Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: Effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide

Abstract

Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outward-facing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971) J. Physiol. (London) 213:647--667; Edwards, P.A. (1973) Biochim. Biophys. Acta 311:123--140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since 14C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inward-facing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carrier-substrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.

Characterization of choline transport at maternal and fetal interfaces of the perfused guinea‐pig placenta.

“…However, it is not possible to speculate further regarding the inhibitory effect of NEM on choline transport in the placenta. Choline transport in erythrocytes (Martin, 1971) and brain synaptosomes (Diamond & Kennedy, 1969) was irreversibly inhibited by sulphydryl reagents, and according to the interpretation of Martin (1971), later confirmed by Deves & Krupka (1981), only the inward-facing conformation of the choline carrier reacts with NEM. An additional factor to be considered in the placenta is the metabolic inhibitory effects of NEM since it has been'shown to inhibit cholineacetyltransferase (Roskoski, 1974).…”

“…In nerves, choline uptake is mainly related to cellular acetylcholine (ACh) synthesis and has been studied in the squid giant axon (Hodgkin & Martin, 1965) and brain synaptosomes (Marchbanks, 1968). In the erythrocyte membrane a choline transport system of unknown function has been very well characterized (see review by Martin, 1977;Deves & Krupka, 1981). In epithelia such as the intestinal mucosa (see review by Rose, 1980) choline is also transported by a specific carrier-mediated process, however, here transepithelial (lumen-to-blood) transfer of choline, rather than cellular utilization for metabolic processes, appears to be dominant.…”

The carrier reorientation step in erythrocyte choline transport: pH effects and the involvement of a carrier ionizing group

“…This conclusion is supported by evidence from three different types of experiments, namely accelerated exchange [5,9,13], noncompetitive inhibition by a nontransported substrate analog present in the trans compartment [5], and inactivation of the system by N-ethylmaleimide in the presence of substrates and substrate analogs [4,6,14]. Taken together, the observations show that (i) dissociation of the substrate is rapid compared with the reorientation of the free carrier or the carrier-substrate complex, and (ii) reorientation of the complex is rapid compared with that of the free carrier; i.e., in terms of the carrier scheme in Fig.…”

“…Several lines of evidence have shown that in the choline transport system of erythrocytes, which depends on a mechanism corresponding to the kinetic scheme in Fig. 1 [4,6,10,14], the slowest step in transport is the reorientation of the free carrier [3,4,6,9,13], with the rate constants in the scheme declining in the following order: k-l, k-2 >> f2, f-2 >> fl, f-1. The choline system would therefore lend itself to an investigation of the problem.…”

The choline carrier of erythrocytes: Location of the NEM-reactive thiol group in the inner gated channel

“…j As the channels exist in f In the choline system, diffusion of a substrate molecule from its binding site into the bulk solution (i.e. dissociation from the substrate site) is a rapid (nonrate-limiting) step in transport [3]. Passage through the channels may therefore be regarded as being relatively unhindered, and as in the model in Fig.…”