Project Details
Description
PROJECT SUMMARY
Interstitial cells of Cajal (ICC) are one of the two known main classes of interstitial cells that regulate
gastrointestinal (GI) motor functions in health and disease. Throughout the GI tract, ICC modulate smooth
muscle excitability, facilitate efferent and afferent neural control, and generate electrical slow waves (SWs),
which drive phasic contractile activity underlying peristalsis and segmentation. Pacemaker activity is
compartmentalized in specific ICC classes in an organ-specific manner. Current models of SW generation
involve spontaneous transient inward currents triggered by Ca2+ release from the endoplasmic reticulum,
activation of other ion channels including voltage-dependent Ca2+ currents, Ca2+-activated Ca2+ release, and
store-operated Ca2+ entry. Previous work supported by this grant demonstrated selective expression of the
regulated isoform of the electrogenic Na+/HCO3− cotransporter SLC4A4 (solute carrier family 4 member 4;
isoform NBCe1b) in pacemaker ICC classes and revealed a role for intracellular HCO3−/pH regulation in SW
activity, adding a new component to the ICC pacemaker apparatus. However, while SLC4A4’s roles in acid-
base regulation and renal tubular acidosis are well established, its significance to GI disease in humans
remains unclear as is its precise role in SW generation and the mechanisms of regulation of its expression and
function. Linking SLC4A4 to GI diseases is a major challenge because of the uncertainty about the phenotypes
associated with its dysfunction or dysregulation. Furthermore, mechanistic evaluation of gene function and
regulation in ICC is severely limited by the phenotypic instability of these cells in culture. Based on strong
preliminary data, in this proposal we aim to overcome these difficulties by screening a large phenome-wide
association study (PheWAS, which starts with SNVs and searches for associated traits) for SNVs associated
with GI disease-related phenotypes that map within the topologically associating domain containing SLC4A4
(i.e., where most cis-regulatory interactions occur) (Specific Aim1). We will predict the significance of these
SNVs by deep epigenomic profiling and validate the predicted functions by genome and epigenome editing
(Specific Aim 2). To enable these experimental manipulations and the detailed analysis of the regulation of
SLC4A4 functions in the context of SW activity, we have validated cell lines derived from gastrointestinal
stromal tumors (GISTs) as models of human pacemaker ICC. In Specific Aim 3, we will subject GIST cells
expressing or lacking SLC4A4 to genetic and pharmacological manipulations along with phosphoproteomics to
dissect the contribution of SLC4A4 to SW activity in the context of cholinergic and KIT receptor tyrosine kinase
signaling. SLC4A4’s role in GI motor functions will be investigated using Slc4a4-deleted mice. Results from this
project will establish genotype-phenotype relationships for SLC4A4 and the mechanisms of epigenetic control
of SLC4A4 expression in the context of GI disorders. We will also determine the contribution of SLC4A4
activity to SWs and the pathways regulating SLC4A4 function, setting the stage for future preclinical work.
Status | Active |
---|---|
Effective start/end date | 4/1/00 → 12/31/24 |
Funding
- National Institute of Diabetes and Digestive and Kidney Diseases: $582,827.00
- National Institute of Diabetes and Digestive and Kidney Diseases: $254,303.00
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