Single-Particle Tracking of Membrane Protein Diffusion in a Potential: Simulation, Detection, and Application to Confined Diffusion of CFTR Cl− Channels

摘要

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 ∼0.004 μm2/s. SPT data fitted closely to a springlike attractive potential, V(r) = kr2, 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/μm, 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.