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

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.The enzyme acetylcholinesterase (AChE) 1 plays a key role in cholinergic neurotransmission (1). Its predominant form at the neuromuscular junction is the collagen-tailed asymmetric form, A 12 , which is located on the extracellular surface positioned for the hydrolysis of acetylcholine. Most of this junctional AChE is associated with the basal lamina (BL), located between the nerve ending and the muscle plasma membrane (2, 3), and can be removed from the cell surface of muscle tissue (4, 5) and mouse myotubes (6) by treatment with collagenase, indicating that the collagenic tail of the enzyme is involved in its anchorage to the BL (7,8). Although 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 A 12 has two heparin-binding consensus sequences in its collagenic tail (11). Asymmetric AChE forms have a high binding affinity for BL components, particularly HSPGs (12) which are themselves major constituents of basement membranes (13,14). Heparin and heparan sulfate have also 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). The demonstration that A 12 could bind and be selectively eluted from heparin-agarose columns, whereas non-collagenous forms and A 12 after collagenase treatment could not, proved the direct interaction of A 12 with heparin in vitro (11,17). Direct interactions with heparin/heparan sulfate moieties in vivo have also been demonstrated. Asymmetric AChE forms were shown to bind the surface of HSPGri...

Acetylcholinesterase Dynamics at the Neuromuscular Junction of Live Animals

“…It has been demonstrated that part of the ColQ-AChE complex is covalently associated in the basal lamina, and it thus seems likely that this enzyme represents a very stable pool of AChE, whereas the nonlinked complex may be more dynamic. To date, the proportion of this covalently linked ColQ-AChE is debated, so it may represent the majority of the enzyme (33) or only a minor proportion (34). From our quantification, we estimate that approximately one-third of the AChEs could be very stable, since the labeling could be found weeks after initial saturation.…”

Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin

“…These workers provided evidence, in vertebrate skeletal muscle and Torpedo electric organ, that only A forms of AChE interact with heparin and\or other proteoglycans, via motifs on their collagen tails [15,21]. We showed previously that approx.…”

“…The A forms are believed to be attached to the basal lamina within the synaptic cleft by interaction with heparan sulphate [15]. The reason for this elaborate polymorphism is, as yet, unknown, but has been ascribed to the functional requirements of different types of synapses [1,16,17].…”

The spectrum of mutations causing end-plate acetylcholinesterase deficiency

“…One mechanism for anchoring ColQ involves electrostatic interactions between clustered positive charges of the two sets of heparan sulfate proteoglycan binding sites in the collagen domain with negatively charged basal lamina molecules, such as perlecan. 15,16 However, complete extraction of asymmetric AChE from the basal lamina requires collagenase digestion, 25,26 and this implies an additional nonelectrostatic anchoring mechanism. Consistent with this, Casanueva and collaborators 27 recently found two collagenous molecules (140 and 195-215 kd) in basal lamina that bind asymmetric AChE.…”