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 α2βδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αβδ subunits (presumed to be α2βδ2 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 α2βδ2 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 α2βδ2 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 α2βδ2 receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.
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 βδγ 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.
Slow channel syndrome (SCS) is an autosomal dominant disease resulting from mutations in muscle acetylcholine receptor (AChR) 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 acetylcholine (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 multi-exponential 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 non-liganded openings points to dual sources for the calcium-dependent myopathy in certain forms of SCS.
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