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P Charnet

Publications and source records attributed to P Charnet.

At least 37 records · Page 2Linked to original sources

A transgenic mouse model of the slow-channel syndrome.

To investigate the effect of acetylcholine receptor (AChR) mutations on neuromuscular transmission and to develop a model for the human neuromuscular disease, the slow-channel syndrome, we generated transgenic mice with abnormal AChRs using a delta subunit with a mutation in the ion channel domain. In three transgenic lines, nerve-evoked end-plate currents and spontaneous miniature end-plate currents (MEPCs) had prolonged decay phases and MEPC amplitudes were reduced by 33%. Single nerve stimuli elicited repetitive compound muscle action potentials in vivo. Transgenic mice were abnormally sensitive to the neuromuscular blocker, curare. These observations demonstrate that we can predictably alter AChR function, synaptic responses, and muscle fiber excitation in vivo by overexpressing subunits containing well-defined mutations. Furthermore these data support the hypothesis that the electrophysiological findings in the neuromuscular disorder, the slow-channel syndrome, are due to mutant AChRs.

Animals↗

Coexpression of the beta2 subunit does not induce voltage-dependent facilitation of the class C L-type Ca channel.

Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of the alpha1 and beta subunits of the alpha1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation in Xenopus oocytes. Here we show that, by expressing the alpha1C together with different beta subunits in oocytes, the beta1, beta3 and beta4, but not the beta2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of the beta2 subunit mRNA, suggest that beta2 may be the beta subunit associated with functional cardiac L-type Ca2+ channels.

Action Potentials↗

cAMP-dependent phosphorylation of the cardiac L-type Ca channel: a missing link?

Cardiac inotropic effects of beta adrenergic agonists occur mainly through an increase in L-type (class C) calcium channel activity. This response has been attributed to phosphorylation of the L-type Ca channel, or a closely associated protein, by the cAMP-dependent protein kinase A (PKA). Among the three subunits forming the cardiac L-type Ca channel (alpha 1, beta and alpha 2-delta), biochemical studies have revealed that two subunits, alpha 1 and beta, are phosphorylated in vitro by protein kinase A, the alpha 1 subunit being the primary target. However, attempts to reconstitute the cAMP-dependent regulation of the expressed class C Ca channel, either in Xenopus oocytes or in cell lines, have provided contradictory results. We were unable to detect cAMP-dependent modulation of class C alpha 1 subunit Ca channels expressed in Xenopus oocytes, even when coinjected with auxiliary subunits beta and alpha 2-delta. Nevertheless, activity of Ca channels recorded from cardiac-mRNA injected oocytes was potentiated by injection of cAMP or PKA, even when expression of the beta subunit was suppressed using antisense oligonucleotide. Taken together, these results indicate that cAMP-dependent regulation does not exclusively involve the alpha 1 and the beta subunits of the Ca channel and suggest that unidentified protein(s), expressed in cardiac tissue, are most likely necessary.

Animals↗

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.

Animals↗

Cloning and functional expression of a neuronal calcium channel beta subunit from house fly (Musca domestica).

The primary structure of a calcium channel beta subunit (beta M) from housefly (Musca domestica) has been deduced by cDNA cloning and sequence analysis. The open reading frame encodes a 441-amino acid polypeptide with a calculated molecular mass of 48,755 Da. Whole-mount in situ hybridization indicates that beta M mRNA is predominantly expressed in neuronal tissues. Transcription of beta M mRNA is evident from stage 13/14 of embryogenesis up to adulthood. Different expression patterns of splice variants were found in larvae and in adult fly heads. Amino acid identity between beta M and mammalian beta subunits is lower (66-68%) than within mammalian beta subunits (74-80%). Calculation of a phylogenetic tree indicates that beta M is an ancestral form of the four distinct beta subunit gene products identified in mammalian tissues so far. Despite these sequence differences, beta M is able to enhance endogenous calcium channel activity in Xenopus laevis oocytes as well as dihydropyridine binding to membranes from COS 7 cells transfected with rabbit heart alpha 1 cDNA in the same manner as was previously shown for mammalian beta subunits.

Alternative Splicing↗

Calcium currents recorded from a neuronal alpha 1C L-type calcium channel in Xenopus oocytes.

Xenopus oocytes expressing neuronal alpha 1C, alpha 2 and beta 1b calcium channel subunit cDNAs were used in this study. During two-electric voltage clamp recording the oocyte was injected with 10-20 nl of a 100 mM BAPTA solution. Under these conditions, the endogenous Ca-activated Cl current was completely suppressed resulting in an alpha 1C Ba current free from Cl current contamination. BAPTA injection also allowed alpha 1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, alpha 1C whole cell Ca currents were smaller in magnitude and showed kinetic and voltage-dependent properties more similar to those for L-type Ca currents recorded in native cells. That Ca-dependent inactivation occurs in BAPTA-buffered cells suggests that the Ca-binding site involved in this type of inactivation is very close to the pore of the channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Regulation by protein kinase-C of putative P-type Ca channels expressed in Xenopus oocytes from cerebellar mRNA.

Xenopus oocytes injected with rat cerebellar mRNA expressed functional voltage-dependent Ca channels detected as an inward Ba current (IBa). The pharmacological resistance to dihydropyridines and omega-conotoxin together with the blockade obtained with Agelenopsis aperta venom suggest that these channels could be somehow assimilated to P-type Ca channels. The precise nature of the transplanted Ca channels was assessed by hybrid-arrest experiments using a specific oligonucleotide antisense-derivated from the recently cloned alpha 1-subunit of P channels (BI-1 clone). In addition, we demonstrate that exogenous Ca channel activity was enhanced by two different PKC activators (a phorbol ester and a structural analog to diacylglycerol). The general electrophysiological and pharmacological properties of the stimulated Ca channels remain unchanged. This potentiation induced by PKC activators is antagonized by a PKC inhibitor (staurosporine) and by a monoclonal antibody directed against PKC. It is concluded that P-type Ca channels are potentially regulated by PKC phosphorylation and the functional relevance of this intracellular pathway is discussed.

Animals↗

Cyclic AMP-dependent regulation of P-type calcium channels expressed in Xenopus oocytes.

Xenopus oocytes injected with rat cerebellum mRNA, express voltage-dependent calcium channels (VDCC). These were identified as P-type Ca2+ channels by their insensitivity to dihydropyridines and omega-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and omega-Aga-IV-A). Coinjection of cerebellar mRNA and antisense oligonucleotide complementary to the dihydropyridine-resistant brain Ca2+ channel, named BI [Mori Y. et al. (1991) Nature 350:398-402] or rbA [Starr T. V. B. et al. (1991) Proc Natl Acad Sci USA 88:5621-5625], strongly reduced the expressed Ba2+ current suggesting that these clones encode a P-type VDCC. The macroscopic Ca2+ channel activity was increased by direct intraoocyte injection of cAMP. This increase in current amplitude was concomitant with a slowing of current inactivation, and was attributed to activation of protein kinase A, since it could be antagonized by a peptidic inhibitor of this enzyme. Positive regulation of P-type VDCC could be of importance in Purkinje neurons and motor nerve terminals where this channel is predominant.

Animals↗

Functional properties of a neuronal class C L-type calcium channel.

The rat brain class C calcium channel alpha 1 subunit cDNA, rbC-II, was subcloned into a vertebrate expression vector and transient expression was assayed following nuclear injection into Xenopus oocytes. Whole cell recordings showed that rbC-II currents (recorded with 40 mM Ba2+ as the charge carrier) had variable activation rates and minimal inactivation over an approximately 700 msec depolarizing step pulse. The pharmacological properties of the rbC-II current were consistent with those of an L-type calcium channel, being sensitive to dihydropyridines (10 microM nifedipine blocked approximately 85% of the current, 10 microM Bay K 8644 enhanced the current between 2- and 10-fold) and not affected by the N- and P-type calcium channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, respectively. Coexpression of rbC-II with cloned rat neuronal calcium channel alpha 2 and beta subunits resulted in several changes to the electrophysiological properties of the rbC-II current including, an increased whole cell peak current, an increased rate of activation and a hyperpolarizing shift in the voltage dependence of activation. Taken together with results showing that the neuronal class D alpha 1 subunit also encodes an L-type calcium channel [Williams M. E., Feldman D. H., McCue A. F., Brenner R., Velicelebi G., Ellis S. B. and Harpold M. M. (1992a) Neuron 8: 71-84], these results indicate that the mammalian nervous system expresses two distinct genes encoding L-type calcium channels.

Animals↗

Interconversion between distinct gating pathways of the high threshold calcium channel in rat ventricular myocytes.

1. High-voltage-activated Ca2+ current (ICa) waveforms in adult rat ventricular myocytes comprise two components, referred to here as ICa(fc) and ICa(sc) to denote the fast and slow components, respectively, of ICa decay. At all test potentials, the two time constants of ICa decay, tau fc and tau sc, differ by approximately an order of magnitude. Neither tau fc nor tau sc varies appreciably with test potential, however, suggesting that current inactivation is not markedly voltage dependent. 2. Current activation at all test potentials follows a sigmoidal time course and is best described by a power function with n = 4. Deactivation of the currents, examined following variable length depolarizations to various test potentials, however, follows a single exponential time course. In addition, the kinetics of activation and deactivation of ICa(fc) and ICa(sc) are indistinguishable. 3. Although both begin to activate at approximately -30 mV, the voltage dependences of ICa(fc) and ICa(sc) are distinct: ICa(fc) peaks at -10 mV and ICa(sc) peaks at +10 mV. 4. The relative amplitudes of ICa(fc) and ICa(sc) vary with the holding potential from which the currents are evoked and with the frequency of current activation: hyperpolarized holding potentials and low stimulation frequencies reveal preferential activation of ICa(fc), whereas depolarized holding potentials and high stimulation frequencies potentiate ICa(sc). In addition, the observed voltage- and frequency-dependent changes in ICa(fc) and ICa(sc) amplitudes are reciprocal. 5. The apparent voltage dependences of steady-state inactivation of ICa(fc) and ICa(sc) are also distinct. ICa(fc) is reduced to approximately 50% of its maximal amplitude at -45 mV, whereas ICa(sc) is approximately 50% inactivated at -30 mV. 6. Recovery of ICa(peak) from steady-state inactivation follows a complex time course. Following inactivation at -10 mV, ICa(peak) recovers at -90 mV to its maximal value over a biexponential time course; ICa(peak) then decreases over the next several seconds to a steady-state level. 7. The time course of recovery from steady-state inactivation of ICa(fc) at -90 mV is best described by the sum of two exponentials; the two time constants of recovery differ by approximately a factor of 25. ICa(sc), in contrast, recovers rapidly and over a single exponential time course to its maximal value. When the recovery time at -90 mV is increased, however, ICa(sc) amplitude decreases slowly and over a single exponential time course to a steady-state level.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Electrophysiological characterization of a TTX-sensitive sodium current in native Xenopus oocytes.

We have studied a fast inward current expressed in oocytes from one Xenopus laevis. This current was characterized as a sodium current. It was activated by depolarizations to -50 mV or higher, peaked within 3-5 ms, and then decayed following a mono-exponential timecourse. When clamped at different holding potentials, the current displayed voltage-dependent inactivation with a V0.5 of -51 mV. The channel responsible for this Na+ entry was blocked by tetrodotoxin with a K0.5 of 8 nM, and was resistant to block by lidocaine at concentrations up to 100 microM. The pharmacological similarities between neuronal and oocyte sodium channels suggest that the two channels share a conserved structure.

Animals↗

Endogenous Xenopus-oocyte Ca-channels are regulated by protein kinases A and C.

Calcium entry into Xenopus oocyte occurs mainly through voltage-dependent calcium channels. These channels were characterized as belonging to a particular type of calcium channel insensitive to dihydropyridines, omega-conotoxin, and Agelenopsis aperta venom, but blocked by divalent cations (Co, Cd, Ni). Intracellular injection of cAMP, or bath application of phorbol ester, induced a marked increase in calcium current amplitude and a slowing of the inactivation time-course. Despite their different pharmacology, endogenous calcium channels, like cardiac or neuronal calcium channels, could be thus regulated by protein kinases A and C.

Animals↗

Protein kinase C regulation of cardiac calcium channels expressed in Xenopus oocytes.

L-Type cardiac Ca2+ channels expressed in Xenopus oocyte were studied following rat heart ribonucleic acid, messenger (mRNA) injection. We demonstrate that exogenous Ca2+ channels are sensitive to intracellular regulation by protein kinase C (PKC). This was performed by using two types of PKC activators [phorbol esters and a structural analogue of diacyl-glycerol (DAG)] and a specific peptidic inhibitor. Ca2+ channel modulation resulted in an initial increase of the inward current, without any modification of the voltage-dependent properties, and a second delayed phase, specifically observed with phorbol esters, characterized by a progressive decrease in current amplitude. Concomitantly, a reduction of membrane capacitance, reflecting a reduction of the total membrane surface area, was observed. We suggest that this phenomenon underlies the irreversible decrease of the expressed Ba2+ current via sequestration of Ca2+ channels and/or PKC. We also demonstrate that regulation of cardiac mRNA-directed Ca2+ channels by PKC activators was strictly dependent on intracellular Ca2+ concentration, and was partially additive with cyclic-adenosine-monophosphate-(cAMP) dependent regulation.

Animals↗

Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

1. Co-injection of RNA synthesized from cloned neuronal acetylcholine receptor (nAChR) subunits (alpha 4 and beta 2) in Xenopus oocytes produced functional receptors. In macroscopic voltage-clamp experiments, the agonist-induced current exhibited a strong inward rectification. 2. Voltage jumps from +50 mV to more negative potentials produced relaxations of the agonist-induced current with a single exponential time course. The relaxation rate constant was only weakly voltage dependent. 3. At the single-channel level, three conductances were recorded of 12, 22 and 34 pS. Their burst durations were similar and varied only weakly with voltage (e-fold for 120 to 370 mV), consistent with the poorly voltage-dependent relaxation rate constants. However, the burst durations were less than 10 ms, or less than 1/5 the value expected from voltage-jump relaxations. 4. Hexamethonium (Hex, 0.5 to 8 microM) inhibited the agonist-induced current and produced voltage-jump relaxations characterized by a rapid conductance increase and a slower conductance decrease. Analysis of these relaxations suggested that the Hex-receptor interaction is open-channel blockade characterized by a forward binding rate of 1 x 10(7) M-1 s-1 and a dissociation rate constant of about 25 s-1. 5. For the relaxations produced by QX222, the slowest phase was a conductance increase, suggesting that the dissociation rate constant for QX222 is 10-30-fold greater than for Hex. 6. Hex but not QX222 produced an additional use-dependent blockade that was manifest during repetitive hyperpolarizing pulses. 7. With mouse muscle ACh receptors expressed in oocytes, the blockade by Hex did not depend strongly on voltage. Neither Hex nor QX222 produced appreciable use-dependent block on muscle ACh receptors. 8. Of the four conditions studied (neuronal and muscle receptors, Hex and QX222), only the blockade of the neuronal AChR by Hex is characterized by a residence time longer than the normal open time. 9. It is concluded that the modest differences in primary amino acid sequence between muscle and neuronal receptors lead to profound changes in their interactions with channels blockers.

Acetylcholine↗

Structure of the gamma-less nicotinic acetylcholine receptor: learning from omission.

The nicotinic acetylcholine receptor can be expressed in Xenopus oocytes by injection of in vitro synthesized RNA for the alpha, beta, gamma, and delta mouse muscle subunits. However, detectable responses can also be obtained by injection of alpha, beta, and delta subunit RNA only. The receptors expressed in this case (gamma-less receptors) share many of the properties of the normal receptor, including relaxation time constants, Hill slope, and relative permeability for Na+, K+, Cs+, and Tris+. The major single-channel conductances of alpha beta gamma delta and alpha beta delta receptors are similar (34.2 +/- 2.9 and 38.5 +/- 0.6 pS, respectively) but clearly different from the major conductances seen after the combined injection of alpha beta delta mouse subunit RNA and Xenopus gamma subunit RNA. Mutations in the second transmembrane segment of the alpha and beta subunits, known to affect open time and blockade by QX-222, are equally effective in the gamma-less receptor. These data strongly suggest that the gamma-less receptor has the same pore diameter as the normal receptor and that alpha, beta, and delta subunits participate in its formation. Injection of alpha beta gamma delta well as alpha beta delta RNA produced additional subconductance states of around 25 pS. The low conductance state was sensitive to mutations introduced in the alpha or beta subunits with or without the gamma subunit, indicating that this channel did not need the gamma subunits but required at least the alpha and beta subunits to be produced. Injection of alpha beta delta and the adult-type epsilon subunit RNA gave rise to channels with conductances of 35 and 55 pS when the stoichiometry of the injection was 2:1:1:1, but only the 55-pS channel was recorded when the epsilon subunit RNA concentration was increased by 10-fold (stoichiometry of 2:1:1:10). The gamma-less receptor can thus be expressed even when the adult epsilon subunit is present. Whether gamma-less receptors are expressed at normal adult neuromuscular junctions remains unknown.

Amino Acid Sequence↗

Modulation of Ca currents in isolated frog atrial cells studied with photosensitive probes. Regulation by cAMP and Ca2+: a common pathway?

We have studied the regulation of cardiac Ca current by intracellular cyclic AMP (cAMP) and Ca2+, using photosensitive, caged compounds and the whole-cell, patch-clamp technique in isolated frog atrial cells. Although both low voltage activated (LVA) and high voltage activated (HVA) Ca channels were found to be present in these cells, only the HVA Ca currents were sensitive to modulation by isoproterenol or dihydropyridines (DHPs). The application of extracellular isoproterenol, as well as the photorelease of intracellular cAMP or Ca2+ at micromolar and submicromolar concentrations, respectively, had no effect on LVA Ca currents. In contrast, these agents: (i) increased the amplitude of currents through HVA channels, carried by either Ca2+ or Ba2+ with a similar time-course, (ii) slowed the decay of the current when Ba2+ was the permeating ion, and (iii) modulated the agonist effect of the DHP Bay-K 8644. The strong similarities between the effects of cAMP and Ca2+ suggest that both of these intracellular messengers might eventually lead to the phosphorylation of HVA Ca channels. It is possible that Ca-dependent phosphorylation of the channels may account for the potentiation of Ca current induced by repetitive stimulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Reverse pharmacology of the nicotinic acetylcholine receptor. Mapping the local anesthetic binding site.

We have been examining the interaction of a local anesthetic derivative, QX-222, with the ion channel pore of the muscle AChR, using a combination of mutagenesis, oocyte expression, and electrophysiology. Single channel recording, together with macroscopic voltage-jump relaxations, provides a measure of the residence time of the open channel blocker within the pore. We have found systematic changes in the apparent affinity of the open channel for QX-222 following amino acid substitutions in the proposed M2 transmembrane helix of each of the four subunits of the AChR. Assigning the number 1' to the residue at the cytoplasmic end of the M2 helix, positions 2',6',10',14', and 18' are modeled as forming the lining of the pore. Polar to nonpolar substitutions at 6' decrease QX-222 residence time, while the opposite effect is seen at position 10'. Nonpolar to polar substitutions have the converse effect. The distance between the aromatic and quaternary amine moieties of QX-222 corresponds almost exactly to the repeat distance of an alpha helix. This structural feature is common to many local anesthetic drugs. We propose a model for the binding of QX-222 within the ion channel of the AChR that is consistent with these observations.

Amino Acid Sequence↗