Design of the ⁶²Gap27 mimetic peptide and its role in the regulation of intercellular communication. (A) Predicted 3-dimensional model of the Cx62 tertiary structure. The ribbon view of the structure is colored using the temperature coloring scheme in which blue indicates ordered regions with low predicted per-residue errors, and red indicates high per-residue errors and more flexibility. (B) Schematic representation of the designed ⁶²Gap27 sequence on Cx62. In the topologic diagram of the Cx62 protomer, the predicted binding site is highlighted in orange. NT, NH₂ terminus; CL, cytoplasmic loop; CT, COOH terminus; T, transmembrane; E, extracellular. (C) Surface representation of Cx62 hemichannels being targeted by ⁶²Gap27 showing the pore cross-section and side views. (D) Inter-protomer interactions. The hemichannel formed by 6 protomers of Cx62 is shown in gray ribbon view, and the side chains in the zoomed views are shown as sticks with brown and yellow colors to differentiate between the residues of interacting protomer pairs. (E) Modeled intercellular interactions between docked hemichannels. In the left-hand panel, a Cx62 gap junction channel is shown. The region enclosed by dashed lines is sectioned perpendicular to the pore axis and is viewed from the pore axis (right-hand panel). The interactions between the 2 docked hemichannels (the first external loop [E1] and the second external loop [E2] regions) are depicted in the close-up images. In region E1, Gln58 forms symmetrical hydrogen bonds with the same residue from the opposite protomer, whereas Asn55 forms a hydrogen bond with Arg57 in the opposite protomer. In region E2, Asn196 and Asp199 form hydrogen bonds with the same residues on the opposite protomer. (F) The efflux of calcein from human platelets was measured using flow cytometric analysis. Calcein-loaded platelets incubated with ⁶²Gap27 or scrambled peptide (100 μg/mL) were stimulated with thrombin (0.1 U/mL). Representative histograms of calcein fluorescence for unstimulated (green) and thrombin-stimulated platelets in the presence of scrambled peptide (blue) or ⁶²Gap27 (100 µg/mL) (orange) (n = 4). (G) Calcein efflux after thrombin stimulation for varying time periods was measured by the rate of fluorescence reduction in platelets. Median fluorescence intensity for unstimulated and stimulated samples treated with scrambled peptide or ⁶²Gap27 was analyzed (n = 4). (H) Calcein-loaded platelets were treated with scrambled peptide or ⁶²Gap27 (100 µg/mL) for 5 minutes before their stimulation on fibrinogen and collagen-coated coverslips, and fluorescence recovery after photobleaching analysis was performed. Images represent fluorescence recovery (Pre-bleach, At-bleach, and Post-bleach) in samples treated with scrambled peptide or ⁶²Gap27. (I) Quantified data show mean fluorescence recovery intensity of scrambled peptide and ⁶²Gap27-treated samples normalized to the level of fluorescence at bleach point (shown in red circles; panel H) (n = 5). Data are mean ± standard error of the mean (SEM). ****P < .0001 (calculated by 2-way ANOVA).