Elsevier

Neuroscience

Volume 102, Issue 4, 14 February 2001, Pages 969-978
Neuroscience

Hemicholinium-3 mustard reveals two populations of cycling choline cotransporters in Limulus

https://doi.org/10.1016/S0306-4522(00)00534-0Get rights and content

Abstract

Cholinergic neurons have both a low-affinity and a high-affinity choline transport process. The high-affinity choline transport is sodium dependent and thus it can be referred to as choline cotransport. Choline cotransport has been shown to be up-regulated by neuronal activity. Protein kinase C has also been shown to regulate choline cotransport. Both forms of regulation appear to modulate transport by altering the numbers of choline cotransporters in the nerve terminal membrane. The present study centers on choline cotransporter trafficking in Limulus brain hemi-slice preparations. The competitive, reversible, non-permeant ligand, [3H]hemicholinium-3, was used in binding studies to estimate the relative number of choline cotransporters in plasma membranes. The hemicholinium-3 mustard derivative has been shown to be an irreversible, highly selective, non-permeant ligand for the choline cotransporter, and was also used. Hemicholinium-3 mustard binding to the choline cotransporter blocked [3H]choline transport and [3H]hemicholinium-3 binding. Antecedent elevated potassium exposure of cholinergic tissues has been shown to up-regulate choline transport by the recruitment of additional choline cotransporters to surface membranes. This treatment was also effective in the recruitment of cotransporters following maximal inhibition by hemicholinium-3 mustard of brain hemi-slices. Long-term washout of hemicholinium-3 mustard in hemi-slices resulted in a time-dependent restoration of choline cotransport. Full recovery occurred within 2 h. In uninhibited slice preparations, both staurosporine and chelerythrine, protein kinase C inhibitors, stimulated choline uptake. However, within a 1-h washout recovery of uptake following hemicholinium-3 mustard inhibition, the staurosporine responsive but not chelerythrine responsive transport had returned.

On the basis of these findings, we hypothesize the existence of two distinct populations of cycling choline cotransporters, which includes inactive or “silent” transporters.

Section snippets

Materials

Horseshoe crabs (Limulus polyphemus) were obtained from Marine Biological Laboratories (Woods Hole, MA) and were maintained in moist excelsior at an ambient temperature of 4–6°C. The pharmacological reagents used in this study, chelerythrine, staurosporine and HC-3, were purchased from Sigma Chemical Company (St. Louis, MO). Methyl-[3H]choline chloride (sp. act.=60–90 Ci/mmol) and methyl-[3H]HC-3 diacetate salt (sp. act.=120 Ci/mmol) were purchased from Dupont New England Nuclear (Wilmington,

Effect of 200 μM hemicholinium-3 mustard on 0.1 and 50 μM [3H]choline transport

Gylys and Jenden16 have shown that HCM selectively inhibits HACU in rat brain synaptosomes. To assess the specificity of HCM inhibition in the Limulus brain hemi-slice preparations, we determined the HCM inhibitory effect at two concentrations of [3H]choline, 0.1 and 50 μM. The high-affinity cotransport component of choline uptake at these two concentrations was determined by subtracting out that transport which occurs in the absence of Na+. At 0.1 μM choline, cotransport activity comprises

Discussion

In an effort to elucidate the mechanisms involved in the regulation of the ChCoT, mustard derivatives of choline and HC-3 have been developed and used as probes in numerous in vitro and in vivo studies.9., 35., 36., 37. One such compound, HCM, has been determined to be an irreversible, highly selective inhibitor of HACU.16., 17., 44. Gylys et al.17 reported that HCM is highly selective for the ChCoT, showing no measurable effect on Na+-independent (i.e. low-affinity) choline transport, nor the

Acknowledgements

This work was supported by NIH grants (MBRS) RR08037 and (RIMI) P20-RR11808.

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