Figure 9. Mechanism of APN-mediated increase in substrate affinity of B⁰AT1.

Oocytes were injected with either 10 ng of B⁰AT1 cRNA, 15 ng of APN cRNA or 2 ng of collectrin cRNA. (A) Oocytes were voltage clamped at −50 mV and subsequently superfused with either 10 mM leucine or 10 mM Leu-Ser-Lys-Leu tetrapeptide. Each tracing is a typical example of currents observed in all oocytes injected with the cRNA indicated. Leucine-induced sodium currents were recorded on day 4 and 5 post-injection for all oocytes. (B) Oocytes were recorded as indicated in (A). Each data point indicates an APN mutant co-expressed with B⁰AT1, or B⁰AT1 co-expressed with collectrin. All peptide-induced sodium currents were normalized to the corresponding leucine-induced Na⁺ current (I_Pep/I_Leu). Each data point represents the mean±S.D. for both peptide-induced sodium currents and apparent K_m values. The trend line was fitted using linear regression (r=−0.84, P=0.035 and n=74). (C) A total of 15 oocytes/sample were incubated in 0.5 mg/ml sulfo-NHS-LC-biotin on day 5 post-injection before being lysed and treated with streptavadin-coated agarose beads. Samples were separated by SDS/PAGE. Subsequently B⁰AT1 was detected by immunoblotting. Molecular masses are indicated to the left-hand side in kDa.