Single-particle tracking of membrane protein diffusion in a potential: simulation, detection, and application to confined diffusion of CFTR Cl- channels.
Confined diffusion of membrane receptors and lipids can result from intramembrane barriers, skeletal interactions, rafts, and other phenomena. We simulated single-particle diffusion in two dimensions in an arbitrary potential, V(r), based on summation of random and potential gradient-driven motions. Algorithms were applied and verified for detection of potential-driven diffusion, and for determination of V(r) from radial particle density distributions, taking into account experimental uncertainties in particle position and finite trajectory recording. Single-particle tracking (SPT) analysis of the diffusion of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels in mammalian cells revealed confined diffusion with diffusion coefficient approximately 0.004 microm(2)/s. SPT data fitted closely to a springlike attractive potential, V(r) = kr(2), but not to other V(r) forms such as hard-wall or viscoelastic-like potentials. The "spring constant", k, determined from SPT data was 2.6 +/- 0.8 pN/microm, and not altered significantly by modulation of skeletal protein architecture by jasplakinolide. However, k was reduced by a low concentration of latrunculin, supporting the involvement of actin in the springlike tethering of CFTR. Confined diffusion of membrane proteins is likely a general phenomenon suitable for noninvasive V(r) analysis of force-producing mechanisms. Our data provide the first measurement of actin elasticity, to the best of our knowledge, that does not involve application of an external force.
Jin
Songwan
S
Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California, USA.
Haggie
Peter M
PM
Verkman
A S
AS
D000818:Animals; D002460:Cell Line; D018118:Chloride Channels / Q000737:chemistry / Q000502:physiology; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology*; D004058:Diffusion; D004285:Dogs; D004594:Electrophysiology; D000882:Haplorhini; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011487:Protein Conformation
D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator