The objective of this proposal is to study the dynamic responses of the inositol 1,4,5-triphosphate (InsP3) receptor (InsP3R) Ca2+-release channel in its native endoplasmic reticulum (ER) membrane to rapid physiological changes in the concentrations of its ligands. Ca2+ release by individual InsP3R channels is the building block of complex intracellular Ca2+ signals that control numerous physiological processes, from apoptosis and secretion to immune responses and memory. Understanding diverse Ca2+ signaling phenomena including quantal Ca2+ release, frequency modulation of Ca2+ oscillations, and propagating Ca2+ waves has been severely hampered by the lack of insights into dynamic ligand regulation of single InsP3R channels. We discovered that InsP3R channels in isolated nuclei of cultured Sf9 cells can be detected regularly in nuclear patch clamp experiments, with properties and regulation that are highly reminiscent of mammalian InsP3R. Furthermore, it is now possible to obtain, with high success rates, excised nuclear membrane patches in all, especially the cytoplasmic-side-out, configurations. With these advances, the time course of single InsP3R channel activity can be monitored, for the first time, during rapid (msec) changes in ligand concentrations on the cytoplasmic or lumenal side. The kinetics of InsP3R channel activity during Ca2+-induced Ca2+ release events will be characterized under physiological conditions. Instrinsic InsP3R channel behaviors that can contribute to quantal Ca2+ release, including time-dependent inactivation, heterogenous InsP3 sensitivity, and regulation of InsP3R channel activity by ER Ca2+ concentration (Ca2+)ER, will be investigated. (Ca2+)ER regulation of InsP3R channel conductance properties, gating behaviors and activity durations will also be examined. These studies represent the first systematic investigation of single InsP3R channel kinetics under various dynamic physiological conditions the channels experience in vivo. They will provide much-needed kinetic information on ligand regulation of single InsP3R channel for future mathematical modeling of Ca2+ signaling, from the molecular to cellular levels.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074999-05
Application #
7646135
Study Section
Special Emphasis Panel (ZRG1-CB-E (90))
Program Officer
Shapiro, Bert I
Project Start
2005-07-01
Project End
2011-07-31
Budget Start
2009-07-01
Budget End
2011-07-31
Support Year
5
Fiscal Year
2009
Total Cost
$253,985
Indirect Cost
Name
University of Pennsylvania
Department
Physiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Vais, Horia; Foskett, J Kevin; Mak, Don-On Daniel (2011) InsP3R channel gating altered by clustering? Nature 478:E1-2; discussion E2-3
Vais, Horia; Foskett, J Kevin; Mak, Don-On Daniel (2010) Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions. J Gen Physiol 136:687-700
Cheung, King-Ho; Mei, Lijuan; Mak, Don-On Daniel et al. (2010) Gain-of-function enhancement of IP3 receptor modal gating by familial Alzheimer's disease-linked presenilin mutants in human cells and mouse neurons. Sci Signal 3:ra22
Vais, Horia; Siebert, Adam P; Ma, Zhongming et al. (2010) Redox-regulated heterogeneous thresholds for ligand recruitment among InsP3R Ca2+-release channels. Biophys J 99:407-16
King Jr, J Darwin; Fitch, Adam C; Lee, Jeffrey K et al. (2009) AMP-activated protein kinase phosphorylation of the R domain inhibits PKA stimulation of CFTR. Am J Physiol Cell Physiol 297:C94-101
Ionescu, Lucian; White, Carl; Cheung, King-Ho et al. (2007) Mode switching is the major mechanism of ligand regulation of InsP3 receptor calcium release channels. J Gen Physiol 130:631-45
Mak, Don-On Daniel; Pearson, John E; Loong, King Pan Campion et al. (2007) Rapid ligand-regulated gating kinetics of single inositol 1,4,5-trisphosphate receptor Ca2+ release channels. EMBO Rep 8:1044-51