CaM-dependent modulation of human CaV1.3 whole-cell and single-channel currents by C-terminal CaMKII phosphorylation site S1475.

Phosphorylation enables rapid modulation of voltage-gated calcium channels (VGCC) in physiological and pathophysiological conditions. How phosphorylation modulates human CaV1.3 VGCC, however, is largely unexplored. We characterized modulation of CaV1.3 gating via S1475, the human equivalent of a phosphorylation site identified in the rat. S1475 is highly conserved in CaV1.3 but absent from all other high-voltage activating calcium channel types co-expressed with CaV1.3 in similar tissues. Further, it is located in the C-terminal EF-hand motif, which binds calmodulin (CaM). This is involved in calcium-dependent channel inactivation (CDI). We used amino acid exchanges that mimic either sustained phosphorylation (S1475D) or phosphorylation resistance (S1475A). Whole-cell and single-channel recordings of phosphorylation state imitating CaV1.3 variants in transiently transfected HEK-293 cells revealed functional relevance of S1475 in human CaV1.3. We obtained three main findings: (1) CaV1.3_S1475D, imitating sustained phosphorylation, displayed decreased current density, reduced CDI and (in-) activation kinetics shifted to more depolarized voltages compared with both wildtype CaV1.3 and the phosphorylation-resistant CaV1.3_S1475A variant. Corresponding to the decreased current density, we find a reduced open probability of CaV1.3_S1475D at the single-channel level. (2) Using CaM overexpression or depletion, we find that CaM is necessary for modulating CaV1.3 through S1475. (3) CaMKII activation led to CaV1.3_WT-current properties similar to those of CaV1.3_S1475D, but did not affect CaV1.3_S1475A, confirming that CaMKII modulates human CaV1.3 via S1475. Given the physiological and pathophysiological importance of CaV1.3, our findings on the S1475-mediated interplay of phosphorylation, CaM interaction and CDI provide hints for approaches on specific CaV1.3 modulation under physiological and pathophysiological conditions. KEY POINTS: Phosphorylation modulates activity of voltage-gated L-type calcium channels for specific cellular needs but is largely unexplored for human CaV1.3 channels. Here we report that S1475, a CaMKII phosphorylation site identified in rats, is functionally relevant in human CaV1.3. Imitating phosphorylation states at S1475 alters current density and inactivation in a calmodulin-dependent manner. In wildtype CaV1.3 but not in the phosphorylation-resistant variant S1475A, CaMKII activation elicits effects similar to constitutively mimicking phosphorylation at S1475. Our findings provide novel insights on the interplay of modulatory mechanisms of human CaV1.3 channels, and present a possible target for CaV1.3-specific gating modulation in physiological and pathophysiological conditions.

Direct, gabapentin-insensitive interaction of a soluble form of the calcium channel subunit α2δ-1 with thrombospondin-4.

The α2δ-1 subunit of voltage-gated calcium channels binds to gabapentin and pregabalin, mediating the analgesic action of these drugs against neuropathic pain. Extracellular matrix proteins from the thrombospondin (TSP) family have been identified as ligands of α2δ-1 in the CNS. This interaction was found to be crucial for excitatory synaptogenesis and neuronal sensitisation which in turn can be inhibited by gabapentin, suggesting a potential role in the pathogenesis of neuropathic pain. Here, we provide information on the biochemical properties of the direct TSP/α2δ-1 interaction using an ELISA-style ligand binding assay. Our data reveal that full-length pentameric TSP-4, but neither TSP-5/COMP of the pentamer-forming subgroup B nor TSP-2 of the trimer-forming subgroup A directly interact with a soluble variant of α2δ-1 (α2δ-1S). Interestingly, this interaction is not inhibited by gabapentin on a molecular level and is not detectable on the surface of HEK293-EBNA cells over-expressing α2δ-1 protein. These results provide biochemical evidence that supports a specific role of TSP-4 among the TSPs in mediating the binding to neuronal α2δ-1 and suggest that gabapentin does not directly target TSP/α2δ-1 interaction to alleviate neuropathic pain.

Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia.

BACKGROUND: Microglia reactivity is a hallmark of neurodegenerative diseases. We have previously identified activated microglia/macrophage whey acidic protein (AMWAP) as a counter-regulator of pro-inflammatory response. Here, we studied its mechanisms of action with a focus on toll-like receptor (TLR) and nuclear factor κB (NFκB) signaling. METHODS: Recombinant AMWAP was produced in Escherichia coli and HEK293 EBNA cells and purified by affinity chromatography. AMWAP uptake was identified by fluorescent labeling, and pro-inflammatory microglia markers were measured by qRT-PCR after stimulation with TLR ligands. NFκB pathway proteins were assessed by immunocytochemistry, Western blot, and immunoprecipitation. A 20S proteasome activity assay was used to investigate the anti-peptidase activity of AMWAP. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. Microglial proliferation was investigated using flow cytometry, and their phagocytosis was monitored by the uptake of 661W photoreceptor debris. RESULTS: AMWAP was secreted from lipopolysaccharide (LPS)-activated microglia and recombinant AMWAP reduced gene transcription of IL6, iNOS, CCL2, CASP11, and TNFα in BV-2 microglia treated with LPS as TLR4 ligand. This effect was replicated with murine embryonic stem cell-derived microglia (ESdM) and primary brain microglia. AMWAP also diminished pro-inflammatory markers in microglia activated with the TLR2 ligand zymosan but had no effects on IL6, iNOS, and CCL2 transcription in cells treated with CpG oligodeoxynucleotides as TLR9 ligand. Microglial uptake of AMWAP effectively inhibited TLR4-dependent NFκB activation by preventing IRAK-1 and IκBα proteolysis. No inhibition of IκBα phosphorylation or ubiquitination and no influence on overall 20S proteasome activity were observed. Functionally, both microglial nitric oxide (NO) secretion and 661W photoreceptor apoptosis were significantly reduced after AMWAP treatment. AMWAP promoted the filopodia formation of microglia and increased the phagocytic uptake of apoptotic 661W photoreceptor cells. CONCLUSIONS: AMWAP is secreted from reactive microglia and acts in a paracrine fashion to counter-balance TLR2/TLR4-induced reactivity through NFκB inhibition. AMWAP also induces a neuroprotective microglial phenotype with reduced neurotoxicity and increased phagocytosis. We therefore hypothesize that anti-inflammatory whey acidic proteins could have a therapeutic potential in neurodegenerative diseases of the brain and the retina.

FOXD4a and FOXD4b, two new winged helix transcription factors, are expressed in human leukemia cell lines.

Winged helix factors are important regulators of embryonal development and tissue differentiation. They are also involved in translocations found in acute leukemias and solid tumors. We have detected transcripts from five known and four novel winged helix genes in leukemia cell lines and CD34(+) blood progenitor cells by reverse trancription-polymerase chain reaction with degenerate primers on the highly conserved DNA binding domain. The genomic clones coding for two new winged helix proteins, FOXD4a and FOXD4b were isolated by high-stringency hybridization of a human phage library. FOXD4a and FOXD4b are encoded by a 1319 and 1250 bp single exon coding for a winged helix DNA binding domain, an amino-terminal acidic region and a carboxy-terminal proline- and alanine-rich region which correspond to putative transcriptional regulatory motifs. TATA box, CCAAT box, and transcription factor binding motifs have been identified in the 5' region of the genes. In addition, foxD4a and foxD4b cDNA has been isolated from NB-4 mRNA. The fox genes are transcribed in a tissue-restricted pattern in adult and fetal human tissues. FoxD4a and foxD4b mRNA was expressed in the leukemia cell lines KG-1, Kasumi, NB-4, HL-60, U937, THP-1, HEL, U266, Jurkat, and Raji. It has already been shown that winged helix factors are also involved in carcinogenesis. Based upon these studies, our results suggest that FOXD4a and FOXD4b may play a role in leukemogenesis.