Pierre Charnet

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.

CHANGE IN MEMBRANE PERMEABILITY INDUCED BY PROTEGRIN 1 : IMPLICATION OF DISULPHIDE BRIDGES FOR PORE FORMATION

Protegrin 1 (PG‐1) is a naturally occurring cationic antimicrobial peptide that is 18 residues long, has an aminated carboxy terminus and contains two disulphide bridges. Here, we investigated the antimicrobial activity of PG‐1 and three linear analogues. Then, the membrane permeabilisation induced by these peptides was studied upon Xenopus laevis oocytes by electrophysiological methods. From the results obtained, we concluded that protegrin is able to form anion channels. Moreover, it seems clear that the presence of disulphide bridges is a prerequisite for the pore formation at the membrane level and not for the antimicrobial activity.

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 diacylglycerol (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.

Coexpression of theβ2 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 theαl andβ subunits of theα1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation inXenopus oocytes. Here we show that, by expressing theα1C together with differentβ subunits in oocytes, theβ1,β3 andβ4, but not theβ2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of theβ2 subunit mRNA, suggest thatβ2 may be theβ subunit associated with functional cardiac L-type Ca2+ channels.

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.

Calcium currents recorded from a neuronal α1C L-type calcium channel in Xenopus oocytes

Xenopus oocytes expressing neuronal α1C, α2 and β1b 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 α1C Ba current free from Cl current contamination. BAPTA injection also allowed α1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, α1C 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.

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 ω-conotoxin and by their blockade by Agelenopsis aperta venom (containing the funnel-web spider toxins: FTX and ω-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.

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 (I Ba). The pharmacological resistance to dihydropyridines and ω‐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 α1‐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.

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.

Modulation of the α1A Ca2+ channel by β subunits at physiological Ca2+ concentration

The class A Ca2+ channel αl subunit (αlA) was expressed in Xenopus oocytes alone or in combination with the βlb, β2a, β3, or β4 subunit. Analysis of voltage‐dependent activation and inactivation in the presence of 1.8 mM external Ca2+ showed an hyperpolarising shift of both relations when compared to similar recordings performed in the presence of 40 mM Ba2+. These shifts, which differed for activation and inactivation, were strongly modulated by the nature of the co‐expressed β subunit. On the other hand, for each combination, the kinetics of inactivation were similar in 1.8 mM Ca2+ and 40 mM Ba2+ (for example co‐expression of the β2a subunit reduced inactivation using either 40 mM Ba2+ or 1.8 mM Ca2+). Thus, modulation of channel properties by the β subunit is different in physiological Ca2+ or high Ba2+ concentrations. These results must be taken into consideration to extrapolate the role of the β subunit in native cells.