Zebrafish model for congenital myasthenic syndrome reveals mechanisms causal to developmental recovery.

Mutations in muscle ACh receptors cause slow-channel syndrome (SCS) and Escobar syndrome, two forms of congenital myasthenia. SCS is a dominant disorder with mutations reported for all receptor subunits except γ. Escobar syndrome is distinct, with mutations located exclusively in γ, and characterized by developmental improvement of muscle function. The zebrafish mutant line, twister, models SCS in terms of a dominant mutation in the α subunit (α(twi)) but shows the behavioral improvement associated with Escobar syndrome. Here, we present a unique electrophysiological study into developmental improvement for a myasthenic syndrome. The embryonic α(twi)ßδγ receptor isoform produces slowly decaying synaptic currents typical of SCS that transit to a much faster decay upon the appearance of adult ε, despite the α(twi) mutation. Thus, the continued expression of α(twi) into adulthood is tolerated because of the ε expression and associated recovery, raising the likelihood of unappreciated myasthenic cases that benefit from the γ-ε switch.

Acetylcholine receptor gating in a zebrafish model for slow-channel syndrome.

Slow-channel syndrome (SCS) is an autosomal-dominant disease resulting from mutations in muscle acetylcholine (ACh) receptor subunits. The associated fatigue and muscle degeneration are proposed to result from prolonged synaptic responses that overload intracellular calcium. Single-channel studies on reconstituted receptors bearing human mutations indicate that the prolonged responses result from an increase in receptor open duration and, in some cases, increased sensitivity to ACh. We show that both of these aberrant receptor properties are recapitulated in heterozygotic zebrafish bearing an L258P mutation in the α subunit, thus affording the unique opportunity to compare the single-channel properties of mutant receptors to the synaptic currents in vivo. Whole-cell recordings revealed synaptic currents that decayed along a multiexponential time course, reflecting receptors containing mixtures of wild-type and mutant α subunits. Treatment with quinidine, an open-channel blocker used to treat the human disorder, restored fast synaptic current kinetics and the ability to swim. Quinidine block also revealed that mutant receptors generate a large steady-state current in the absence of ACh. The spontaneous openings reflected a destabilization of the closed state, leading to an apparent increase in the sensitivity of these receptors to ACh. The effective block by quinidine on synaptic currents as well as nonliganded openings points to dual sources for the calcium-dependent myopathy in certain forms of SCS.

An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses.

Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned α(2)ßδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αßδ subunits (presumed to be α(2)ßδ(2) receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of γ/ε-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed α(2)ßδ(2) receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for α(2)ßδ(2) receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down ε subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the α(2)ßδ(2) receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.