Human neuronal stargazin-like proteins, γ_2, γ_3 and γ_4; an investigation of their specific localization in human brain and their influence on Ca_V2.1 voltage-dependent calcium channels expressed in Xenopus oocytes

Background: Stargazin (γ2) and the closely related γ3, and γ4 transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ2, γ3, and γ4 in the human central nervous system (CNS). In addition, we investigated whether human γ2 or γ4 could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes. Results: The mRNA encoding human γ2 is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ3 is abundant in cerebral cortex and amygdala and γ4 in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ2 and γ4 are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ2 is clearly detected in granule cells. The hippocampus stains for γ2 and γ4 throughout the layers of the every CA region and the dentate gyrus, whilst γ3 appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a CaV2.1/β4 VDCC complex, either in the absence or presence of an α2δ2 subunit, neither γ2 nor γ4 significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation. Conclusion: The human γ2, γ3 and γ4 stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither γ2 nor γ4 modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function

Capsaicin-induced endocytosis of endogenous presynaptic Ca_{V}2.2 in DRG-spinal cord co-cultures inhibits presynaptic function

The N-type calcium channel, CaV2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their post-synaptic targets in the spinal cord. In this study we have utilized CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. CaV2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in CaV2.2_HA expression and increased association with the post-synaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca2+ influx. The mechanism of this down-regulation of CaV2.2_HA involves a Rab 11a-dependent trafficking process, since dominant-negative Rab11a(S25N) occludes the effect of capsaicin on presynaptic CaV2.2_HA expression, and also prevents the effect of capsaicin on action potential induced Ca2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway

The Asymptotic Giant Branch and the Tip of the Red Giant Branch as Probes of Star Formation History: The Nearby Dwarf Irregular Galaxy KKH 98

We investigate the utility of the asymptotic giant branch (AGB) and the red giant branch (RGB) as probes of the star formation history (SFH) of the nearby (D=2.5 Mpc) dwarf irregular galaxy, KKH 98. Near-infrared (IR) Keck Laser Guide Star Adaptive Optics (AO) images resolve 592 IR bright stars reaching over 1 magnitude below the Tip of the Red Giant Branch. Significantly deeper optical (F475W and F814W) Hubble Space Telescope images of the same field contain over 2500 stars, reaching to the Red Clump and the Main Sequence turn-off for 0.5 Gyr old populations. Compared to the optical color magnitude diagram (CMD), the near-IR CMD shows significantly tighter AGB sequences, providing a good probe of the intermediate age (0.5 - 5 Gyr) populations. We match observed CMDs with stellar evolution models to recover the SFH of KKH 98. On average, the galaxy has experienced relatively constant low-level star formation (5 x 10^-4 Mo yr^-1) for much of cosmic time. Except for the youngest main sequence populations (age < 0.1 Gyr), which are typically fainter than the AO data flux limit, the SFH estimated from the the 592 IR bright stars is a reasonable match to that derived from the much larger optical data set. Differences between the optical and IR derived SFHs for 0.1 - 1 Gyr populations suggest that current stellar evolution models may be over-producing the AGB by as much as a factor of three in this galaxy. At the depth of the AO data, the IR luminous stars are not crowded. Therefore these techniques can potentially be used to determine the stellar populations of galaxies at significantly further distances.Comment: 15 pages, 14 figs, accepted for publication in Ap

HST Survey of Clusters in Nearby Galaxies. II. Statistical Analysis of Cluster Populations

We present a statistical system that can be used in the study of cluster populations. The basis of our approach is the construction of synthetic cluster color-magnitude-radius diagrams (CMRDs), which we compare with the observed data using a maximum likelihood calculation. This approach permits a relatively easy incorporation of incompleteness (a function of not only magnitude and color, but also radius), photometry errors and biases, and a variety of other complex effects into the calculation, instead of the more common procedure of attempting to correct for those effects. We then apply this procedure to our NGC 3627 data from Paper I. We find that we are able to successfully model the observed CMRD and constrain a number of parameters of the cluster population. We measure a power law mass function slope of alpha = -1.50 +/- 0.07, and a distribution of core radii centered at r_c = 1.53 +/- 0.15 pc. Although the extinction distribution is less constrained, we measured a value for the mean extinction consistent with that determined in Paper I from the Cepheids.Comment: 21 pages, 3 figures accepted for publication by A

Optical identification of the companion to PSR J1911-5958A, the pulsar binary in the outskirts of NGC 6752

We report on the identification of the optical counterpart of the binary millisecond pulsar PSR J1911-5958A, located in the outskirts of the globular cluster NGC 6752. At the position of the pulsar we find an object with V=22.08, B-V=0.38, U-B=-0.49. The object is blue with respect to the cluster main sequence by 0.8 magnitudes in B-V. We argue that the object is the white dwarf companion of the pulsar. Comparison with white dwarf cooling models shows that this magnitude and colors are consistent with a low-mass white dwarf at the distance of NGC 6752. If associated with NGC 6752, the white dwarf is relatively young, <2 Gyr, which sets constraints on the formation of the binary and its ejection from the core of the globular cluster.Comment: Accepted for publication in A&A Letters (September 1st, 2003

Human neuronal stargazin-like proteins, γ(2), γ(3 )and γ(4); an investigation of their specific localization in human brain and their influence on Ca(V)2.1 voltage-dependent calcium channels expressed in Xenopus oocytes.

BACKGROUND: Stargazin (γ(2)) and the closely related γ(3), and γ(4 )transmembrane proteins are part of a family of proteins that may act as both neuronal voltage-dependent calcium channel (VDCC) γ subunits and transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazoleproponinc (AMPA) receptor regulatory proteins (TARPs). In this investigation, we examined the distribution patterns of the stargazin-like proteins γ(2), γ(3), and γ(4 )in the human central nervous system (CNS). In addition, we investigated whether human γ(2 )or γ(4 )could modulate the electrophysiological properties of a neuronal VDCC complex transiently expressed in Xenopus oocytes. RESULTS: The mRNA encoding human γ(2 )is highly expressed in cerebellum, cerebral cortex, hippocampus and thalamus, whereas γ(3 )is abundant in cerebral cortex and amygdala and γ(4 )in the basal ganglia. Immunohistochemical analysis of the cerebellum determined that both γ(2 )and γ(4 )are present in the molecular layer, particularly in Purkinje cell bodies and dendrites, but have an inverse expression pattern to one another in the dentate cerebellar nucleus. They are also detected in the interneurons of the granule cell layer though only γ(2 )is clearly detected in granule cells. The hippocampus stains for γ(2 )and γ(4 )throughout the layers of the every CA region and the dentate gyrus, whilst γ(3 )appears to be localized particularly to the pyramidal and granule cell bodies. When co-expressed in Xenopus oocytes with a Ca(V)2.1/β(4 )VDCC complex, either in the absence or presence of an α2δ(2 )subunit, neither γ(2 )nor γ(4 )significantly modulated the VDCC peak current amplitude, voltage-dependence of activation or voltage-dependence of steady-state inactivation. CONCLUSION: The human γ(2), γ(3 )and γ(4 )stargazin-like proteins are detected only in the CNS and display differential distributions among brain regions and several cell types in found in the cerebellum and hippocampus. These distribution patterns closely resemble those reported by other laboratories for the rodent orthologues of each protein. Whilst the fact that neither γ(2 )nor γ(4 )modulated the properties of a VDCC complex with which they could associate in vivo in Purkinje cells adds weight to the hypothesis that the principal role of these proteins is not as auxiliary subunits of VDCCs, it does not exclude the possibility that they play another role in VDCC function

Involvement of CaV2.2 channels and α2δ-1 in homeostatic synaptic plasticity in cultured hippocampal neurons

In the mammalian brain, presynaptic CaV2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV2.2 channels at plastic hippocampal synapses remains to be elucidated. Here, we assess CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV2.2 channels at the presynaptic site. This work focussed further on the role of α2δ-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. We find that α2δ-1-overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous α2δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for α2δ-1 during synaptic plastic adaptation