A Major Portion of Synaptic Basal Lamina Acetylcholinesterase Is Detached by High Salt- and Heparin-containing Buffers from Rat Diaphragm Muscle and Torpedo Electric Organ

Olivia I Casanueva,Tea Garcı́A-Huidobro,Eliseo O Campos,Rebeca Aldunate,Jorge Garrido,Nibaldo C Inestrosa

doi:10.1074/jbc.273.7.4258

Olivia I Casanueva, Tea Garcı́A-Huidobro + Show 4 more

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https://doi.org/10.1074/jbc.273.7.4258

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Abstract

Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.

Highlights

The enzyme acetylcholinesterase (AChE)1 plays a key role in cholinergic neurotransmission [1]
Despite the overwhelming evidence supporting the notion that asymmetric AChE forms are associated with the basal lamina (BL) through electrostatic bonds, it was recently suggested that the enzyme displaced by high ionic strength and heparin buffers was non-junctional and originated in intracellular compartments [20, 21]
Asymmetric AChE Is Associated with BL-enriched Preparations—Since it was recently reported that neither high salt- nor heparin-containing buffers detached asymmetric collagentailed forms of AChE from the BL of the vertebrate neuromuscular junction [20, 21], we wanted to re-evaluate the solubilization properties of the asymmetric forms present in BLenriched preparations, derived from both diaphragm muscle

Summary

Introduction

The enzyme acetylcholinesterase (AChE) plays a key role in cholinergic neurotransmission [1]. The precise mechanisms by which asymmetric AChE forms are anchored to the BL remain elusive [9], there is compelling evidence to suggest that heparan sulfate proteoglycans (HSPGs) or related proteoglycans are involved [7, 10]. This evidence includes the recent finding that A12 has two heparin-binding consensus sequences in its collagenic tail [11]. Heparin and heparan sulfate have been shown to release asymmetric AChE activity from rat muscle end plate regions [15] and BL sheets purified from the electric organ of Discopyge [16]. Strongly suggest that both high salt and heparin buffers solubilize a major portion of extracellular collagen-tailed AChE and demonstrate the capacity of these buffers to dissolve AChE-rich regions from neuromuscular junctions, as detected by histochemical staining

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