A Uniquely Selective Inhibitor of the Mammalian Fetal Neuromuscular Nicotinic Acetylcholine Receptor

Abstract

We have purified and characterized a novel conotoxin from the venom of Conus obscurus, which has the unique property of selectively and potently inhibiting the fetal form of the mammalian neuromuscular nicotinic acetylcholine receptor (nAChR) (␣1␤1␥␦-subunits). Although this conotoxin, ␣A-conotoxin OIVB (␣A-OIVB), is a high-affinity antagonist (IC 50 of 56 nM) of the fetal muscle nAChR, it has Ͼ1800-fold lower affinity for the adult muscle nAChR (␣1␤1⑀␦-subunits) and virtually no inhibitory activity at a high concentration on various neuronal nAChRs (IC 50 Ͼ 100 M in all cases). The peptide (amino acid sequence, CCGVONAACPOCVCNKTCG), with three disulfide bonds, has been chemically synthesized in a biologically active form. Although the neuromuscular nAChRs are perhaps the most extensively characterized of the receptors/ion channels of the nervous system, the precise physiological roles of the fetal form of the muscle nAChR are essentially unknown. ␣A-OIVB is a potentially important tool for delineating the functional roles of ␣1␤1␥␦ receptors in normal development, as well as in various adult tissues and in pathological states. In addition to its potential as a research tool, ␣A-OIVB may have some direct biomedical applications.

α-Conotoxins Identify theα3β4* Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells

“…Binding of a single a-Ctx molecule to a nAChR subunit interface is sufficient to block the allosteric transitions required for ion channel opening (Talley et al, 2006), and binding to a target site can occur with high affinity without regard to composition of other binding sites in the receptor complex. Examples include inhibition of one of five sites in an a7 homopentamer, binding to the a-« or a-d interface in a muscle type nAChR, or binding to the a6-b2 interface in an a6b2a4b2b3 nAChR (Groebe et al, 1995;Sine et al, 1995;Jacobsen et al, 1999;Gotti et al, 2005;Teichert et al, 2005). Thus, the affinities of the LvIA and BuIA analogs for their respective binding would be expected to be similar for a mixed b2 and b4 nAChR subtype.…”

Prolongation of Evoked and Spontaneous Synaptic Currents at the Neuromuscular Junction after Activity Blockade Is Caused by the Upregulation of Fetal Acetylcholine Receptors

“…However, another possible cause of EPC prolongation after block of synaptic activity is postsynaptic upregulation of fetal-type AChRs (Kues et al, 1995), which have a longer mean open time (Sakmann and Brenner, 1978;Fischbach and Schuetze, 1980). To examine the contribution of fetal-type AChRs to prolongation of EPC decay, we selectively blocked fetal-type AChRs using a recently discovered toxin that is selective for fetal-type AChRs (␣A-conotoxin OIVA[K15N]) (Teichert et al, 2005(Teichert et al, , 2006. Before using ␣A-conotoxin OIVA[K15N] on muscle after blockade of synaptic activity, we confirmed that it had no effect on control muscle.…”

“…Determination of the contribution of fetal-type AChRs to EPCs and miniature EPCs (MEPCs) was performed by measuring EPCs and MEPCs in individual muscles before and after application ␣A-conotoxin OIVA[K15N] [synthesized as described previously (Teichert et al, 2005(Teichert et al, , 2006]. After recording from endplates in control Ringer's solution, ␣A-conotoxin OIVA[K15N] was added to 20 ml of Ringer's solution at a final concentration of 1 M, and this solution was bubbled continuously with 95% O 2 /5% CO 2 and recirculated throughout the bath.…”

Essential roles of the acetylcholine receptor γ-subunit in neuromuscular synaptic patterning

“…To determine whether the ε-subunit was assembled into functional AChRs during embryonic stages, we measured EPPs in response to distinct toxins that specifically recognize the γ-AChRs [αA-conotoxin OIVB or αA-OIVB (Teichert et al, 2005)] and ε-AChRs [waglerin 1 (McArdle et al, 1999)]. As shown in Fig.…”