Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function (original) (raw)
2004, Nature Neuroscience
L-type Ca 2+ channels are involved in neuronal differentiation and outgrowth and in synaptic plasticity 1,2. At many ribbon synapses, Ca 2+ influx through L-type Ca 2+ channels triggers neurotransmitter release 3-5. The α 1-subunit of the L-type Ca v 1.4 channel (Ca v 1.4α1) is specific to photoreceptors and is present at highest density in the synaptic terminals 5,6. Compared with other L-type Ca 2+ channels, Ca v 1.4 channels are activated at relatively more negative voltages and show slow inactivation 7-9 , important properties for the ability of photoreceptors to sustain continual glutamate release in the dark 4,10. Null mutations in Ca v 1.4α1 are responsible for an X-linked disorder, CSNB2 (refs. 11,12). ERGs of these patients indicate that a deficit may occur in transmission of signals from rod photoreceptors to bipolar cells. In mice, deletion of the β 2-subunit, another component of the photoreceptor L-type channel, alters the expression of Ca v 1.4 and produces a phenotype similar to that seen in CSNB2 patients 13. CaBPs, a subfamily of calmodulin (CaM)-like neuronal Ca 2+-binding proteins 14 , modulate voltage-dependent Ca 2+ channels (VDCCs) and inositol triphosphate receptors 15-17. Here we show that CaBP4, which has only been partially characterized in silico 14 , is found specifically in photoreceptor synaptic terminals, is important for the normal function of photoreceptor synapses and colocalizes with and modulates the activity of expressed Ca v 1.4 channels. These results indicate that CaBP4 may be an important regulator of Ca 2+ influx and transmitter release in photoreceptor synaptic terminals. RESULTS CaBP4 is a CaM-related CaBP CaBP4 was identified by polymerase chain reaction (PCR) cloning using primers based on the sequence of its closest relative, CaBP2 (ref. 14). The full-length cDNA of human CaBP4 contains an open reading frame of 825 bp, predicting a protein of 275 amino acids and a calculated molecular mass of 30,432 Da. Human, bovine, mouse and rat CaBP4 are highly homologous in their C-terminal regions (∼90%) and are less conserved in their N-terminal regions (∼60%) with interspecies differences occurring in the first exon (Fig. 1a). CaBP4 contains four EF-hand motifs, although the second motif cannot coordinate Ca 2+ because the Lys residue in position 1 is not suitable for Ca 2+ coordination (Fig. 1a). The Cabp4 gene is short (∼4 kb) with a six-exon structure similar to CaM (see Supplementary Fig. 1 online). The human genome data at the NCBI predict that Cabp4 is in the opposite orientation and is separated by four genes and ∼60 kb from Cabp2 on chromosome 11 at q13.1, a region with conserved synteny with mouse chromosome 19. CaBP4 is localized in rod and cone photoreceptor synapses Northern blot analyses with the CaBP4 cDNA probe showed two transcripts of 1.6 kb and 3.8 kb in the retina (Fig. 1b). CaBP4 products from PCR with reverse transcription (RT-PCR) were observed in the retina but not in other locations (Fig. 1c). The CaBP4 antisense