title: A high-throughput assay for connexin 43 (Cx43, GJA1) gap junctions using codon-optimized aequorin.
abstract: Gap junctions (GJs) are intercellular channels which are composed of the connexin family of proteins that allow electrical and chemical communications and synchronization in tissue ensembles. Evidence suggests that pharmaceutical modulators of these channels may have therapeutic potential or carry undesired liability. In this report, we exogenously expressed human connexin 43 (Cx43, GJA1) and demonstrated functionality in a 96-well flow cytometry assay detecting intercellular transfer of the calcein dye. We have designed a 384-well high-throughput method for detecting the transfer of calcium between HeLa cells expressing Cx43. In this assay, donor cells coexpress Cx43 and the α1A adrenergic Gα-coupled receptor, while recipient cells coexpress Cx43 and the cytoplasmic version of the calcium-sensitive luminescent protein aequorin enhanced by codon optimization (cytoAeq). The two cell populations were mixed, dispensed to 384-well plates, and incubated for 3 h to allow the formation of GJs. Activation of α1A by epinephrine in donor cells led to dose-dependent calcium increases in recipient cells, which were detected by measuring the intensity of aequorin luminescence. The response was dependent on the expression of Cx43 and inhibited by the GJ blocker 18α-glycyrrhetinic acid, suggesting Cx43 GJ-mediated activity. In a parallel experiment with capsaicin and the TrpV1 ion channel in place of phenylephrine and α1A, a similar magnitude of difference in the maximal calcium response was detected in both donor and recipient cells, suggesting that calcium is likely the permeant ion through the GJ. This assay may pave the way for high-throughput screening of GJ modulators for drug discovery.
authors.affiliation: Biological Reagents and Assay Development, GlaxoSmithKline R&D, Stevenage, United Kingdom.
authors.forename: Nazia
authors.initials: N
authors.lastname: Haq
authors.forename: David
authors.initials: D
authors.lastname: Grose
authors.forename: Emma
authors.initials: E
authors.lastname: Ward
authors.forename: Oiyee
authors.initials: O
authors.lastname: Chiu
authors.forename: Natalie
authors.initials: N
authors.lastname: Tigue
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Dowell
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Powell
authors.forename: Mao Xiang
authors.initials: MX
authors.lastname: Chen
mesh_terms: D000331:Aequorin; D001681:Biological Assay / Q000295:instrumentation*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002469:Cell Separation / Q000295:instrumentation; D003062:Codon / Q000235:genetics; D018031:Connexin 43 / Q000032:analysis* / Q000378:metabolism*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D017022:Flow Injection Analysis / Q000295:instrumentation; D006367:HeLa Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D049408:Luminescent Agents
chemical_list: D003062:Codon; D018031:Connexin 43; D049408:Luminescent Agents; D000331:Aequorin; D002118:Calcium