Voltage-dependent Ca 2ϩ channels (Ca V ) are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1-7) that are classified into three main subfamilies according to their high voltageactivated (HVA) 2 gating (Ca V 1 and Ca V 2) or low voltage-activated gating (Ca V 3). In addition to the transmembrane poreforming Ca V ␣1 subunit, Ca V 1 and Ca V 2 channels arise from the multimerization of three other proteins (7): a cytoplasmic Ca V ␤ subunit, a mostly extracellular Ca V ␣2␦ subunit, and calmodulin constitutively bound to the C terminus of Ca V ␣1 (8 -12).A considerable body of work documents the interaction and modulation of the Ca V ␣1 subunit of Ca V 1 and Ca V 2 channels (13-18) by the auxiliary Ca V ␤. The high affinity Ca V ␣1-Ca V ␤ interaction site on the pore-forming Ca V ␣1 subunit is a conserved 18-residue sequence in the I-II linker called the ␣ interaction domain (AID) (19,20) that has been structurally resolved by high resolution x-ray crystallography (21-23). Structural work showed that the AID forms a ␣-helix that binds to the ␣ binding pocket (ABP) in the Ca V ␤ nucleotide kinase (NK) domain. It has been proposed that the MMQKAL cluster of residues within the latter determines the high affinity nanomolar interaction between the two proteins (24 -29). Numerous mutational analyses of the AID residues have correlated the Ca V ␤-induced biophysical modulation with the high affinity binding of Ca V ␤ to the AID peptide in a variety of Ca V ␣1 isoforms for Ca V 1 and Ca V 2 channels (25, 29 -32).The association of Ca V ␣1 and Ca V ␤ subunits is also critical for proper channel maturation and cell surface expression of Ca V 2.2 (17), Ca V 1.2 (33, 34), and Ca V 2.3 (35). In Ca V 2.2, the I-II linker is presumed to play a role in this process (17,18), and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by Ca V ␤1b and Ca V ␤3 (32). In addition, the Ca V ␤2-induced increase in Ca V 1.2 whole cell currents was abolished with the AID-defective YWI/AAA mutant (29), suggesting that high affinity binding of Ca V ␤ onto AID is required to traffic Ca V ␣1 to the plasma membrane. Nonetheless, the unique character of the high affinity AID-ABP interface in the membrane targeting of Ca V ␣1 has been questioned (27, 36 -40). In particular, it has been suggested that Ca V ␤-mediated plasma membrane targeting could be uncoupled from Ca V ␤-mediated modulation of channel gating (26, 41) with important contributions from other intracellular regions (33, 39,(42)(43)(44).In addition to Ca V ␤, the ancillary subunit Ca V ␣2␦ and the ubiquitous calmodulin (CaM) protein have also been proposed to modulate HVA channel maturation and targeting (9). For instance, co-expression of Ca V ␣2␦ promoted the trafficking of the Ca V ␣1 subunit of Ca V 2.2 in COS-7 cells (45), suggesting that Ca V ␣2...

Voltage-dependent Ca 2ϩ channels are membrane proteins that play a critical role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells (1-6). The Ca V ␣1 subunits of voltage-dependent Ca 2ϩ channels are evolutionarily related to the ␣ subunit of K V channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1-S6). Although the overall identity at the primary sequence level is quite low between Ca V and K V channels, it improves significantly for transmembrane segments. By homology with the three-dimensional structures of KcsA, MthK, K V AP, KirBac, and K V 1.2 channels (7-11), the four S4 transmembrane segments are thus believed to act as the voltage sensor, whereas the S6 helices line the channel pore of Ca V ␣1.Both in K V and Ca V channels, the question as to how the S4 motion is transmitted to the S6 helix to open the gate upon depolarization remains heavily studied. In the electromechanical coupling model based upon the K V 1.2 crystal structure (12), the S4S5 linker, which is located within atomic proximity (4 -5 Å) of the S6 helix, interacts with the latter in the closed state of the channel. The movement of the S4 voltage sensor is likely to induce a conformational change that pulls the S4S5 linker away from the S6 inner helix ultimately resulting in ions flowing through the pore. The closed conformation of the channel is postulated to be stabilized by hydrophobic interactions at the level of the "closed bundle" in S6 (13-15).Experimental evidence supporting a role for the S4S5 linker in the voltage dependence of activation of K V channels is steadily mounting. Introduction of the S4S5 linker from KcsA disrupts the voltage-dependent activation of K V 2.1 (16, 17). In hERG, cross-linking studies have shown that residues located in the S4S5 linker and in the S6 helix are in atomic proximity and that gating currents were greatly affected as a result of disulfide bridge formation (18). Furthermore, mutations of a unique glycine residue (Gly-546) within the N-terminal end of the S4S5 linker were shown to stabilize the closed state by ϳ1.6 -4.3 kcal/mol and restricted the voltage sensor movement (19).The potential coupling mechanism between the S4S5 linker and the S6 helix has yet to be explored in Ca V channels. In contrast to K V channels, Ca V channels are structurally asymmetrical with four distinct albeit homologous S4S5 linkers and four distinct S6 helices. We have already shown that glycine substitutions in the distal S6 regions of domains I, II, III, and IV altered the relative stability between the open and closed states (20). Mutations within IIS6 were found to impact most significantly on the activation gating of Ca V 2.3. In particular, I701G shifted by Ϫ35 mV the voltage dependence of activation while slowing down inactivation and deactivation kinetics (20). These data indicated that IIS6 plays a unique role in the channel equilibrium between the closed and the open state(s) in Ca V 2.3.To determine whether the S4S5 linker of Domai...

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Large-conductance, voltage-and Ca 2þ -activated K þ channels (MaxiK, BK) are broadly expressed ion channels typically observed as a plasma membrane protein in various cell types. In murine astrocyte primary cultures, which are more indicative of in-vivo reactive astrocytes rather than resting astrocytes, our previous results using high-resolution confocal microscopy have revealed the novel finding that MaxiK pore-forming a subunit (MaxiKa) is distributed intracellularly, colocalized along the microtubule network. This MaxiKa association with microtubules was further confirmed by in vitro His-tag pulldown assays, co-immunoprecipitation assays from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca 2þ elicited by general pharmacological agents, caffeine (20mM) or thapsigargin (1mM), resulted in increased MaxiKa labeling at the plasma membrane. More notably U46619, a stable analog of thromboxane A2 (TXA2) which triggers Ca 2þ -release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKa surface labeling. We now show using whole-cell patch clamp recordings that U46619 stimulated cells develop a ~3-fold increase in current amplitude. This data indicates that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. These changes in MaxiKa plasma membrane distribution are effectively blocked by preincubating astrocytes with a cell permeable Ca 2þ -chelator, BAPTA-AM, or by microtubule disruption prior to stimulation. While microtubules are largely absent in mature astrocytes, our immunohistochemistry results in brain slices show that cortical astrocytes in the developing newborn mouse brain (P1) have a robust expression of microtubules that significantly colocalize with MaxiKa. The results of this study provide the novel insight that suggests Ca 2þ released from intracellular stores, may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiKa stores to the plasma membrane of reactive astrocytes. Supported by NIH.

La réalité virtuelle, qui implique un environnement généré par un système informatique donnant une impression de réalité, de présence et d'engagement (Pellas et al., 2020), a connu des développements dans le domaine de l’éducation (Freina et Ott, 2015; Jensen et Konradsen, 2018). Les avantages qu’elle présente, notamment pour la visualisation des concepts abstraits, pour la réalisation de tâches expérimentales difficiles ou impossibles à réaliser dans la réalité ainsi que pour la motivation, l’engagement et le transfert des apprentissages la rendent particulièrement utile pour l’apprentissage des sciences (Dalgarno et Lee, 2010; Lewis et al., 2021; Shin, 2017). En nous ancrant dans une démarche adaptée de l’analyse de la valeur pédagogique (Rocque et al., 1998), du modèle ADDIE, de l’art de la conception des jeux sérieux (Ryerson University, 2018) et d’un modèle de conception d’applications en réalité virtuelle (Vergara et al., 2017), nous avons développé de manière itérative différents jeux sérieux en réalité virtuelle en sciences au collégial (biologie, chimie et physique) pour finalement les mettre à l’essai en classe à l’automne 2022. Cet article vise à partager le processus expérimenté pour le développement, les résultats de chacune des étapes de ce processus ainsi que les principes qui en sont ressortis. Le tout sera utile aux acteurs du milieu de l’éducation désirant développer des jeux sérieux en réalité virtuelle.

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