Figure 2.

A cholestanol lipid anchor increases the relative affinity and potency of an NK₁R antagonist.A and B, the affinity of Span compared to Span-Chol was assessed by competition with fluorescent SP-TAMRA in HEK-NK₁R cells by high-content imaging (n = 5). HEK-NK₁R cells were preincubated with vehicle (0.1% v/v DMSO; total binding) or increasing concentrations of Span or Span-Chol for a total of 30 min (A) or 4 h (B) at 37°C prior to addition of 0.5 nM SP-TAMRA. Data are expressed as a percentage of the fluorescent intensity measured in the presence of 10 nM Span or Span-Chol (%FLUOR_Max). Symbols show means, and error bars S.E.M. of five independent experiments performed in triplicate. C and D, Calcium transients were measured in HEK-NK₁R cells in response to 1 nM SP following short (30 min; C) or long (4 h; D) preincubation with increasing concentrations of Span or Span-Chol (n = 3). Four-h preincubation experiments compared continuous exposure to antagonist (4 h) versus a “pulsed” exposure (0.5 h exposure, wash [W], 3.5 h rest). Symbols show means, and error bars S.E.M. of three independent experiments performed in triplicate.