that the new conductance passed K+, Rb+, and Cs+ equally well but was much less permeable to Na+, Li+ and protons. Single-channel measurements revealed a linear current-voltage relationship and three unitary conductance states of 15, 11, and 8 pS, using symmetric 150 mM KCl solutions. Measured macroscopic K+ fluxes varied considerably among different preparations, suggesting some unknown regulation of the channel; the variability appeared to arise from variation in the channel's open probability, not the unit conductance or the number of channels present. The recorded single-channel events and the selectivity data are remarkably similar to those reported for antibiotic channel-forming ionophore gramicidin. We believe that the variability in both macroscopic permeability experiments and single-channel experiments may reflect a variable contamination with gramicidin applied to the animals as the topical antibiotic V-Sporin.

Animals↗
PubMed1993

Role of vanadium in nutrition: metabolism, essentiality and dietary considerations.

Vanadium is a pervasive element of biological systems, being widely distributed across the food supply. Food refining and processing appear to increase vanadium content. At higher intakes, it accumulates in body tissues such as liver, kidney and bone. Essentiality of the nutrient has been established in lower life forms but the significance and extent of vanadium's role in humans has been overshadowed by the absence of deficiency symptoms in man. While the pharmacological properties of vanadium have stimulated much interest, knowledge of basic metabolic processes regulating vanadium remains incomplete. Ultimate determination of essentiality for humans will depend on greater understanding of the fundamental biochemical roles of vanadium.

Animal Nutritional Physiological Phenomena↗
PubMed1993

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.

Animals↗
PubMed1993

State-dependent block underlies the tissue specificity of lidocaine action on batrachotoxin-activated cardiac sodium channels.

We have identified two kinetically distinct modes of block, by lidocaine, of cardiac sodium channels, activated by batrachotoxin and incorporated into planar lipid bilayers. Here, we analyze the slow blocking mode which appears as a series of nonconducting events that increase in frequency and duration with increasing lidocaine concentrations. This type of block occurred rarely, if at all, for the skeletal muscle sodium channel subtype. Kinetic analysis showed that a linear open-closed-blocked model is sufficient to account for the major features of our data. Slow block occurs from a long closed state that is a distinguishing characteristic of cardiac channels under these conditions. Slow block showed no significant voltage dependence in the range of -60 to -20 mV for which the detailed kinetic analysis was performed, and was not elicited by application of the permanently charged lidocaine derivative QX-314. By contrast, the fast block, described in the companion paper, results from drug binding to the open state, and is similar for cardiac and skeletal muscle sodium channels. Application of trypsin to the cytoplasmic end of the channel eliminates both the spontaneous, long, gating closures and slow block. Thus, the lidocaine-sensitive closed state of batrachotoxin-activated cardiac sodium channels exhibits a protease susceptibility resembling that of the inactivated state of unmodified sodium channels. It is the slow block caused by lidocaine binding to this closed state that underlies the channel-subtype specificity of lidocaine action in our experiments.

Animals↗
PubMed1993

Dual actions of procainamide on batrachotoxin-activated sodium channels: open channel block and prevention of inactivation.

We have investigated the action of procainamide on batrachotoxin (BTX)-activated sodium channels from bovine heart and rat skeletal muscle. When applied to the intracellular side, procainamide induced rapid, open-channel block. We estimated rate constants using amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157). Membrane depolarization increased the blocking rate and slowed unblock. The rate constants were similar in both magnitude and voltage dependence for cardiac and skeletal muscle channels. Qualitatively, this block resembled the fast open-channel block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80), but procainamide was about sevenfold less potent. Molecular modeling suggests that the difference in potency between procainamide and lidocaine might arise from the relative orientation of their aromatic rings, or from differences in the structure of the aryl-amine link. For the cardiac channels, procainamide reduced the frequency of transitions to a long-lived closed state which shows features characteristic of inactivation (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys J. 65:91). Mean durations of kinetically identified closed states were not affected. The degree of fast block and of inhibition of the slow closures were correlated. Internally applied QX-314, a lidocaine derivative and also a fast blocker, produced a similar effect. Thus, drug binding to the fast blocking site appears to inhibit inactivation in BTX-activated cardiac channels.

Animals↗
PubMed1993

Dissecting lidocaine action: diethylamide and phenol mimic separate modes of lidocaine block of sodium channels from heart and skeletal muscle.

We have investigated block of sodium channels by diethylamide and phenol, which resemble the hydrophilic tertiary amine head and the hydrophobic aromatic tail of the lidocaine molecule, respectively. Diethylamide and phenol separately mimicked the fast and slow modes of block caused by lidocaine. Experiments were performed using single batrachotoxin-activated bovine cardiac and rat skeletal muscle sodium channels incorporated into neutral planar lipid bilayers. Diethylamide, only from the intracellular side, caused a voltage-dependent reduction in apparent single channel amplitude ('fast' block). Block was similar for cardiac and skeletal muscle channels, and increased in potency when extracellular sodium was replaced by N-methylglucamine, consistent with an intrapore blocking site. Thus, although occurring at 15-fold higher concentrations, block by diethylamide closely resembles the fast mode of block by lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:80-90). For cardiac sodium channels, phenol bound to a closed state causing the appearance of long blocked events whose duration increased with phenol concentration. This slow block depended neither on voltage nor on the side of application, and disappeared upon treatment of the channel with trypsin. For skeletal muscle channels, slow phenol block occurred with only very low probability. Thus, phenol block resembles the slow mode of block observed for lidocaine (Zamponi, G. W., D. D. Doyle, and R. J. French. 1993. Biophys. J. 65:91-100). Our data suggest that there are separate sites for fast lidocaine block of the open channel and slow block of the "inactivated" channel. Fast block by diethylamide inhibited the long, spontaneous, trypsin-sensitive (inactivation-like) closures of cardiac channels, and hence secondarily antagonized slow block by phenol or lidocaine. This antagonism would potentiate shifts in the balance between the two modes of action of a tertiary amine drug caused by changes in the relative concentrations of the charged (fast blocking) and neutral (slow blocking) forms of the drug.

Animals↗
PubMed1992

Action of derivatives of mu-conotoxin GIIIA on sodium channels. Single amino acid substitutions in the toxin separately affect association and dissociation rates.

We have studied binding and block of sodium channels by 12 derivatives of the 22-residue peptide mu-conotoxin GIIIA (mu-CTX) in which single amino acids were substituted as follows: Arg or Lys by Gln, Gln-18 by Lys, Asp by Asn, and HO-Pro by Pro. Derivatives were synthesized as described by Becker et al. [(1989) Eur. J. Biochem. 185, 79]. Binding was measured by displacement of labeled saxitoxin from eel electroplax membranes (100 mM choline chloride, 10 mM HEPES-NaOH, pH 7.4). Blocking kinetics were evaluated from steady-state, single-channel recordings from rat skeletal muscle sodium channels incorporated into planar, neutral phospholipid/decane bilayers (200 mM NaCl, 10 mM HEPES-NaOH, pH 7.0). Blocking events generally appeared as periods of seconds to minutes in which current through the single channel was completely eliminated. A notable exception was seen for the substitution Arg-13-Gln for which the "blocked" events showed measurable conductances of about 20-40% of the open state. The substitution of Arg-13 reduced binding to electroplax membranes to undetectable levels and increased the apparent dissociation constant determined for skeletal muscle channels by greater than 80-fold compared with the native peptide. Other substitutions caused smaller decreases in affinity. The decreased potency of the toxin derivatives resulted both from increases in the rates of dissociation from the channel, and from decreases in association rates. Our data support the suggestion by Sato et al. [(1991) J. Biol. Chem. 265, 16989] that Arg-13 associates intimately with the binding site on the channel. In addition, our results suggest that certain residues affect almost exclusively the approach and docking of the toxin with its binding site, others appear to be important only to the strength of the association once binding has taken place, and yet others affect both.

Amino Acid Sequence↗