Michèle Ildefonse

Tityusγ toxin, a high affinity effector of the Na+ channel in muscle, with a selectivity for channels in the surface membrane

Toxin γ from the venom ofTityus serrulatus scorpion produces a partial block of the surface Na+ channel in frog muscle. This block occurs with no change in the voltage-dependence or in the kinetics of the remaining surface Na+ current. The partial blockade of Na+ channel activity occurs with no change in tubular Na+ currents nor in twitch tension. The maximum effect of the toxin is attained at concentrations as low as 3×10−10 M. Hyperpolarization to potentials more negative than the resting potential (E=−90 mV) reduces or abolishes the effect of the toxin.Radioiodinated toxin γ binds to frog muscle membranes with a very high affinity corresponding to a dissociation constant of about 1×10−11 M. Data obtained with both rabbit and frog muscle indicate that toxin γ is specific for Na+ channels in surface membranes. Toxin γ does not seem to bind to Na+ channels in T-tubule membranes. The biochemical data are in good agreement with electrophysiological studies and data on contraction. There is oneTityus γ toxin binding site per tetrodotoxin binding site in surface membranes. Competition experiments have confirmed thatTityus γ toxin binds to a new toxin receptor site on the Na+ channel structure. This site is the same that the toxin II fromCentruroides suffusus binding site, but this toxin has 100 times less affinity for the Na+ channel thanTityus γ toxin.

Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers

SummaryUnitary currents through cGMP-dependent channels of retinal rods are observed following incorporation into planar lipid bilayers of native vesicles from purified rod outer segment membranes washed free of soluble and peripheral proteins. The influence of the concentration of cGMP, inhibitors (cis-diltiazem, tetracaine and Ag+) and divalent cations (Ca2+, Mg2+, and Co2+) on the conductance and open probability of the channel is described, as well as the voltage dependence of these effects. The cGMP dependence suggests the existence of four binding sites for cGMP and reveals that sequential binding of four cGMP molecules corresponds to the opening of four discrete conductance levels. Finally, we provide conclusive evidence that activated G-protein does not directly inactivate the cGMP-dependent channels of bovine retinal rods.

Excitation-Contraction Coupling in Skeletal Muscle

A historical approach is a simple way for reviewing the background necessary for discussions of the current concerns. 1947 is a convenient starting point; in this field, as well as in others, a number of contributions appeared two to three years after the war, that defined the field for years to come. Heilbrunn and Wiercinski [1] essentially demonstrated Ca2+ to be the second messenger that controls contraction, and proposed that Ca2+ entered the myoplasm from outside; A.V. Hill [2] demonstrated, with a simple diffusion calculation, the need for a propagation mechanism of activation, other than diffusion from the outside. This system was identified in the fifties: in 1958 Huxley and Taylor [3] suceeded in depolarizing localized regions of the membrane of a fiber with an extracellular electrode placed very close to the fiber, a predecessor of patch clamp. The depolarization only induced contractions (localized to the underlying zone) when the pipette was placed over the I band, but not over the A band. It is interesting that C. Franzini — Armstrong, when reviewing the field, always starts at this time. This result obviously motivated her, as it did other morphologists, to search for a structure underlying these localized effects. Her celebrated studies have a predecessor, closer to home, E. Veratti [4] who had described the T tubular system in 1902, using the Golgi staining technique. Apparently his contemporaries, when presented with the pictures, only said se non e vero e ben trovato, and ignored the result, wich was only exploited in the sixties and seventies by Franzini — Armstrong [4 bis], B. Eisenberg [5], L. Peachey [6] and others. Figure 1 shows a view of a thick transversal cut of a fiber bundle, with Golgi stain (Peachey and Eisenberg [7]), demonstrating how the T system effectively reduces the (diffusion) distances in the transversal direction to under a micrometer. As the morphological description of the T system progressed, so did that of the sarcoplasmic reticulum (Sr).

Gating of retinal rod cation channel by different nucleotides : comparative study of unitary currents

SummarySingle channels are observed after incorporation of native vesicles from bovine rod outer segment membranes into planar lipid bilayers. The activity of a single channel in the presence of cGMP is compared to that induced by the analog 8-bromo-cGMP and by cAMP. At +80 mV, K0.5 is about 3 μm for 8Br-cGMP, 18 μm for cGMP and 740 μm for cAMP. In cAMP, the amplitude of the current is smaller than in cGMP or 8Br-cGMP and depends on the filter cut-off frequency. The open/closed transition rates of the channel are slightly slower with 8Br-cGMP than with cGMP while they are 5 to 10 times faster with cAMP. Addition of Ni2+ ions to either cGMP or cAMP increases the open probability: the open/closed transition rates and amplitude of the current in cAMP are then comparable to those in cGMP. A dual effect of the addition of cAMP on the cGMPor 8Br-cGMP dependent activity previously reported (Furman, R.E., Tanaka, J.C. 1989. Biochemistry28:2785–2788) is observed with a single channel: addition of subthreshold cAMP concentrations to cGMP (or to 8Br-cGMP) markedly increases Po; addition of cAMP concentrations higher than about 70 μm progressively accelerates the kinetics and reduces the amplitude to values observed in cAMP alone. The results are discussed in relation with the model previously proposed to account for the existence of four current levels (Ildefonse, M., Bennett, N. 1991. J. Membrane Biol.123:133–147).

Existence of a sodium current in the tubular membrane of frog twitch muscle fibre; Its possible role in the activation of contraction

Abstract1.The membrane current of frog twitch muscle fibre has been recorded together with contraction in the test gap of a double sucrose gap apparatus.2.In Ringer, the inward current sometimes shows 2 phases with the same threshold and the same reversal potential: a rapid one (early inward current) and a slower one (late inward current). The mechanical threshold is near the inward current threshold. The amplitude of the contraction increases progressively with depolarizations, without modification of its time to peak. Experiments with variation of the pulse duration and with conditioning depolarizations show that a part of the contraction seems to be correlated with the late inward current.3.Experiments in a sodium free solution, with low TTX concentration, and on glycerol treated fibres show that the late inward current corresponds to a tubular sodium current.4.A method is described to separate the two phases of inward current. The smooth development of the current-voltage relation of the late inward current, its diminution without modification in time to peak under the action of TTX, and the exponential decay of its tail current all suggest that the tubular membrane potential is sufficiently well controlled.5.In the experiments where the tubular membrane potential seems to be controlled, a part of the contraction depends on the tubular sodium current, perhaps involving a mechanism of sodium induced calcium release.

Block by Cs of K currentiK1 and of carbachol induced K currentiCch in frog atrium

Abstract1. In frog atrium, Cs ions block both the inward rectifieriK1 and the carbachol induced K currentiCch. 2. BothiK1 andiCch display a high affinity for Cs with a K0.5 of 4×10−5 M forik1 and of 8×10−5 M foriCch atV=−50 mV. 3. Block of bothiK1 andiCch is strongly voltage dependent. When fitted by the block model of Woodhull (1973), δ is >1 for the two currents. 4. From these similarities, action of Cch on frog atrium K permeability could be interpreted as a modification ofiK1.

Effects of cysteine modification on the activity of the cGMP-gated channel from retinal rods

The effect of sulfhydryl reagents on the activity of the cGMP-gated channel from bovine retinal rods was studied by measurements of 8-Br-cGMP-(cGMP)-induced calcium efflux from rod membrane vesicles and records of 8-Br-cGMP-dependent sodium currents through channels incorporated into planar lipid bilayers. N-ethylmaleimide and mersalyl (thiol blockers) as well as diamide (dithiol-disulfide conversion agent) have a dual effect on the channels activity: at low concentration, they increase the apparent affinity for cyclic nucleotide (“activation”) at the same time inducing a loss of cooperativity for nucleotide binding; at higher concentration, N-ethylmaleimide and diamide produce a reduction of the amplitude and initial rate of the calcium release at saturating nucleotide concentration, while mersalyl is shown to reduce the activity of the channels in bilayer experiments (“inhibition”). Nitric oxide precursors have no effect. The results suggest that blocking at least 1 of the 3 cytoplasmic cysteine residues situated close to the cGMP-binding site in each channel subunit by N-ethylmaleimide, mersalyl, or diamide (forming a dimer between 2 subunits) increases the affinity for the nucleotide. Inhibition is produced by blocking at least one of the 2 other cytoplasmic sulfhydryl groups (N-ethylmaleimide, mersalyl, oxidized glutathione) or the 2 others (diamide, intrasubunit bridge), and may concern a process of channel inactivation. The 3 cytoplasmic sulfhydryl groups are accessible when the channels are in the open state, but not (or much less) accessible when the channels are in the closed state.

Coexpression of α and β Subunits of the Rod Cyclic GMP-Gated Channel Restores Native Sensitivity to Cyclic AMP: Role of D604/N1201

Abstract Coexpression of the βwt and αwt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the β subunit as previously suggested (Varnum et al., 1995, Neuron. 15:619–625), we compared currents from wild-type ( αwt and αwt / βwt ) and from mutated channels ( α D604N, α D604N/ βwt, and αwt/β N1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the β subunit. The D604N mutation in the α subunit and, to a lesser extent, coexpression of the βwt subunit with the αwt subunit reduce the open probability for cGMP compared to that of the αwt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux; Monod et al., 1965, J. Mol. Biol. 12:88–118) suggests that the D604N mutation in the α subunits and coassembly of α and β subunits alter the free energy of gating by cAMP more than that of cAMP binding.

Basis for Intracellular Retention of a Human Mutant of the Retinal Rod Channel a Subunit

A mutant of the a subunit of the retinal rod cyclic GMP-gated channel, [Arg654(1-bp del)], corresponding to a truncated alphaR654Dstop subunit, was previously described in patients with retinitis pigmentosa: when expressed in HEK-293 cells, this mutated a subunit was retained inside the cell, but had normal channel activity in one case where it reached the plasma membrane, indicating that the mechanism of targeting is altered by the mutation, but not the function of the channel. The corresponding mutants of the bovine rod channel (alphaR656D stop), and of the closely related olfactory neuron channel (alphaR632Dstop) alpha subunits were expressed in Xenopus oocytes and their activity was analyzed by patch-clamp. Like their human homologue, these two channels have no activity, and we show that their GFP fusion proteins are accumulated into intracellular compartments. The truncation alone or the R/D mutation alone do not prevent or modify channel activity, indicating that neither the R656 residue nor the C-terminal domain downstream of R656 is necessary for homomeric channel targeting and function. Several mutations of R656 and of the preceding residues in the R656Dstop mutant disclose that the motif responsible for the absence of channel activity is an endoplasmic reticulum retention signal (KXKXXstop) in which the nature of the residues in positions -1 and -4 is determinant.

Evidence for an action of sodium ions in the activation of contraction of twitch muscle fibre

Simultaneous records in current clamp conditions of potential and of contraction of frog skeletal twitch muscle fibre have shown that a part of the contraction directly depends on the intervention of sodium ions (increase in presence of veratrine, decrease in lithium Ringer). The results confirm previous voltage clamp data and suggest a mechanism of sodium induced calcium release.