Identification and characterization of novel human Ca(v)2.2 (alpha 1B) calcium channel variants lacking the synaptic protein interaction site.

The physical interaction between the presynaptic vesicle release complex and the large cytoplasmic region linking domains II and III of N-type (Ca(v)2.2) calcium channel alpha(1)B subunits is considered to be of fundamental importance for efficient neurotransmission. By PCR analysis of human brain cDNA libraries and IMR32 cell mRNA, we have isolated novel N-type channel variants, termed Ca(v)2.2-Delta1 and Delta2, which lack large parts of the domain II-III linker region, including the synaptic protein interaction site. They appear to be widely expressed across the human CNS as indicated by RNase protection assays. When expressed in tsA-201 cells, both novel variants formed barium-permeable channels with voltage dependences and kinetics for activation that were similar to those observed with the full-length channel. All three channel types exhibited the hallmarks of prepulse facilitation, which interestingly occurred independently of G-protein betagamma subunits. By contrast, the voltage dependence of steady-state inactivation seen with both Delta1 and Delta2 channels was shifted toward more depolarized potentials, and recovery from inactivation of Delta1 and Delta2 channels occurred more rapidly than that of the full-length channel. Moreover, the Delta1 channel was dramatically less sensitive to both omega-conotoxin MVIIA and GVIA than either the Delta2 variant or the full-length construct. Finally, the domain II-III linker region of neither variant was able to effectively bind syntaxin in vitro. These results suggest that the structure of the II-III linker region is an important determinant of N-type channel function and pharmacology. The lack of syntaxin binding hints at a unique physiological function of these channels.

The Na/Ca-K exchanger gene family.

Ca(2+) extrusion driven by both the inward Na(+) gradient as well as the outward K(+) gradient is essential for visual transduction in retinal rod and cone photoreceptors because it removes Ca(2+) that enters photoreceptors via the cGMP-gated and light-sensitive channels. We have cloned rod and cone Na/Ca-K exchanger (NCKX) cDNAs from several species, and we have cloned NCKX cDNAs from lower organisms that lack vertebrate-type vision. Although in situ NCKX physiology has only been documented for vertebrate photoreceptors, it is now clear that NCKX gene products have a much broader distribution pattern. Here, we review some of the structural and functional features that have emerged from our studies on different members of the NCKX gene family.

The retinal rod and cone Na+/Ca2+-K+ exchangers.

The past few years has seen significant progress in our understanding of the retinal rod and cone Na+/Ca2+-K+ exchanger (NCKX) genes. The human rod and cone NCKX genes were localized to chromosomes 15q22 and 9p22, respectively. In situ hybridization localized the rod and cone NCKX transcripts in both human and chicken retinas: rod NCKX transcripts were found only in the inner segments of rods, whereas cone NCKX transcripts were found in a subset of retinal ganglion cells as well as in the inner segments of cones. We identified two sets of putative transmembrane spanning segments (TM's) as the only sequence elements strongly conserved between the rod and cone NCKX cDNAs, as well as between mammalian NCKX cDNAs and NCKX cDNAs cloned from lower organisms (C. elegans and Drosophila). The two sets of TM's make up less than onethird of the rod NCKX sequence and less than half of the cone NCKX sequence. Basic cation binding properties as inferred from an analysis of 45Ca transport rates and NCKX currents were very similar for all our NCKX clones, implying that conserved residues within the two sets of TM's contain all the residues involved in cation binding and cation transport.

Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons.

Sequences in the cytoplasmic II-III loop of CaV2 voltage-gated calcium channels, termed the synaptic protein interaction (synprint) site, are considered important for the functional incorporation of presynaptic calcium channels into the synaptic vesicle fusion apparatus. Two novel CaV2.2 splice variants lack large parts of the cytoplasmic II-III loop (Delta1 R756-L1139, Delta2 K737-A1001) including the synprint protein-protein interaction domain. Here we expressed green fluorescent protein (GFP)-alpha1B subunit fusion constructs of CaV2.2 splice variants in mouse hippocampal neurons to study their distribution in distinct neuronal compartments and to address the question of whether and how the synprint site functions in the presynaptic targeting of N-type calcium channels. Similar to full-length GFP-alpha1B but divergent from the somatodendritic alpha1C-HA (CaV1.2) channel type, the splice variants GFP-alpha1B-Delta1 and GFP-alpha1B-Delta2 were targeted into the axons. Nevertheless, their ability to form bona fide presynaptic clusters was almost abolished for GFP-alpha1B-Delta1 and significantly reduced for GFP-alpha1B-Delta2. Thus, the synprint site is important for normal synaptic targeting of CaV2.2 but not essential. Conversely, insertion of the synprint site into the II-III loop of alpha1C-HA did not restore axonal targeting or synaptic clustering. Together these results indicate that protein-protein interactions with the synprint site must cooperate with other targeting mechanisms in the incorporation of CaV2.2 into presynaptic specializations of hippocampal neurons but are neither necessary nor sufficient for axonal targeting. The unique targeting properties of the splice variants lacking the synprint site are suggestive of specific functions of these calcium channels apart from activating fast synaptic transmission.