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IP3 Receptor Calcium Imaging - Parker Lab

Position: Assistant Project Scientist (2010-2016) Location: Department of Neurobiology & Behavior, UC Irvine PI: Dr. Ian Parker

Overview

Utilized super-resolution and TIRF fluorescence microscopy to study intercellular calcium signals and signaling proteins at the single-cell and single-molecule level in cultured neuroblastoma cells.

Research Focus

Investigation of calcium puffs - localized Ca²⁺ signals mediated by inositol trisphosphate receptors (IP3Rs). This work pioneered optical patch-clamp techniques to resolve individual IP3R channel openings during calcium puffs.

Key Questions

  • How do IP3R channels cluster and coordinate their activity?
  • What determines recruitment of channels during calcium puffs?
  • How are calcium puffs terminated?
  • What is the probability of triggering puffs based on cluster size?

Technical Innovation

Optical Patch-Clamp

Ian Parker developed single-channel calcium imaging using TIRF microscopy to: - Resolve openings of individual IP3R channels - Measure channel gating kinetics during puffs - Quantify numbers of functional channels in clusters - Study coupled channel closings

This technique provided unprecedented resolution of calcium channel dynamics.

Major Findings

Channel Recruitment

  • Puff triggering probability is linearly related to number of IP3Rs in a cluster
  • Channels recruit additional channels through calcium-induced calcium release (CICR)
  • Individual puffs show wide amplitude variability even at sites with similar cluster size

Puff Termination

  • Multiple channels tend to close in near-synchrony
  • Termination cannot be explained solely by Ca²⁺-inhibition
  • Coupled gating of adjacent IP3Rs likely contributes to robust termination

IP3 Diffusion

  • IP3 diffusion is hindered in cytoplasm
  • Diffusion coefficient is 10-fold lower than in water
  • Findings impact understanding of spatial calcium signaling

Temperature Dependence

  • IP3-mediated calcium signals show strong temperature dependence
  • Both local (puffs) and global signals affected
  • Important for physiological relevance of in vitro studies

Techniques

Microscopy

  • TIRF (Total Internal Reflection Fluorescence)
  • Confocal microscopy
  • Single-molecule imaging
  • Ca²⁺ fluorescence indicators (Fluo-4, etc.)

Experimental Methods

  • SH-SY5Y neuroblastoma cell culture
  • Caged IP3 photolysis
  • Optical patch-clamp recordings
  • Temperature-controlled imaging

Analysis

  • Custom image analysis software (FLIKA development)
  • Stochastic modeling
  • Channel gating kinetics analysis
  • Spatial statistics

Publications (8 total)

  1. Dickinson GD, Ellefsen K, Dawson D, Pearson J, Parker I. (2016) Hindered diffusion of inositol trisphosphate. Science Signaling 9(453):ra108. PMID: 27919026

  2. Smith IF, Swaminathan D, Dickinson GD, Parker I. (2014) Single-molecule tracking of inositol trisphosphate receptors reveals different motilities and distributions. Biophysical Journal 107(4):834-45. PMID: 25140418

  3. Wiltgen SM, Dickinson GD, Swaminathan D, Parker I. (2014) Termination of calcium puffs and coupled closings of inositol trisphosphate receptor channels. Cell Calcium 56(3):157-68. PMID: 25016315

  4. Dickinson GD, Parker I. (2013) Factors determining the recruitment of inositol trisphosphate receptor channels during calcium puffs. Biophysical Journal 105(11):2474-84. PMID: 24314078

  5. Dickinson GD, Parker I. (2013) Temperature dependence of IP3-mediated local and global Ca²⁺ signals. Biophysical Journal 104(2):386-95. PMID: 23442860

  6. Neely Kayala KM, Dickinson GD, Minassian A, Walls KC, Green KN, Laferla FM. (2012) Presenilin-null cells have altered two-pore calcium channel expression and lysosomal calcium. Brain Research 1489:8-16. PMID: 23103503

  7. Dickinson GD, Swaminathan D, Parker I. (2012) The probability of triggering calcium puffs is linearly related to the number of inositol trisphosphate receptors in a cluster. Biophysical Journal 102(8):1826-36. PMID: 22768938

  8. Swaminathan D, Dickinson GD, Demuro A, Parker I. (2020) Noise analysis of cytosolic calcium image data. Cell Calcium 86:102152. PMID: 31918030

View all publications →

Software Development

This research drove development of FLIKA (Fluorescence Image Analysis) by Kyle Ellefsen and Brett Settle, which later became the foundation for numerous analysis plugins.

View FLIKA projects →

Impact

This work established optical patch-clamp as a method for studying ion channel dynamics and contributed fundamental insights into IP3R channel function and calcium puff generation/termination.

Awards

Poster Prize - Gordon Research Conference on Calcium Signaling (2015)

Page last updated: December 2024