Matthieu Rousset

Functional roles of γ2, γ3 and γ4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes

1 Stargazin or γ2, the product of the gene mutated in the stargazer mouse, is a homologue of the γ1 protein, an accessory subunit of the skeletal muscle L‐type Ca2+ channel. γ2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to γ1. Two new members of the γ family expressed in the brain have recently been identified: γ3 and γ4. 2 We have co‐expressed, in Xenopus oocytes, the human γ2,γ3 and γ4 subunits with the P/Q‐type (CaV2.1) Ca2+ channel and different regulatory subunits (α2‐δ; β1, β2, β3 or β4). 3 Subcellular distribution of the γ subunits confirmed their membrane localization. 4 Ba2+ currents, recorded using two‐electrode voltage clamp, showed that the effects of the γ subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing β subunits displayed an unusual slow‐inactivating Ba2+ current. Expression of both β and γ subunits increased the appearance of the slow‐inactivating current. 5 Our data support a role for the γ subunit as a brain Ca2+ channel modulatory subunit and suggest that β and γ subunits are involved in a switch between two regulatory modes of the P/Q‐type channel inactivation.

Puroindolines form ion channels in biological membranes.

Wheat seeds contain different lipid binding proteins that are low molecular mass, basic and cystine-rich proteins. Among them, the recently characterized puroindolines have been shown to inhibit the growth of fungi in vitro and to enhance the fungal resistance of plants. Experimental data, using lipid vesicles, suggest that this antimicrobial activity is related to interactions with cellular membranes, but the underlying mechanisms are still unknown. This paper shows that extracellular application of puroindolines on voltage-clamped Xenopus laevis oocytes induced membrane permeabilization. Electrophysiological experiments, on oocytes and artificial planar lipid bilayers, suggest the formation, modulated by voltage, of cation channels with the following selectivity: Cs(+) > K(+) > Na(+) > Li(+) > choline = TEA. Furthermore, this channel activity was prevented by addition of Ca(2+) ions in the medium. Puroindolines were also able to decrease the long-term oocyte viability in a voltage-dependent manner. Taken together, these results indicate that channel formation is one of the mechanisms by which puroindolines exert their antimicrobial activity. Modulation of channel formation by voltage, Ca(2+), and lipids could introduce some selectivity in the action of puroindolines on natural membranes.

Ca2+ Channel Inactivation Heterogeneity Reveals Physiological Unbinding of Auxiliary β Subunits

Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations.

Molecular Determinant for Specific Ca/Ba Selectivity Profiles of Low and High Threshold Ca2+ Channels

Voltage-gated Ca2+ channels (VGCC) play a key role in many physiological functions by their high selectivity for Ca2+ over other divalent and monovalent cations in physiological situations. Divalent/monovalent selection is shared by all VGCC and is satisfactorily explained by the existence, within the pore, of a set of four conserved glutamate/aspartate residues (EEEE locus) coordinating Ca2+ ions. This locus however does not explain either the choice of Ca2+ among other divalent cations or the specific conductances encountered in the different VGCC. Our systematic analysis of high- and low-threshold VGCC currents in the presence of Ca2+ and Ba2+ reveals highly specific selectivity profiles. Sequence analysis, molecular modeling, and mutational studies identify a set of nonconserved charged residues responsible for these profiles. In HVA (high voltage activated) channels, mutations of this set modify divalent cation selectivity and channel conductance without change in divalent/monovalent selection, activation, inactivation, and kinetics properties. The CaV2.1 selectivity profile is transferred to CaV2.3 when exchanging their residues at this location. Numerical simulations suggest modification in an external Ca2+ binding site in the channel pore directly involved in the choice of Ca2+, among other divalent physiological cations, as the main permeant cation for VGCC. In LVA (low voltage activated) channels, this locus (called DCS for divalent cation selectivity) also influences divalent cation selection, but our results suggest the existence of additional determinants to fully recapitulate all the differences encountered among LVA channels. These data therefore attribute to the DCS a unique role in the specific shaping of the Ca2+ influx between the different HVA channels.

Ca2+-dependent interaction of BAPTA with phospholipids

Starting from a comparative study of different Ca2+ chelators on the G‐protein‐induced inhibition of the CaV2.1 Ca channels, we demonstrate that BAPTA and DM‐nitrophen are able to interact, in a Ca2+‐ and lipid‐dependent manner, with phospholipid monolayers. Critical insertion pressure and sensitivity to charged lipids indicated that insertion in the lipid film may occur in biological membranes as those found on Xenopus oocytes. This novel property is not found for EGTA and EDTA and may participate to the unusual ability of BAPTA‐related molecules to chelate Ca2+ ions in the very close vicinity of the plasma membrane, where most of the Ca2+‐dependent signalling triggered by voltage‐gated Ca2+ currents occurs.

Biophysical characterization of the honeybee DSC1 orthologue reveals a novel voltage-dependent Ca2+ channel subfamily: CaV4

Insect DSC1 channels have sequences that are intermediate between voltage-gated Na+ and Ca2+ channels but have hitherto been classified as the former. Gosselin-Badaroudine et al. clone and characterize honeybee DSC1, revealing high selectivity for Ca2+ and suggesting reclassification of DSC1 homologues as Ca2+ channels.

Characterization of the honeybee AmNaV1 channel and tools to assess the toxicity of insecticides.

Pollination is important for both agriculture and biodiversity. For a significant number of plants, this process is highly, and sometimes exclusively, dependent on the pollination activity of honeybees. The large numbers of honeybee colony losses reported in recent years have been attributed to colony collapse disorder. Various hypotheses, including pesticide overuse, have been suggested to explain the disorder. Using the Xenopus oocytes expression system and two microelectrode voltage-clamp, we report the functional expression and the molecular, biophysical, and pharmacological characterization of the western honeybee’s sodium channel (Apis Mellifera NaV1). The NaV1 channel is the primary target for pyrethroid insecticides in insect pests. We further report that the honeybee’s channel is also sensitive to permethrin and fenvalerate, respectively type I and type II pyrethroid insecticides. Molecular docking of these insecticides revealed a binding site that is similar to sites previously identified in other insects. We describe in vitro and in silico tools that can be used to test chemical compounds. Our findings could be used to assess the risks that current and next generation pesticides pose to honeybee populations.

Two sets of amino acids of the domain I of Cav2.3 Ca 2+ channels contribute to their high sensitivity to extracellular protons

Extracellular acidification decreases Ca2+ current amplitude and produces a depolarizing shift in the activation potential (Va) of voltage-gated Ca2+ channels (VGCC). These effects are common to all VGCC, but differences exist between Ca2+ channel types and the underlying molecular mechanisms remain largely unknown. We report here that the changes in current amplitude induced by extracellular acidification or alkalinisation are more important for Cav2.3 R type than for Cav2.1 P/Q-type Ca2+ channels. This difference results from a higher shift of Va combined with a modification of channel conductance. Although involved in the sensitivity of channel conductance to extracellular protons, neither the EEEE locus nor the divalent cation selectivity locus could explain the specificity of the pH effects. We show that this specificity involves two separate sets of amino acids within domain I of the Cavα subunit. Residues of the voltage sensor domain and residues in the pore domain mediate the effects of extracellular protons on Va and on channel conductance, respectively. These new insights are important for elucidating the molecular mechanisms that control VGCC gating and conductance and for understanding the role of extracellular protons in other channels or membrane-tethered enzymes with similar pore and/or voltage sensor domains.

Role of β Subunits in Voltage-Gated Calcium Channel Functions

Ca2+ channel auxiliary subunits have been suspected to play fundamental roles in channel function from their early biochemical characterization (see Chapter 4). Their strong association with the main α1 subunits and their role in the reconstitution of proper channel activity, were strong evidences for key functions. Later, the discovery of a whole family of β subunit genes, now counting four members, with regions of high similarity including channel binding sites as well as more ubiquitous protein-protein interaction domains, and expression studies challenging different combinations of calcium channel subunits, confirmed their modulatory functions in every aspect of channel activity, from expression and targeting to regulation by G-proteins. The discovery of neuropathologies linked to genetic defects in the β subunit gene constitutes one more argument for assigning to this auxiliary subunit a central role in channel function and regulation.

RGK Small GTPases and Regulation of CaV2 Channels

About 10 years ago, a yeast two-hybrid screen highlighted the unexpected interaction between the regulatory subunit of the voltage–gated Ca2+ channels, CaVβ, and Kir/Gem, a member of the recently identified Ras-related GTP-binding protein family RGK (Rad-Gem-Kir). It soon appeared that all the members of this family, Gem, Rad, Rem and Rem2, were able to inhibit high-voltage activated Ca2+ channels, thus opening new fields of research to understand the molecular mechanisms leading to channel inhibition and to analyze their potential physiological signification. While much of these works were first concentrated on L-type CaV1.2 channels, it is clear now that presynaptic CaV2.1 and CaV2.2 channels are also sensitive to RGK inhibition. Recent data suggest that multiple routes are used by the RGK proteins to inhibit Ca2+ channels, including modifications of channel targeting and recycling, gating-charge mobility and/or open-channel probability. A direct RGK-CaVβ interaction appears to be absolutely necessary, but additional interactions with the channel protein itself have been highlighted and suggest a finely tuned specificity at the channel level. Whether these interactions also play a role in other channel CaVα or CaVβ functions, such as synaptic transmission or transcriptional regulation, still needs to be investigated.