The retinal rod Na/Ca-K exchanger (NCKX) is a unique calcium extrusion protein utilizing both inward sodium gradient and outward potassium gradient. Three mammalian rod NCKX cDNAs have been cloned to date, but quantitative analysis of NCKX function in heterologous systems has proven difficult. Here, we describe a simple system for quantitative analysis of NCKX function; stable transformation of cultured insect cells with the novel pEA1/153A vector containing NCKX cDNAs was combined with measurements of potassium-dependent 45 Ca uptake in sodium-loaded cells. We carried out structure-function studies on NCKX with the following results: 1) two-thirds of the full-length sequence of bovine NCKX could be deleted without affecting potassiumdependent calcium transport and without affecting key properties of the potassium binding site; 2) the affinity of NCKX for potassium was about 10-fold greater in choline medium when compared with lithium medium; this shift was observed in rod outer segments or in cells expressing full-length rod NCKX, the above deletion mutant, or a distantly related NCKX paralog cloned from Caenorhabditis elegans. We conclude that the potassium binding site is highly conserved among members of the NCKX family and is formed by residues located within the two sets of transmembrane spanning segments in the NCKX sequence.Calcium extrusion across the plasma membrane of cells is vital to all cells, in view of the ubiquitous role of calcium as second messenger and since sustained elevated calcium levels rapidly lead to cell death (1). Calcium extrusion against a large electrochemical calcium gradient is mediated by two classes of plasma membrane proteins, an ATP-driven calcium pump and Na/Ca exchangers. Two groups of plasma membrane Na/Ca exchangers can be distinguished: those that neither require nor transport potassium (the NCX family) and those that require and, in the case of the rod photoreceptor NCKX1, have been demonstrated to transport potassium (the NCKX family) (for recent reviews, see Refs. 2 and 3). To date, three NCKX1 cDNAs have been cloned from mammalian rod photoreceptors (4 -6) and one NCKX2 cDNA from rat brain (7). Furthermore, several potential NCKX paralogs present in lower organisms have been identified on the basis of analysis of sequences obtained from genomic sequencing projects (2, 8). Studies on functional properties of the "in situ" Na/Ca-K exchanger have been limited thus far to NCKX1 found in the plasma membrane of the outer segments of retinal rod photoreceptors (reviewed in Refs. 9 -11). Sequence comparison of the three mammalian NCKX1 orthologs cloned to date reveals a remarkably low sequence identity (ϳ65%) in contrast to sequence identities of Ͼ90% observed for other sodium-coupled transporters. We examined functional activity of heterologously expressed dolphin, bovine, and human NCKX1 in several cell systems and only observed consistent and robust functional expression with the dolphin NCKX1 cDNA (6). Comparing the mammalian rod NCKX1 sequences with the sequence fro...

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.

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.

cDNAs of human and bovine retinal rod Na+-Ca2++K+ exchanger (NCKX1) have previously been cloned, but potassium-dependent Na-Ca exchange activity upon heterologous expression has not been demonstrated. We have cloned NCKX1 cDNA from dolphin, examined function upon transfection in HEK293 cells, and compared the dolphin sequence encoded by the cDNA with those of human and bovine. The dolphin NCKX1 cDNA encodes 1013 amino acid residues. Comparison to bovine and human NCKX1 revealed strong conservation in the transmembrane domains (>95%), but relatively low conservation in the large extracellular ( approximately 50%) and cytosolic (<50%) domains. The dolphin cytosolic domain differs from the bovine sequence by the absence of a stretch of 114 amino acids. HEK293 cells transfected with dolphin NCKX1 cDNA showed K+-dependent Na-Ca exchange in >95% of the experiments, whereas transfection with bovine NCKX1 yielded no function. The bovine NCKX1 phenotype was imparted on dolphin NCKX1 when the dolphin cytosolic loop was replaced by that from bovine. Conversely, deletion of 114 amino acids from the bovine sequence to match the dolphin sequence resulted in a mutant bovine NCKX1 which performed K+-dependent Na-Ca exchange. These results suggest that domains within the large cytosolic loop of NCKX1 control functional activity when expressed in heterologous systems.

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the ␣ 1B calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein ␤ subunits on both wild type and mutant ␣ 1B calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of G␤ 1 on channel activity, whereas G␤ 2 , G␤ 3 , and G␤ 4 are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the G␤ 1 subunit.The modulation of calcium channel activity by activation of intracellular messenger pathways is a key mechanism for fine tuning calcium entry into neurons. For example, the activation of protein kinase C has been shown to mediate an up-regulation of N-type calcium currents in intact neurons (1, 2) and in transient expression systems (3,4). In contrast, the direct 1:1 binding of G protein ␤␥ subunits to the domain I-II linker region of N-type, P/Q-type, and possibly R-type calcium channels results in a depression of current activity (5-8) (reviewed in Refs. 9 and 10), which can be reversed by strong membrane depolarization (10 -12). Different types of calcium channels are modulated by G-proteins to different extents, such that N-type channels are typically inhibited more effectively than P/Q-type channels (13-16). There is also increasing evidence that the degree of inhibition is dependent on the G-protein ␤ subunit species (16 -18). Finally, it has been shown that protein kinase C-dependent phosphorylation of the N-type calcium channel ␣ 1 subunit antagonizes receptor-mediated G-protein inhibition of the channel (1,2,12,19). This phenomenon (termed PKC 1 /Gprotein cross-talk) appears to be mediated by a single threonine residue in the ␣ 1B domain I-II linker region (4). For somatostatin receptor-induced G-protein inhibition of N-type calcium channels, mutation of Thr-422 to glutamic acid mimics the antagonistic effect of protein kinase C on G protein inhibition, whereas a switch to alanine precludes the occurrence of P...