Figure 6.

Only Span-Chol, and not Span, inhibits NK₁R signaling from endosomes. After a 4 h preincubation Span-Chol, but not Span, blocks SP-stimulated sustained increases in PKC and cAMP (n = 3). A and B, HEK cells transfected with HA-NK₁R and cytoCKAR (A) or cytoEpac2 (B) were stimulated with vehicle (0.0001% v/v MilliQ H2O) or 1 nM SP and signaling was measured over 20 min. C, cartoon of the sequence of events in endosomes following NK₁R stimulation. β-arr, β-arrestin. The orange circle indicates the time at which internalization starts, and the vertical orange line indicates when it reaches a plateau. Signaling is represented by black lines. D–H, HEK cells transfected with HA-NK₁R and cytoCKAR (D–E) or cytoEpac2 (F–G) were pretreated with 1 μM Span (D, F) or Span-Chol (E, G) for 4 h prior to addition of vehicle (0.0001% v/v MilliQ H2O) or 1 nM SP. Symbols show the mean, and error bars represent S.E.M. of grouped cells from three independent experiments. H, smoothed time course traces showing the change in effectiveness of Span versus Span-Chol at blocking SP-stimulated PKC signaling as the NK₁R transitions from the plasma membrane (data taken from Fig. 5A) to endosomes (data from Fig. 6A). Data are expressed as the FRET relative to the baseline FRET (F/F₀). Arrows indicate time of vehicle/SP addition.