Figure 12. Persistent PKA activation... | Rockefeller University Press

Figure 12.

$Persistent PKA activation promoted trafficking of NaV1.5 from a cytosolic reservoir to the plasma membrane. (A) Biotinylation experiments in HEK293 cells expressing GFP-NaV1.5. Top: Representative immunoblot images of WCL and biotinylated fraction (Biot’) from HEK293 cells expressing GFP-NaV1.5 and native EB1, exposed to PKA for 4–6 h or without PKA for the same duration, without or with pretreatment with chloroquine 100 µM (chloroquine was present during incubation with PKA or CON). Abs used in immunoblotting are listed on the left. NKA = Na/K pump α-subunit as the loading control. The absence of EB1 in Biot’ lanes confirms the lack of contamination from cytosolic proteins. Bottom left: Summary of WCL GFP-NaV1.5 (normalized to CON) and cell surface GFP-NaV1.5 (Biot’/WCL), shown as mean + SE with individual data points. Right: PKA:CON ratio of cell surface GFP-NaV1.5 in “No chloroquine” and “With chloroquine” groups (n =10 and 4, respectively; t test between the two groups: P = 0.046). (B) Immunofluorescence images of native NaV1.5 or GFP-NaV1.5, RyR2, and fluorescence images of wheat germ agglutinin (WGA, marker of plasma membrane and t-tubules) from the types of myocytes listed above. A detailed description is in the Discussion section. LS: lateral surface. The myocyte image second from right is a duplicate from Fig. 3 A, NaV1.5 immunofluorescence image from a control myocyte after culture for 1 h. (C) Cartoon of working hypothesis. Scale bars in B are 10 mm. Source data are available for this figure: SourceData F12.$

Persistent PKA activation promoted trafficking of Na _V 1.5 from a cytosolic reservoir to the plasma membrane. (A) Biotinylation experiments in HEK293 cells expressing GFP-Na_V1.5. Top: Representative immunoblot images of WCL and biotinylated fraction (Biot’) from HEK293 cells expressing GFP-Na_V1.5 and native EB1, exposed to PKA for 4–6 h or without PKA for the same duration, without or with pretreatment with chloroquine 100 µM (chloroquine was present during incubation with PKA or CON). Abs used in immunoblotting are listed on the left. NKA = Na/K pump α-subunit as the loading control. The absence of EB1 in Biot’ lanes confirms the lack of contamination from cytosolic proteins. Bottom left: Summary of WCL GFP-Na_V1.5 (normalized to CON) and cell surface GFP-Na_V1.5 (Biot’/WCL), shown as mean + SE with individual data points. Right: PKA:CON ratio of cell surface GFP-Na_V1.5 in “No chloroquine” and “With chloroquine” groups (n =10 and 4, respectively; t test between the two groups: P = 0.046). (B) Immunofluorescence images of native Na_V1.5 or GFP-Na_V1.5, RyR2, and fluorescence images of wheat germ agglutinin (WGA, marker of plasma membrane and t-tubules) from the types of myocytes listed above. A detailed description is in the Discussion section. LS: lateral surface. The myocyte image second from right is a duplicate from Fig. 3 A, Na_V1.5 immunofluorescence image from a control myocyte after culture for 1 h. (C) Cartoon of working hypothesis. Scale bars in B are 10 mm. Source data are available for this figure: SourceData F12.