Emmanuel Bourinet

Splicing of α 1A subunit gene generates phenotypic variantsof P- and Q-type calcium channels

P-type and Q-type calcium channels mediate neurotransmitter release at many synapses in the mammalian nervous system. The α1A calcium channel has been implicated in the etiologies of conditions such as episodic ataxia, epilepsy and familial migraine, and shares several properties with native P- and Q-type channels. However, the exact relationship between α1A and P- and Q-type channels is unknown. Here we report that alternative splicing of the α1A subunit gene results in channels with distinct kinetic, pharmacological and modulatory properties. Overall, the results indicate that alternative splicing of the α1A gene generates P-type and Q-type channels as well as multiple phenotypic variants.

Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception

Analgesic therapies are still limited and sometimes poorly effective, therefore finding new targets for the development of innovative drugs is urgently needed. In order to validate the potential utility of blocking T‐type calcium channels to reduce nociception, we explored the effects of intrathecally administered oligodeoxynucleotide antisenses, specific to the recently identified T‐type calcium channel family (CaV3.1, CaV3.2, and CaV3.3), on reactions to noxious stimuli in healthy and mononeuropathic rats. Our results demonstrate that the antisense targeting CaV3.2 induced a knockdown of the CaV3.2 mRNA and protein expression as well as a large reduction of ‘CaV3.2‐like’ T‐type currents in nociceptive dorsal root ganglion neurons. Concomitantly, the antisense treatment resulted in major antinociceptive, anti‐hyperalgesic, and anti‐allodynic effects, suggesting that CaV3.2 plays a major pronociceptive role in acute and chronic pain states. Taken together, the results provide direct evidence linking CaV3.2 T‐type channels to pain perception and suggest that CaV3.2 may offer a specific molecular target for the treatment of pain.

Specific contribution of human T‐type calcium channel isotypes (α1G, α1H and α1I) to neuronal excitability

In several types of neurons, firing is an intrinsic property produced by specific classes of ion channels. Low‐voltage‐activated T‐type calcium channels (T‐channels), which activate with small membrane depolarizations, can generate burst firing and pacemaker activity. Here we have investigated the specific contribution to neuronal excitability of cloned human T‐channel subunits. Using HEK‐293 cells transiently transfected with the human α1G (CaV3.1), α1H (CaV3.2) and α1I (CaV3.3) subunits, we describe significant differences among these isotypes in their biophysical properties, which are highlighted in action potential clamp studies. Firing activities occurring in cerebellar Purkinje neurons and in thalamocortical relay neurons used as voltage clamp waveforms revealed that α1G channels and, to a lesser extent, α1H channels produced large and transient currents, while currents related to α1I channels exhibited facilitation and produced a sustained calcium entry associated with the depolarizing after‐potential interval. Using simulations of reticular and relay thalamic neuron activities, we show that α1I currents contributed to sustained electrical activities, while α1G and α1H currents generated short burst firing. Modelling experiments with the NEURON model further revealed that the α1G channel and α1I channel parameters best accounted for T‐channel activities described in thalamocortical relay neurons and in reticular neurons, respectively. Altogether, the data provide evidence for a role of α1I channel in pacemaker activity and further demonstrate that each T‐channel pore‐forming subunit displays specific gating properties that account for its unique contribution to neuronal firing.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel–ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Alternatively Spliced α1G (CaV3.1) Intracellular Loops Promote Specific T-Type Ca2+ Channel Gating Properties

Abstract At least three genes encode T-type calcium channel α 1 subunits, and identification of cDNA transcripts provided evidence that molecular diversity of these channels can be further enhanced by alternative splicing mechanisms, especially for the α 1G subunit (Ca V 3.1). Using whole-cell patch-clamp procedures, we have investigated the electrophysiological properties of five isoforms of the human α 1G subunit that display a distinct III-IV linker, namely, α 1G-a , α 1G-b , and α 1G-bc , as well as a distinct II-III linker, namely, α 1G-ae , α 1G-be , as expressed in HEK-293 cells. We report that insertion e within the II-III linker specifically modulates inactivation, steady-state kinetics, and modestly recovery from inactivation, whereas alternative splicing within the III-IV linker affects preferentially kinetics and voltage dependence of activation, as well as deactivation and inactivation. By using voltage-clamp protocols mimicking neuronal activities, such as cerebellar train of action potentials and thalamic low-threshold spike, we describe that inactivation properties of α 1G-a and α 1G-ae isoforms can support channel behaviors reminiscent to those described in native neurons. Altogether, these data demonstrate that expression of distinct variants for the T-type α 1G subunit can account for specific low-voltage-activated currents observed in neuronal tissues.

Interaction of SNX482 with Domains III and IV Inhibits Activation Gating of α1E (CaV2.3) Calcium Channels

We have investigated the action of SNX482, a toxin isolated from the venom of the tarantula Hysterocrates gigas, on voltage-dependent calcium channels expressed in tsa-201 cells. Upon application of 200 nM SNX482, R-type alpha(1E) calcium channels underwent rapid and complete inhibition, which was only poorly reversible upon washout. However, upon application of strong membrane depolarizations, rapid and complete recovery from inhibition was obtained. Tail current analysis revealed that SNX482 mediated an approximately 70 mV depolarizing shift in half-activation potential, suggesting that the toxin inhibits alpha(1E) calcium channels by preventing their activation. Experiments involving chimeric channels combining structural features of alpha(1E) and alpha(1C) subunits indicated that the presence of the domain III and IV of alpha(1E) is a prerequisite for a strong gating inhibition. In contrast, L-type alpha(1C) channels underwent incomplete inhibition at saturating concentrations of SNX482 that was paralleled by a small shift in half-activation potential and which could be rapidly reversed, suggesting a less pronounced effect of the toxin on L-type calcium channel gating. We conclude that SNX482 does not exhibit unequivocal specificity for R-type channels, but highly effectively antagonizes their activation.

Determinants of voltage-dependent inactivation affect Mibefradil block of calcium channels

The voltage gated calcium channel family is a major target for a range of therapeutic drugs. Mibefradil (Ro 40-5967) belongs to a new chemical class of these molecules which differs from other Ca2+ antagonists by its ability to potently block T-type Ca2+ channels. However, this molecule has also been shown to inhibit other Ca2+ channel subtypes. To further analyze the mechanism governing the Ca2+ channel-Mibefradil interaction, we examined the effect of Mibefradil on various recombinant Ca2+ channels expressed in mammalian cells from their cloned cDNAs, using Ca2+ as the permeant ion at physiological concentration. Expression of alpha1A, alpha1C, and alpha1E in tsA 201 cells resulted in Ca2+ currents with functional characteristics closely related to those of their native counterparts. Mibefradil blocked alpha1A and alpha1E with a Kd comparable to that reported for T-type channels, but had a lower affinity (approximately 30-fold) for alpha1C. For each channel, inhibition by Mibefradil was consistent with high-affinity binding to the inactivated state. Modulation of the voltage-dependent inactivation properties by the nature of the coexpressed beta subunit or the alpha1 splice variant altered block at the Mibefradil receptor site. Therefore, we conclude that the tissue and sub-cellular localization of calcium channel subunits as well as their specific associations are essential parameters to understand the in vivo effects of Mibefradil.

Multiple determinants in voltage‐dependent P/Q calcium channels control their retention in the endoplasmic reticulum

Surface expression level of voltage‐dependent calcium channels is tightly controlled in neurons to avoid the resulting cell toxicity generally associated with excessive calcium entry. Cell surface expression of high voltage‐activated calcium channels requires the association of the pore‐forming subunit, Cavα, with the auxiliary subunit, Cavβ. In the absence of this auxiliary subunit, Cavα is retained in the endoplasmic reticulum (ER) through mechanisms that are still poorly understood. Here, we have investigated, by a quantitative method based on the use of CD8α chimeras, the molecular determinants of Cavα2.1 that are responsible for the retention, in the absence of auxiliary subunits, of P/Q calcium channels in the ER (referred to here as ‘ER retention’). This study demonstrates that the I–II loop of Cavα2.1 contains multiple ER‐retention determinants beside the β subunit association domain. In addition, the I–II loop is not the sole domain of calcium channel retention as two regions identified for their ability to interact with the I–II loop, the N‐ and C‐termini of Cavα2.1, also produce ER retention. It is also not an obligatory determinant as, similarly to low‐threshold calcium channels, the I–II loop of Cavα1.1 does not produce ER retention in COS7 cells. The data presented here suggests that ER retention is suppressed by sequential molecular events that include: (i) a correct folding of Cavα in order to mask several internal ER‐retention determinants and (ii) the association of other proteins, including the Cavβ subunit, to suppress the remaining ER‐retention determinants.

Multiple structural elements contribute to voltage-dependent facilitation of neuronal α1C (CaV1.2) L-type calcium channels

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that alpha(1C) L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal alpha(IC) L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032-5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between facilitable alpha(1C) and non facilitable alpha(1E) calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of alpha(1C) contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of alpha(1C) is a complex process which involves multiple structural elements were domains I and III play the major role.

A Bacterial Toxin with Analgesic Properties: Hyperpolarization of DRG Neurons by Mycolactone

Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT2 receptors (angiotensin II type 2 receptors; AT2R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT2R, with this action being not affected by known ligands of AT2R. This result points towards novel AT2R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics.