Les canaux ioniques permettent le passage des ions continuel ou en réponse à des stimuli, jouant un rôle important dans le fonctionnement cellulaire. Les mutations génétiques dans les canaux ioniques peuvent causer plusieurs troubles neurodéveloppementaux et cardiovasculaires, collectivement appelés canalopathies. Bien que les altérations génétiques des canaux ioniques modifient l’expression des canaux, les mécanismes cellulaires régulant l'expression des canaux à la surface restent inconnus. Pour répondre à cette question, nous avons étudié l'expression en surface des variantes zH4IR et W434F du canal potassique régulé par le voltage (Kv) Shaker. La variante W434F s'exprime près de 300 fois plus que zH4IR. La coexpression des deux variantes indique la formation d'un complexe hétéromérique entre les deux variantes. Pour vérifier si les propriétés non conductrices de la variante W434F modulent positivement son trafic du réticulum endoplasmique (ER) vers la surface, nous avons introduit un courant de K+ dans la membrane plasmique ou l'ER en utilisant soit des variantes zH4IR, soit KDEL d'un canal potassique rectifiant inward Kir7.1, la séquence KDEL entraînant la rétention de Kir7.1 dans l'ER. La coexpression avec Kir7.1-KDEL, et non avec Kir7.1, a diminué le nombre de canaux W434F à la surface, tandis que l'expression de zH4IR est restée inchangée. L'expression des variantes de canaux en présence de 4-Aminopyridine (4-AP), un bloqueur de Kv perméable, a augmenté le nombre de canaux zH4IR, mais pas ceux de W434F. Cela suggère que la conductance de K+ à travers l'ER lors de l'assemblage des canaux régule le nombre de canaux Kv à la surface de la cellule. / Ion channels permeate ions either constitutively or in response to stimuli playing a vital role in cellular function and homeostasis. Genetic variations in ion channels lead to several neurodevelopmental and cardiovascular disorders collectively referred to as channelopathies. While genetic alterations in ion channels alter functional channel expression, cellular mechanisms regulating surface channel expression remain elusive. To address this question, we examined the surface expression of the Shaker voltage-gated potassium (Kv) channel variant with the W434F mutation introduced into the inactivation-removed (zH4IR) background. The latter variant expresses nearly 300 times the level of zH4IR, with the number of zH4IR channels determined from ionic currents and the variant's expression level determined from gating currents. Coexpression of both variants indicate heteromeric complex formation between the two variants. To check if the non-conducting properties of the W434F variant positively modulates its trafficking from the endoplasmic reticulum (ER) to the surface, we introduced K+ leak into the plasma membrane or the ER using either zH4IR or KDEL variants of an inward rectifying potassium Kir7.1 channel, respectively with the KDEL sequence leading to ER retention of KiR7.1. Coexpressing with Kir7.1-KDEL, and not Kir7.1, decreased the number of surface W434F channels, while the zH4IR expression remained unchanged. Expressing the channel variants in the presence of 4-Aminopyridine (4-AP), a permeable Kv blocker, increased the number of zH4IR channels, but not W434F channels. This suggests that K+ conductance through the ER on assembly of the channels regulates the number of Kv channels on the cell surface.

Ca2+ is a universal intracellular messenger that regulates many cellular responses. Most cells express inositol 1,4,5-trisphosphate receptors (IP3R) that mediate Ca2+ release from the endoplasmic reticulum (ER) when they bind IP3 produced after activation of cell-surface receptors. Vertebrate genomes encode three closely related subtypes of IP3R (IP3R1-3). High-resolution optical analyses have revealed a hierarchy of IP3-evoked Ca2+ signals that are thought to arise from the co-regulation of IP3Rs by IP3 and Ca2+. The smallest events ('blips') report the opening of single IP3Rs, Ca2+ 'puffs' report the almost simultaneous opening of a few clustered IP3Rs, and as stimulus intensities increase further Ca2+ signals propagate regeneratively as Ca2+ waves. The aim of this study was to establish whether all three IP3R subtypes can generate Ca2+ puffs. I first used a haploid cell line (HAP1 cells) to generate, using CRISPR/Cas9, a line lacking all endogenous IP3Rs. However, for analyses of Ca2+ puffs, I used HEK cells that had been engineered, using CRISPR/Cas9 to disrupt endogenous genes, to express single IP3R subtypes. Local Ca2+ signals evoked by flash-photolysis of caged- IP3 were recorded using Cal520 and total internal reflection fluorescence (TIRF) microscopy in human embryonic kidney (HEK) cells. The Flika algorithm was used, and validated, for automated detection of Ca2+ puffs and to measure their properties. IP3 evoked Ca2+ puffs in wild-type HEK cells and in cells expressing single IP3R subtypes. In wild-type cells, the Ca2+ signals invariably propagated regeneratively to give global increases in cytosolic [Ca2+]. This occurred less frequently in cells expressing single IP3R subtypes, commensurate with their lower overall levels of IP3R expression. The properties of the Ca2+ puffs, including their rise and decay times, durations, the size of the unitary fluorescence steps as channels closed channel during the falling phase, and the estimated number of active IP3Rs in each Ca2+ puff, were broadly similar in each of the four cell lines. The latter observation suggests that despite lower overall levels of IP3R expression (~30%) in cells with single subtypes relative to WT cells, there is a mechanism that ensures formation of similarly sized IP3R clusters. The only significant differences between cell lines were the slower kinetics of the Ca2+ puffs evoked by IP3R2, which may suggest dissociation of IP3 from its receptor contributes to the termination of Ca2+ puffs. My results demonstrate, for the first time, that all three IP3R subtypes can generate Ca2+ puffs. I conclude that Ca2+ puffs are fundamental building blocks of all IP3-evoked Ca2+ signals.