Cells in most tissues are interconnected by cell-cell channels which allow the passage of electrolytes and small molecules from cell to cell. Aggregates of these channels are contained in the clustered intramembranous particles of gap junctions. In excitable cells these channels are instrumental for impulse propagation, in other tissues they are thought to pass signal molecules and to synchronize the cells metabolically. The objective of this research project is to understand the process of formation of cell-cell channels. This problem is addressed by functional expression of cell-cell channels in paired oocytes from cloned gap junction (connexin) cDNA. The oocyte assay will be used in combination with mutagenesis, site-specific reagents, tracer flux measurements, and the patch clamp technique to obtain information about molecular domains involved in various aspects of the channel formation process like the docking of the extracellular domains to each other and the associated docking gate. The pore lining will be assayed with a movable probe, using the cysteine scanning mutagenesis approach. Another aspect of this research project deals with the inhibition of gap junction channel formation by the testis-specific connexin33. Using the oocyte assay the mechanism of inhibition is to be investigated with the expectation to obtain clues about the channel formation process. Using transgenic mice the physiological role of cx33 inhibition will be studied. The mice will carry reporter genes for cx33 or gene constructs where the cx33 promoter drives the expression of a connexin (cx32) that is uninhibited by cx33. Expression of cx32 under the control of the cx33 promoter is expected to override the inhibitory role of cx33 and the ensuing phenotypic changes should reveal the physiological role of cx33 inhibition.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM048610-05
Application #
2022665
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1993-01-01
Project End
2000-12-31
Budget Start
1997-01-01
Budget End
1997-12-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Samuels, Stuart E; Lipitz, Jeffrey B; Wang, Junjie et al. (2013) Arachidonic acid closes innexin/pannexin channels and thereby inhibits microglia cell movement to a nerve injury. Dev Neurobiol 73:621-31
Dahl, Gerhard; Qiu, Feng; Wang, Junjie (2013) The bizarre pharmacology of the ATP release channel pannexin1. Neuropharmacology 75:583-93
Dahl, Gerhard; Keane, Robert W (2012) Pannexin: from discovery to bedside in 11±4 years? Brain Res 1487:150-9
Qiu, Feng; Wang, Junjie; Spray, David C et al. (2011) Two non-vesicular ATP release pathways in the mouse erythrocyte membrane. FEBS Lett 585:3430-5
Ambrosi, Cinzia; Gassmann, Oliver; Pranskevich, Jennifer N et al. (2010) Pannexin1 and Pannexin2 channels show quaternary similarities to connexons and different oligomerization numbers from each other. J Biol Chem 285:24420-31
Samuels, Stuart E; Lipitz, Jeffrey B; Dahl, Gerhard et al. (2010) Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury. J Gen Physiol 136:425-42
Wang, Junjie; Dahl, Gerhard (2010) SCAM analysis of Panx1 suggests a peculiar pore structure. J Gen Physiol 136:515-27
Qiu, Feng; Dahl, Gerhard (2009) A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am J Physiol Cell Physiol 296:C250-5
Bunse, Stefanie; Locovei, Silviu; Schmidt, Matthias et al. (2009) The potassium channel subunit Kvbeta3 interacts with pannexin 1 and attenuates its sensitivity to changes in redox potentials. FEBS J 276:6258-70
Ransford, George A; Fregien, Nevis; Qiu, Feng et al. (2009) Pannexin 1 contributes to ATP release in airway epithelia. Am J Respir Cell Mol Biol 41:525-34

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