Luminal transport system for choline⁺ in relation to the other organic cation transport systems in the rat proximal tubule

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 · 1⁻¹, maximal flux, J _max = 0.43 pmol · cm⁻¹· s⁻¹ and a permeability term = 38.0 μm² · s⁻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 · 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 N ¹-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,1,choline ⁺ values of between 0.01 and 0.07 mmol·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,1,choline ⁺ values of 0.3–1.0 mmol · 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.