Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

The potential of metallic copper as an intrinsically antibacterial material is gaining increasing attention in the face of growing antibiotics resistance of bacteria. However, the mechanism of the so-called "contact killing" of bacteria by copper surfaces is poorly understood and requires further investigation. In particular, the influences of bacteria-metal interaction, media composition, and copper surface chemistry on contact killing are not fully understood. In this study, copper oxide formation on copper during standard antimicrobial testing was measured in situ by spectroscopic ellipsometry. In parallel, contact killing under these conditions was assessed with bacteria in phosphate buffered saline (PBS) or Tris-Cl. For comparison, defined Cu2O and CuO layers were thermally generated and characterized by grazing incidence X-ray diffraction. The antibacterial properties of these copper oxides were tested under the conditions used above. Finally, copper ion release was recorded for both buffer systems by inductively coupled plasma atomic absorption spectroscopy, and exposed copper samples were analyzed for topographical surface alterations. It was found that there was a fairly even growth of CuO under wet plating conditions, reaching 4-10 nm in 300 min, but no measurable Cu2O was formed during this time. CuO was found to significantly inhibit contact killing, compared to pure copper. In contrast, thermally generated Cu2O was essentially as effective in contact killing as pure copper. Copper ion release from the different surfaces roughly correlated with their antibacterial efficacy and was highest for pure copper, followed by Cu2O and CuO. Tris-Cl induced a 10-50-fold faster copper ion release compared to PBS. Since the Cu2O that primarily forms on copper under ambient conditions is as active in contact killing as pure copper, antimicrobial objects will retain their antimicrobial properties even after oxide formation.

In postnatal rodent brain, certain NG2-expressing oligodendroglial precursor cells (OPCs) are contacted by synaptic terminals from local neurons. However, it has remained elusive whether and when NG2(+) cells are integrated into neuronal circuits. Here we use patch-clamp recordings from mitotic cells in murine brain slices to show that, unlike any other cell in the central nervous system (CNS), cortical NG2(+) cells divide and relocate while being linked to synaptic junctions. Together with bromodeoxyuridine (BrdU) labeling, our recordings imply that cellular processes that bear synaptic junctions are surprisingly kept during cytokinesis and are inherited by the daughter cells. Cell cycle time (78 h) and relocation speed (5 microm/day) are slowed, and NG2(+) cells largely divide symmetrically. Inheritance of synapses enables newborn glial cells to establish synaptic connections much faster than newborn neurons and ensures that the entire population of NG2(+) cells is exposed to synaptic signals from local axons. The results suggest that synapses do not only transmit neuronal activity but also act as environmental cues for the development of glial cells. Inheritance of synapses allows for the direct transfer of environmental interactions to clonal descendants of OPCs, which might be important for effective colonization and myelination of the developing brain.

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity.P rotein kinases and their substrates represent the largest signaling network that regulates protein-protein interactions, subcellular localization, and ultimately cellular functions (1, 2). Deregulation of the signaling network often leads to disease states such as human malignancies, diabetes, and immune disorders. Although many kinases are excellent therapeutic targets, the precise connection between protein kinases and their direct substrates has not been fully elucidated for a majority of protein kinases. Besides classical genetic and biochemical methods, there have been a number of high throughput approaches for the identification of potential kinase substrates. Common methods include in vitro kinase assays using libraries of synthetic peptides (3), phase expression libraries (4), protein/peptide arrays (5-7), or cell extracts (8, 9), but these methods can often be misleading and provide many false positive results. The discovery of physiological substrates for specific protein kinases has remained challenging, even with recent advances in mass spectrometry.Mass spectrometry-based proteomics has become a powerful tool and been applied to map protein interaction networks, including kinase/phosphatase-substrate networks (10). Large-scale phosphoproteomics, however, does not typically reveal precise connections between protein kinases and their direct substrates (11,12). In recent years, there have been increasing attempts to develop mass spectrometry-based proteomic strategies for the identification of elusive kinase substrates (7,13,1...

Abstract:We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca 2ϩ channel ␣ 1 subunit, ␣ 1H , from a human medullary thyroid carcinoma cell line. The ␣ 1H subunit is structurally similar to previously described ␣ 1 subunits. Northern blot analysis indicates that ␣ 1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba 2ϩ currents recorded from human embryonic kidney 293 cells transiently expressing ␣ 1H activated at relatively hyperpolarized potentials (Ϫ50 mV), rapidly inactivated ( ϭ 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing ␣ 1H . Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ϳ9 pS. These channels are blocked by Ni 2ϩ (IC 50 ϭ 6.6 M) and the T-type channel antagonists mibefradil (ϳ50% block at 1 M) and amiloride (IC 50 ϭ 167 M). Thus, ␣ 1H -containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca 2ϩ channels.