Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Abstract

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium-binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq-like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin- and dynamin-like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq-like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium-binding regions (EF hands) are conserved. The widespread occurrence of frq-like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons.

Long‐term in vivo modulation of synaptic efficacy at the neuromuscular junction of Rana pipiens frogs

“…We performed a Western blot analysis on sartorius and CP muscles, with quantal content of CP NMJs being 3-4 times greater than that of the sartorius (Grinnell & Herrera, 1980), to compare their level of frequenin expression. Consistent with the data obtained from crayfish terminals (Jeromin et al 1999), we found a greater amount of frequenin protein in CP muscles (Fig. 7A).…”

“…For example, frequenin, a well-conserved Ca 2+ -binding protein is known to enhance transmitter release in Drosophila and Xenopus nerve terminals (Pongs et al 1993;Olafsson et al 1995). Frequenin is also likely to play a role in synaptic transmission of the crayfish, since it is more prominently expressed in phasic nerve terminals compared to tonic (Jeromin et al 1999).…”

“…A potential candidate for that function could be the Ca 2+ -binding protein frequenin. Frequenin is believed to modulate neurotransmitter release (Pongs et al 1993;Olafsson et al 1995) and is differentially expressed in tonic and phasic motoneurones of the crayfish (Jeromin et al 1999). However, we found no statistically significant changes in frequenin immunoreactivity and protein levels in stimulated preparations.…”

“…New evidence at the crayfish NMJ suggests a difference in the Ca 2+ dependance of vesicle priming (Millar et al 2005). Among the priming molecules possibly involved, there is the neuronal calcium sensor-1 (Zucker, 2003) who's homologue, frequenin, in crayfish phasic terminals is known to facilitate evoked neurotransmission (Jeromin et al 1999). Although many studies have used invertebrates to investigate the differences in synaptic efficacy between phasic and tonic synapses, little has been done in vertebrate models.…”

The neuronal calcium sensor family of Ca2+-binding proteins

“…The KChIPs 2 and 3(b) have a variable N-terminal extension (yellow), while the other members of the family have an Nterminal myristoylation consensus sequence (a). Gly 42 and Gly 96 , which are conserved in all members of the family, are the sites of rotation following Ca 2 + binding in recoverin.…”