Gap junctions are formed of two hemichannels (or connexons), one from each of the apposed cells. Hemi- channels are formed in the ER or a post ER compartment and inserted into the surface with little localization. They diffuse over the surface to dock with a partner in an apposed membrane and then open. Non-junctional surface hemichannels are for the most part closed, which is reasonable in view of their large conductance and relatively non-specific permeability. However some hemichannels open in physiological or pathological conditions. Cx43, a prevalent connexin in many tissues, has been little characterized in respect to hemi- channel opening. This application proposes to ameliorate that deficiency. Techniques include time lapse recording of dye uptake, recording of single channel activity, isolation of surface Cx43 by biotinylation/ NeutrAvidin pull down, Western blot analysis and site directed mutagenesis.
Aim 1 is to analyze gating of Cx43 hemichannels as a function of voltage and reduced divalent ion concentration.
Aim 2 is to identify sites of modification of Cx43 by oxidizing and reducing agents and by metabolic inhibition (Ml), treatments that affect voltage dependence and open probability. Ml and NO donors induce S-nitrosylation of Cx43, an effect blocked by reducing agents such as DTT. Truncation that removes all cytoplasmic cysteines greatly atten- uates the effect of metabolic inhibition. Now we will remove the cysteines individually and in combination. We will assay phosphorylation of surface hemichannels (isolated by biotinylation) to determine relation to effects of metabolic inhibition. Phosphorylation at several sites modulates gating but does not affect responses to Ml.
Aim 3 is to localize the relative position of the gate closed by acidification with the H3O+ binding site. Preliminary data indicate that the site on the cytoplasmic side of the gate, i.e. weak, membrane permeant acids rapidly and reversibly block the hemichannels, and strong, relatively membrane impermeant acids do not block hemichannels until they open.
Aim 4 is to extend these data to astrocytes, both in culture and in brain slices. Our preliminary data indicate high degree of similarity in culture. These studies should clarify controls of Cx43 hemichannel opening in physiological and pathological conditions. Cx43 is the primary connexin expressed by astrocytes; responses to metabolic challenge will relate to the clinical conditions of focal and global ischemia in the CNS, where the role of astrocytes remains largely unexplored. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS055363-02
Application #
7487052
Study Section
Neural Degenerative Disorders and Glial Biology Study Section (NDGB)
Program Officer
Silberberg, Shai D
Project Start
2007-09-01
Project End
2012-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
2
Fiscal Year
2008
Total Cost
$326,813
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Neurosciences
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Garré, Juan Mauricio; Yang, Guang; Bukauskas, Feliksas F et al. (2017) An Acute Mouse Spinal Cord Slice Preparation for Studying Glial Activation ex vivo. Bio Protoc 7:
Garré, Juan Mauricio; Yang, Guang; Bukauskas, Feliksas F et al. (2016) FGF-1 Triggers Pannexin-1 Hemichannel Opening in Spinal Astrocytes of Rodents and Promotes Inflammatory Responses in Acute Spinal Cord Slices. J Neurosci 36:4785-801
Palacios-Prado, Nicolás; Hoge, Gregory; Marandykina, Alina et al. (2013) Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36. J Neurosci 33:4741-53
Majoul, Irina V; Gao, Liang; Betzig, Eric et al. (2013) Fast structural responses of gap junction membrane domains to AB5 toxins. Proc Natl Acad Sci U S A 110:E4125-33
Schalper, Kurt A; Riquelme, Manuel A; Branes, Maria C et al. (2012) Modulation of gap junction channels and hemichannels by growth factors. Mol Biosyst 8:685-98
Flores, Carmen E; Nannapaneni, Srikant; Davidson, Kimberly G V et al. (2012) Trafficking of gap junction channels at a vertebrate electrical synapse in vivo. Proc Natl Acad Sci U S A 109:E573-82
Bennett, Michael V L; Garre, Juan M; Orellana, Juan A et al. (2012) Connexin and pannexin hemichannels in inflammatory responses of glia and neurons. Brain Res 1487:3-15
Eugenin, Eliseo A; Basilio, Daniel; Sáez, Juan C et al. (2012) The role of gap junction channels during physiologic and pathologic conditions of the human central nervous system. J Neuroimmune Pharmacol 7:499-518
Yu, Yufei; Santos, Laura M; Mattiace, Linda A et al. (2012) Reentrant spiral waves of spreading depression cause macular degeneration in hypoglycemic chicken retina. Proc Natl Acad Sci U S A 109:2585-9
Noh, Kyung-Min; Hwang, Jee-Yeon; Follenzi, Antonia et al. (2012) Repressor element-1 silencing transcription factor (REST)-dependent epigenetic remodeling is critical to ischemia-induced neuronal death. Proc Natl Acad Sci U S A 109:E962-71

Showing the most recent 10 out of 16 publications