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Abstract

The efflux of [³H] choline⁺ from the proximal tubular lumen was measured by using the stop-flow microperfusion method. The 2-s efflux of [³H] choline⁺ follows kinetics with a Michaelis constant, K_m = 0.18 mmol x l^-1, maximal flux, J_max = 0.43 pmol x cm^-1 x s^-1 and a permeability term = 38.0 micron² (small middle dot)^-1. Replacement of Na⁺by N-methyl-D-glucamine⁺ or Li⁺, or a change of luminal pH do not alter choline⁺ efflux. Replacement of Na⁺ by Cs⁺ inhibits 2-s choline⁺ (0. 01 mmol x l^-1) efflux by 22% and replacement by K⁺ inhibits by 49%, indicating that the electrical potential difference across the brush border membrane acts as driving force for choline⁺ transport. Comparing the apparent luminal inhibitory constant values for choline (app. K_{i,l,choline⁺}) with the chemical structure of inhibiting substrates, it was found that the inhibitory potency of amines with high pK_a values, i.e. high basicity, and of quaternary ammonium compounds (tetraethyl to tetrahexylammonium) increases with their hydrophobicity in a similar manner as was observed previously against the contraluminal N¹-methylnicotinamide (NMeN⁺) transporter and the luminal H⁺ /organic cation (N-methyl-4-phenylpyridinium) (MPP⁺) exchanger. Independently of their hydrophobicity, an increase in the inhibitory potency of the homologous series of aminoquinolines against the choline⁺ transporter was observed with increasing pK_a values, i.e. increasing basicity, as was found previously against the two other organic cation transporters. A third parameter influencing the interaction with the choline⁺ transporter is the presence of two amino groups with high pK_a values or one amino group and a permanent positive charge, as is documented with the two-ring aminostyryl and rhodamine compounds, as well as three-ring aminoacridine, aminophenanthrene and cyanine compounds. Thus with the aminostyryl, pyridinium⁺, rhodamine, phenanthridium⁺ and cyanine⁺ dyes app.K_{i,l,choline⁺} values of between 0.01 and 0.07 mmol x l^-1 have been found. A fourth parameter influencing the choline⁺ transporter is the presence of an OH group on the C atom next to that bearing the N atom (as in choline⁺) or an ester-OCOR group (acetylcholine⁺, butyrylcholine⁺) or a thioester-SCOR-group (acetylthiocholine⁺, butyrylthiocholine⁺); or an -OP(OH)₂(OR) group (glycerylphosphoryl-choline⁺), resulting in app.K_{i,l,choline⁺} values of 0.3-1.0 mmol x l^-1. Thus the substrates for the luminal choline⁺ transporter have general features in common with the luminal H⁺/organic cation exchanger and the contraluminal organic cation transporter, i.e. hydrophobicity and basicity. Additional parameters for interaction are an OH (or similar) group positioned a favourable distance from the N atom or a second amino/ammonium group in multi-ring compounds.