The epithelial sodium channel/degenerin (ENaC/Deg) family of ion channels is constituted of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume and airway surface liquid volume. ENaC/Deg channels are gated by diverse stimuli including neuropeptides, mechanical forces, extracellular protons, and some of them are constitutively active, such as ENaCs. These channels are likely organized as homo- or hetero- trimers and are composed of subunits that have a similar topology with two transmembrane domains (TMs) connected by a large extracellular domain with short intracellular N- and C- termini. The first high resolution structure of the extracellular and membrane-spanning domains of an ENaC/Deg channel, Gallus gallus (chicken) acid-sensing ion channel 1 (cASIC1), was recently reported. ASICs are proton-gated channels expressed throughout neurons of mammalian central and peripheral nervous systems that exist in a resting, functionally inactive state, but undergo a rapid activation and desensitization following extracellular acidification. cASIC1 has a chalice-like shape with a large extracellular domain protruding from the plane of the membrane. The extracellular region is organized in discrete subdomains named the "palm, knuckle, 2-ball, finger and thumb". The transition between the TMs and extracellular domain constitute the wrist. While the crystal structure provides great molecular details of cASIC1 in the desensitized state, the mechanism of gating of ASICs and other ENaC/Deg channels remains largely undefined. The first specific aim of this proposal will define conformational rearrangements in the wrist and pore of mouse ASIC1a associated with activation and desensitization. In the second specific aim we will address questions regarding the role of the thumb and palm subdomains in activation, desensitization and recovery from desensitization. Conformational changes that occur in restricted areas of the channel following extracellular acidification will be investigated by voltage clamp fluorometry, a technique that provides information regarding local protein motion associated with specific gating steps. The substituted- cysteine-accessibility method will be used to characterize the structure of specific regions in the closed and desensitized states. Studies proposed in this application address fundamental questions regarding the underlying mechanism of gating and desensitization of mouse ASIC1a. Information derived from this work will be valuable to comprehend the molecular mechanisms of regulation of Deg/ENaC channels.

Public Health Relevance

The epithelial sodium channel/degenerin (ENaC/Deg) family of ion channels is constituted of proteins that are implicated in mechanosensation, pain sensation, regulation of extracellular fluid volume and airway surface liquid volume. These channels have evolved to sense a broad variety of stimuli with homologous structures. Information derived from this work will be valuable to comprehend the molecular mechanisms of regulation of these channels.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK084060-03
Application #
8286392
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Ketchum, Christian J
Project Start
2010-07-20
Project End
2015-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
3
Fiscal Year
2012
Total Cost
$311,219
Indirect Cost
$105,794
Name
University of Pittsburgh
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Carattino, Marcelo D; Mueller, Gunhild M; Palmer, Lawrence G et al. (2014) Prostasin interacts with the epithelial Na+ channel and facilitates cleavage of the ?-subunit by a second protease. Am J Physiol Renal Physiol 307:F1080-7
Raghavan, Venkatesan; Rbaibi, Youssef; Pastor-Soler, NĂºria M et al. (2014) Shear stress-dependent regulation of apical endocytosis in renal proximal tubule cells mediated by primary cilia. Proc Natl Acad Sci U S A 111:8506-11
Della Vecchia, Margaret C; Rued, Anna C; Carattino, Marcelo D (2013) Gating transitions in the palm domain of ASIC1a. J Biol Chem 288:5487-95
Carattino, Marcelo D; Prakasam, H Sandeep; Ruiz, Wily G et al. (2013) Bladder filling and voiding affect umbrella cell tight junction organization and function. Am J Physiol Renal Physiol 305:F1158-68
Shi, Shujie; Carattino, Marcelo D; Hughey, Rebecca P et al. (2013) ENaC regulation by proteases and shear stress. Curr Mol Pharmacol 6:28-34
Carattino, Marcelo D; Della Vecchia, Margaret C (2012) Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity. J Biol Chem 287:12927-34
Kashlan, Ossama B; Blobner, Brandon M; Zuzek, Zachary et al. (2012) Inhibitory tract traps the epithelial Na+ channel in a low activity conformation. J Biol Chem 287:20720-6
Carattino, Marcelo D (2011) Structural mechanisms underlying the function of epithelial sodium channel/acid-sensing ion channel. Curr Opin Nephrol Hypertens 20:555-60
Tolino, Lindsey A; Okumura, Sora; Kashlan, Ossama B et al. (2011) Insights into the mechanism of pore opening of acid-sensing ion channel 1a. J Biol Chem 286:16297-307
Carattino, Marcelo D; Passero, Christopher J (2011) Clues to renal sodium retention. Am J Physiol Renal Physiol 300:F639-40