We have identified a novel interaction between huntingtin (htt) and N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis. Htt is a widely expressed 350-kDa cytosolic protein bearing an N-terminal polyglutamine tract. Htt is proteolytically cleaved by calpains and caspases and the resultant htt N-terminal fragments have been proposed to be biologically active; however, the cellular function of htt and/or the htt fragments remains enigmatic. We show that N-terminal fragments of htt (consisting of exon1) and full-length htt associate with the synaptic protein interaction (synprint) region of the N-type calcium channel. Given that synprint has previously been shown to bind syntaxin 1A and that this association elicits inhibition of N-type calcium channels, we tested whether htt(exon1) affects the modulation of these channels. Our data indicate that htt(exon1) enhances calcium influx by blocking syntaxin 1A inhibition of N-type calcium channels and attributes a key role for htt N-terminal fragments in the fine tuning of neurotransmission.

Cysteine string protein (CSPalpha) is a synaptic vesicle protein that displays unique anti-neurodegenerative properties. CSPalpha is a member of the conserved J protein family, also called the Hsp40 (heat shock protein of 40 kDa) protein family, whose importance in protein folding has been recognized for many years. Deletion of the CSPalpha in mice results in knockout mice that are normal for the first 2-3 weeks of life followed by an unexplained presynaptic neurodegeneration and premature death. How CSPalpha prevents neurodegeneration is currently not known. As a neuroprotective synaptic vesicle protein, CSPalpha represents a promising therapeutic target for the prevention of neurodegenerative disorders.

The fidelity of synaptic transmission depends on the integrity of the protein machinery at the synapse. Unfolded synaptic proteins undergo refolding or degradation in order to maintain synaptic proteostasis and preserve synaptic function, and buildup of unfolded/toxic proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, cysteine string protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated proteins, polyglutamine expanded protein 72Q huntingtinex°n1 or superoxide dismutase-1 (SOD-1G93A) leads to the cellular export of both 72Q huntingtinex°n1 and SOD-1G93A via EV's. In contrast, the inactive CSPαHPD-AAA mutant does not facilitate elimination of misfolded proteins. Furthermore, CSPα-mediated export of 72Q huntingtinex°n1 is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.

Light-dependent changes in cytoplasmic free Ca(2+) are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na(+)/Ca(2+) exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca(2+) measured with optical probes. The exchanger in cones is K(+) dependent and operates both in forward and reverse modes. In the reverse mode, the K(+) dependence of the exchanger is described by binding to a single site with K(1/2) about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods.

An extracellular network of molecular chaperones protects a diverse array of proteins that reside in or pass through extracellular spaces. Proteins in the extracellular milieu face numerous challenges that can lead to protein misfolding and aggregation. As a checkpoint for proteins that move between cells, extracellular chaperone networks are of growing clinical relevance. J-domain proteins (JDPs) are ubiquitous molecular chaperones that are known for their essential roles in a wide array of fundamental cellular processes through their regulation of heat shock protein 70s. As the largest molecular chaperone family, JDPs have long been recognized for their diverse functions within cells. Some JDPs are elegantly selective for their "client proteins," some do not discriminate among substrates and others act cooperatively on the same target. The realization that JDPs are exported through both classical and unconventional secretory pathways has fueled investigation into the roles that JDPs play in protein quality control and intercellular communication. The proposed functions of exported JDPs are diverse. Studies suggest that export of DnaJB11 enhances extracellular proteostasis, that intercellular movement of DnaJB1 or DnaJB6 enhances the proteostasis capacity in recipient cells, whereas the import of DnaJB8 increases resistance to chemotherapy in recipient cancer cells. In addition, the export of DnaJC5 and concurrent DnaJC5-dependent ejection of dysfunctional and aggregation-prone proteins are implicated in the prevention of neurodegeneration. This review provides a brief overview of the current understanding of the extracellular chaperone networks and outlines the first wave of studies describing the cellular export of JDPs.

NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.

Large-conductance, calcium-activated-K(+) (BK) channels are widely distributed throughout the nervous system, where they regulate action potential duration and firing frequency, along with presynaptic neurotransmitter release. Our recent efforts to identify chaperones that target neuronal ion channels have revealed cysteine string protein (CSPα) as a key regulator of BK channel expression and current density. CSPα is a vesicle-associated protein and mutations in CSPα cause the hereditary neurodegenerative disorder, adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL). CSPα null mice show 2.5 fold higher BK channel expression compared to wild type mice, which is not seen with other neuronal channels (i.e. Cav2.2, Kv1.1 and Kv1.2). Furthermore, mutations in either CSPα's J domain or cysteine string region markedly increase BK expression and current amplitude. We conclude that CSPα acts to regulate BK channel expression, and consequently CSPα-associated changes in BK activity may contribute to the pathogenesis of neurodegenerative disorders, such as ANCL.

Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aβ(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aβ(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aβ(42) toxicity. In this study we show that neither extracellular Aβ(42) nor Aβ(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aβ(42), Western analysis of Aβ(42) was performed following external Aβ(42) application. Five hours after a conditioning heat shock, Aβ(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aβ(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aβ(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aβ(42) indicating that Hsp40 modulation of Aβ(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aβ(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aβ(42). Our data reveal a biochemical link between Hsp40 expression and Aβ(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.

In response to a conditioning stress, the expression of a set of molecular chaperones called heat shock proteins is increased. In neurons, stress-induced and constitutively expressed molecular chaperones protect against damage induced by ischemia and neurodegenerative diseases, however the molecular basis of this protection is not known. Here we have investigated the crosstalk between stress-induced chaperones and cysteine string protein (CSPalpha). CSPalpha is a constitutively expressed synaptic vesicle protein bearing a J domain and a cysteine rich "string" region that has been implicated in the long term functional integrity of synaptic transmission and the defense against neurodegeneration. We have shown previously that the CSPalpha chaperone complex increases isoproterenol-mediated signaling by stimulating GDP/GTP exchange of Galpha(s). In this report we demonstrate that in response to heat shock or treatment with the Hsp90 inhibitor geldanamycin, the J protein Hsp40 becomes a major component of the CSPalpha complex. Association of Hsp40 with CSPalpha decreases CSPalpha-CSPalpha dimerization and enhances the CSPalpha-induced increase in steady state GTP hydrolysis of Galpha(s). This newly identified CSPalpha-Hsp40 association reveals a previously undescribed coupling of J proteins. In view of the crucial importance of stress-induced chaperones in the protection against cell death, our data attribute a role for Hsp40 crosstalk with CSPalpha in neuroprotection.

Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene.

Large-conductance, calcium-activated K(+) (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.

The Na+/Ca2+ -K+ exchanger (NCKX) utilizes the inward Na+ gradient and the outward K+ gradient to promote Ca2+ extrusion from cells. Here, we have characterized a second NCKX from Drosophila. Based on its chromosomal location (X chromosome) we have named it Ncxk-x. Three splice variants were isolated with three distinct N-terminal sequences. NCKX-X differs from NCKX proteins described so far in other species by lacking an N-terminal signal peptide. Heterologous expression of the respective cDNA's resulted in NCKX-X protein expression and K+ -dependent Na+/Ca2+ exchange activity for two of the three splice variants. Transcript localization of Nckx-x was investigated and compared with that previously described by us for Nckx30C.

Cysteine string protein (CSPalpha) is a member of the cellular folding machinery that is located on regulated secretory vesicles. We have previously shown that CSPalpha in association with Hsc70 (70kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) is a guanine nucleotide exchange factor (GEF) for G(alphas). Association of this CSPalpha complex with N-type calcium channels, a channel key in coupling calcium influx with synaptic vesicle exocytosis, triggers tonic G protein inhibition of the channels. Syntaxin 1A, a plasma membrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) critical for neurotransmission, coimmunoprecipitates with the CSPalpha/G protein/N-type calcium channel complex, however the significance of syntaxin 1A as a component of this complex remains unknown. In this report, we establish that syntaxin 1A interacts with CSPalpha, Hsc70 as well as the synaptic protein interaction (synprint) region of N-type channels. We demonstrate that huntingtin(exon1), a putative biologically active fragment of huntingtin, displaces both syntaxin 1A and CSPalpha from N-type channels. Identification of the protein components of the CSPalpha/GEF system is essential in establishing its precise role in synaptic transmission.