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R J French

Publications and source records attributed to R J French.

At least 55 records · Page 3Linked to original sources
PubMed1991

Ion channels in transit: voltage-gated Na and K channels in axoplasmic organelles of the squid Loligo pealei.

Ion channels that give rise to the excitable properties of the neuronal plasma membrane are synthesized, transported, and degraded in cytoplasmic organelles. To determine whether plasma membrane ion channels from these organelles could be physiologically activated, we extruded axoplasm from squid giant axons, dissociated organelles from the cytoskeletal matrix, and fused the free organelles with planar lipid bilayers. Three classes of ion channels normally associated with the plasma membrane were identified based on conductance, selectivity, and gating properties determined from steady-state single-channel recordings: (i) voltage-dependent Na channels, (ii) voltage-dependent delayed rectifier K channels, and (iii) large, voltage-independent K channels. The identity of the delayed rectifier channels was confirmed by reconstructing the time course of activation from single-channel responses to depolarizing voltage steps applied across the bilayer. These observations suggest that several classes of plasma membrane ion channels are transported in cytoplasmic organelles in physiologically active forms.

Animals↗
PubMed1990

Optimizing planar lipid bilayer single-channel recordings for high resolution with rapid voltage steps.

We describe two enhancements of the planar bilayer recording method which enable low-noise recordings of single-channel currents activated by voltage steps in planar bilayers formed on apertures in partitions separating two open chambers. First, we have refined a simple and effective procedure for making small bilayer apertures (25-80 micrograms diam) in plastic cups. These apertures combine the favorable properties of very thin edges, good mechanical strength, and low stray capacitance. In addition to enabling formation of small, low-capacitance bilayers, this aperture design also minimizes the access resistance to the bilayer, thereby improving the low-noise performance. Second, we have used a patch-clamp headstage modified to provide logic-controlled switching between a high-gain (50 G omega) feedback resistor for high-resolution recording and a low-gain (50 M omega) feedback resistor for rapid charging of the bilayer capacitance. The gain is switched from high to low before a voltage step and then back to high gain 25 microseconds after the step. With digital subtraction of the residual currents produced by the gain switching and electrostrictive changes in bilayer capacitance, we can achieve a steady current baseline within 1 ms after the voltage step. These enhancements broaden the range of experimental applications for the planar bilayer method by combining the high resolution previously attained only with small bilayers formed on pipette tips with the flexibility of experimental design possible with planar bilayers in open chambers. We illustrate application of these methods with recordings of the voltage-step activation of a voltage-gated potassium channel.

Animals↗
PubMed1988

Effects of membrane surface charge and calcium on the gating of rat brain sodium channels in planar bilayers.

The voltage-dependent gating of single, batrachotoxin-activated Na channels from rat brain was studied in planar lipid bilayers composed of negatively charged or neutral phospholipids. The relationship between the probability of finding the Na channel in the open state and the membrane potential (Po vs. Vm) was determined in symmetrical NaCl, both in the absence of free Ca2+ and after the addition of Ca2+ to the extracellular side of the channel, the intracellular side, or both. In the absence of Ca2+, neither the midpoint (V0.5) of the Po vs. Vm relation, nor the steepness of the gating curve, was affected by the charge on the bilayer lipid. The addition of 7.5 mM Ca2+ to the external side caused a depolarizing shift in V0.5. This depolarizing shift was approximately 17 mV in neutral bilayers and approximately 25 mV in negatively charged bilayers. The addition of the same concentration of Ca2+ to only the intracellular side caused hyperpolarizing shifts in V0.5 of approximately 7 mV (neutral bilayers) and approximately 14 mV (negatively charged bilayers). The symmetrical addition of Ca2+ caused a small depolarizing shift in Po vs. Vm. We conclude that: (a) the Na channel protein possesses negatively charged groups on both its inner and outer surfaces. Charges on both surfaces affect channel gating but those on the outer surface exert a stronger influence. (b) Negative surface charges on the membrane phospholipid are close enough to the channel's gating machinery to substantially affect its operation. Charges on the inner and outer surfaces of the membrane lipid affect gating symmetrically. (c) Effects on steady-state Na channel activation are consistent with a simple superposition of contributions to the local electrostatic potential from charges on the channel protein and the membrane lipid.

Animals↗
PubMed1986

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium.

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.

Animals↗
PubMed1986

Block of sodium channels in planar lipid bilayers by guanidium toxins and calcium. Are the mechanisms of voltage dependence the same?

The block of single, batrachotoxin-activated sodium channels by saxitoxin (STX), tetrodotoxin (TTX), and Ca2+ has been investigated in planar bilayers. All three substances block in a voltage-dependent manner with hyperpolarizing potentials favoring block. Extracellular Ca2+ competitively inhibits binding of STX and relieves STX block. Trimethyloxonium, a carboxyl-methylating agent, eliminates block by STX and TTX and dramatically reduces block by Ca2+. These results suggest that STX, TTX, and Ca2+ compete for a negative site on the outside of the channel. The voltage dependence of block by STX (divalent cation) and TTX (monovalent) was similar (40 mV/e-fold), suggesting that voltage dependence is due to a conformational change in the channel rather than to the toxins entering the membrane electric field to block. A physical model, with an external binding site for toxins and Ca2+ and another site deeper within the electric field (associated with the "selectivity filter") that is accessible to Ca2+ but not toxins, predicts voltage-dependence Ca2+ block without invoking the conformational change needed to explain the voltage dependence of block by TTX and STX.

Animals↗
PubMed1985

An ion's view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions.

We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two.

Animals↗
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.

Animals↗
PubMed1981

Blockage of squid axon potassium conductance by internal tetra-N-alkylammonium ions of various sizes.

We have studied the effects of the tetra-n-alkylammonium (TAA) ions, (CnH2n+1)4N+, n = 1-6, on the potassium conductance of voltage-clamped squid giant axons. Studies using tetrahexylammonium were not quantitatively analyzed as its effect was insufficiently reversible. Each in this series of symmetric ions of graded size blocks the potassium conductance when added to the internal perfusion fluid. There is a general trend for blocking potency to increase with increasing size. We attribute this to stronger interactions of the longer alkyl side chains with hydrophobic regions of the membrane near the channels. Steady-state block by the TAA ions, n = 2-5, showed identical voltage dependence, apparently sensing about 15% of the transmembrane voltage, and kinetics block onset were qualitatively similar. We conclude that the site of action for these ions is the same. Block by TMA is about twice as steeply dependent on voltage. In its action, TMA resembles the alkali cations (French et al., 1979, Biophys, J. 25(2, pt. 2):307a) more than the larger TAA ions. Our results suggest that access to the inner mouth of the K channel is even less restricted than has been previously thought. A calculation indicates that the lumen of the channel cannot be both wide enough to admit the TAA ions and long enough to account for the voltage dependence of block. We consider possible ways to resolve this paradox.

Animals↗
PubMed1981

Receptor mechanisms for 5-hydroxytryptamine in rabbit arteries.

1 Previous investigations into the vascular actions of biogenic amines implicated in migraine have shown that the contractile effects of both 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rabbit ear artery are mediated by a direct sympathomimetic action at alpha-adrenoceptors, while in the rabbit aorta, 5-HT and NA act on pharmacologically distinct receptors. The purpose of the present investigation was to determine whether the absence of 5-HT receptors in rabbit ear arteries is characteristic of distributing arteries in general, or is confined to particular regional circulations.2 Agonist-antagonist interactions were studied in various rabbit vascular preparations (common carotid, external carotid and femoral arterial strips, and perfused ear arteries) by determining pA(2) values for pizotifen and phentolamine against 5-HT- and NA-induced contractile responses.3 In common carotid and femoral arteries, pizotifen was a potent competitive antagonist of 5-HT, but weak against NA. The converse applied to phentolamine. In external carotid and ear arteries, pizotifen was a weak competitive antagonist of both 5-HT and NA, whereas phentolamine was a potent competitive antagonist of both. Cocaine did not influence pA(2) values against NA.4 5-HT and NA were of similar potency in common carotid and femoral arteries, but 5-HT was much less potent than NA in external carotid and ear arteries.5 The results indicate that rabbit common carotid and femoral arteries contain both D-type 5-HT receptors and alpha-adrenoceptors, as does the aorta. However, external carotid arteries, like ear arteries, do not contain specific 5-HT receptors. The action of 5-HT in the external carotid artery is mediated by alpha-adrenoceptors; this is a direct sympathomimetic action since it was not inhibited by cocaine or reserpine-pretreatment.6 The absence of 5-HT receptors in the rabbit extracranial circulation may limit the usefulness of this species as a model for research relating to migraine.

Animals↗
PubMed1980

Incomplete inactivation of sodium currents in nonperfused squid axon.

Perfused squid axons in which K-conductance is blocked show, under voltage clamp, incomplete inactivation of the sodium conductance. The presence of this phenomenon in nonperfused axons was found by comparing membrane current records before and after tetrodotoxin addition to the bathing solution. Sodium currents in nonperfused axons are comparable in behavior at positive potentials to those seen in Cs-perfused axons.

Animals↗
PubMed1978

Blocking of the squid axon potassium channel by external caesium ions.

1. The blocking by external Cs of inward tail currents though the K channel of the squid giant axon, as seen by the effect of Cs on the 'instantaneous' I-V curve is described. 2. Block onset is complete within 50--100 musec of a step in voltage. The block produces a negative slope region in the inward current quadrants of the I-V plots. 3. The experiments were performed in the presence of external concentrations of 240 mM-K and 0-200 mM-Cs, with the external ionic strength maintained by substituting Tris or Na for the Cs. Essentially the same results were obtained with either Tris or Na as the substitute ion. 4. The concentration of Cs required to block 50% of the K channels at zero transmembrane voltage, in intact axons, was estimated to be approximately 1 M. 5. The slope of the dose-response curve is steeper than expected for a blocking reaction with 1:1 stoichiometry when membrane voltage is in the neighbourhood of 100 mV. 6. With Cs concentrations greater than or approximately 50 mM, the voltage dependence of the block is too steep to be accounted for by the binding of a single Cs ion per channel within the membrane electric field. 7. In perfused axons, the block occurred at less negative internal voltage when internal K concentration was reduced. 8. These observations are qualitatively consistent with a multisite channel showing single file properties.

Animals↗
PubMed1977

Unstirred layer effects on calculations of the potential difference across an ion exchange membrane.

The potential difference between two solutions of the same 1:1 electrolyte bathing an ion exchange membrane has been calculated as the sum of the following components: (a) a Donnan potential at each membrane-solution interface, (b) a diffusion potential within the membrane phase, and (c) a diffusion potential in the unstirred layer on each side of the membrane. For a highly charged ion exchange membrane with at least one surface in contact with a dilute solution, calculated transmembrane potential differences are extremely sensitive to the assumed thickness of the unstirred layers. This sensitivity to unstirred layer thickness is primarily due to changes in the Donnan components of the potential difference. By this approach, it was possible to fit membrane potential data from Gunn and Curran (1971, Biophys. J. 11:559) for a range of bathing solution concentrations from 0.0016 to 4.0 M. If no effort was made to account for the modification of the Donnan potentials by the presence of unstirred layers, the data appeared incompatible with an electrodiffusion equation description. Suggestions for a more stringent experimental test and a brief discussion of possible implications for electrical measurements on fresh-water giant algal cells are presented.

Diffusion↗
PubMed1977

Sodium ions as blocking agents and charge carriers in the potassium channel of the squid giant axon.

Instantaneous K channel current-voltage (I-V) relations were determined by using internally perfused squid axons. When K was the only internal cation, the I-V relation was linear for outward currents at membrane potentials up to +240 mV inside. With 25-200 mM Na plus 300 mM K in the internal solution, an N-shaped I-V curve was seen. Voltage-dependent blocking of the K channels by Na produces a region of negative slope in the I-V plot (F. Bezanilla and C. M. Armstrong. 1972. J. Gen Physiol, 60: 588). At higher voltages (greater than or equal to 160 mV) we observed a second region of increasing current and a decrease in the fraction of the K conductance blocked by Na. Internal tetraethylammonium (TEA) ions blocked currents over the whole voltage range. In a second series of experiments with K-free, Na-containing internal solutions, the I-V curve turned sharply upward about +160 mV. The current at high voltages increased with increasing internal Na concentration was largely blocked by internal TEA. These data suggest that the K channel becomes substantially more permeable to Na at high voltages. This change is apparently responsible for the relief, at high transmembrane voltages, of the blocking effect seen in axons perfused with Na plus K mixtures. Each time a Na ion passed through, vacating the blocking site, the channel would transiently allow K ions to pass through freely.

Animals↗