Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier

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Abstract
Summary The neuronal noradrenaline uptake mechanism (uptake1) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K m) and the maximal initial transport rate (V max) were determined. Furthermore, the dissociation constants (K D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V max, K m, and K D. Neither K m and V max nor K D and V max were found to be correlated. However, the discrepancy between K m and K D expressed as the ratio, Km/KD, was negatively correlated with V max (r = − 0.9315, n = 7, p < 0.01). For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake1. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K D). (2) The discrepancy between K m and K D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V max). The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K m/K D and Vmax supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.

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