Thierry Cens

Voltage and Calcium Use the Same Molecular Determinants to Inactivate Calcium Channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+(Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the α1ACa2+ channel) and the Ca2+-dependent inactivation (of the α1C Ca2+ channel) are similarly influenced by Ca2+ channel β subunits. The same molecular determinants of the β subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different α1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.

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.

Promotion and Inhibition of L-type Ca2+Channel Facilitation by Distinct Domains of the β Subunit

Ca2+ current potentiation by conditioning depolarization is a general mechanism by which excitable cells can control the level of Ca2+ entry during repetitive depolarizations. Several types of Ca2+ channels are sensitive to conditioning depolarization, however, using clearly distinguishable mechanisms. In the case of L-type Ca2+ channels, prepulse-induced current facilitation can only be recorded when the pore-forming α1C subunit is coexpressed with the auxiliary β1, β3, or β4, but not β2, subunit. These four β subunits are composed of two conserved domains surrounded by central, N-terminal, and C-terminal variable regions. Using different deleted and chimeric forms of the β1 and β2subunits, we have mapped essential sequences for L-type Ca2+ channel facilitation. A first sequence, located in the second conserved domain of all β subunits, is responsible for the promotion of current facilitation by the β subunit. A second sequence of 16 amino acids, located on the N-terminal tail of the β2 subunit, induces a transferable block ofL-type current facilitation. Site-specific mutations reveal the essential inhibitory role played by three positive charges on this segment. The lack of prepulse-induced current facilitation recorded with some truncated forms of the β2 subunit suggests the existence of an additional inhibitory sequence in the β2subunit.

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.

Regulation of Ca-sensitive inactivation of a l-type Ca2+ channel by specific domains of β subunits

Ca2+ channel auxiliary β subunits have been shown to modulate voltage‐dependent inactivation of various types of Ca2+ channels. The β1 and β2 subunits, that are differentially expressed with the L‐type α1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+‐dependent inactivation kinetics. Their expression in Xenopus oocytes with the α1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the β2 subunit. Using a series of β subunit deletion mutants and chimeric constructs of β1 and β2 subunits, we show that the inhibitory site located on the amino‐terminal region of the β2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the β2 subunit can modulate, in a specific way, the Ca2+ entry through L‐type Ca2+ channels in different brain or heart regions.

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.

Expression of β subunit modulates surface potential sensing by calcium channels

Abstractu2002We have expressed the α1A calcium channel subunit alone, and in combination with different β subunits, and investigated the effect of the external Ba2+ concentration on the voltage dependence of activation. Increasing the external Ba2+ concentration from 2.5 to 40xa0mM induced in all cases a depolarising shift of the potential for half-activation. The magnitude of this shift however, was different depending on whether the α1A subunit was expressed alone or with a β subunit. Consistently, calculated external surface-charge density and potential were larger when a β subunit was expressed. These results suggest that expression of an auxiliary subunit can influence calcium channel gating by modifying the sensitivity of the voltage sensor to the membrane potential profile.

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.