MOLECULAR-CLONING AND FUNCTIONAL-CHARACTERIZATION OF THE XENOPUS CA2-BINDING PROTEIN FREQUENIN()

Frequenin was originally identified in Drosophila melanogaster as a Ca 2+-binding protein facilitating transmitter release at the neuromuscul ar junction. We have cloned the Xenopus frequenin (Xfreq) by PCR using degenerate primers combined with low-stringency hybridization. The de duced protein has 70% identity with Drosophila frequenin and about 38- 58% identity with other Ca2+-binding proteins. The most prominent feat ures ari: the four EF-hands, Ca2+-binding motifs. Xfreq mRNA is abunda nt in the brain and virtually nondetectable from adult muscle. Western blot analysis indicated that Xfreq is highly concentrated in the adul t brain and is absent from nonneural tissues such as heart and kidney. During development, the expression of the protein correlated well wit h the maturation of neuromuscular synapses. To determine the function of Xfreq at the developing neuromuscular junction, the recombinant pro tein was introduced into Xenopus embryonic spinal neurons by early bla stomere injection. Synapses made by spinal neurons containing exogenou s Xfreq exhibited a much higher synaptic efficacy. These results provi de direct evidence that frequenin enhances transmitter release at the vertebrate neuromuscular synapse and suggest its potential role in syn aptic development and plasticity.