Membrane lateral diffusion and capture of CFTR within transient confinement zones

¹ McGill University

^* To whom correspondence should be addressed. E-mail: ian.bates{at}mcgill.ca.

Submitted on March 10, 2006
Revised on April 20, 2006
Accepted on 8 May 2006

	Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts with scaffolding and other proteins that are expected to restrict its lateral movement, yet previous studies have reported predominantly free diffusion. We examined the lateral mobility of CFTR channels on live baby hamster kidney cells using three complementary methods. Channels bearing an extracellular biotinylation target sequence were labelled with streptavidin conjugated with fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605). Fluorescence recovery after photobleaching (FRAP) and image correlation spectroscopy (ICS) of the dye-labelled channels revealed a significant immobile population (~50%), which was confirmed by direct single particle tracking (SPT) of qDot605-labelled CFTR. Adding 10 Histidine residues at the C-terminus of CFTR to mask the PDZ binding motif abolished its association with EBP50/NHERF1, reduced the immobile fraction, and increased mobility. Other interactions that are not normally detected on this time scale became apparent when binding of PDZ domain proteins was disrupted. SPT revealed that CFTR_His10 channels diffuse randomly, become immobilized for periods lasting up to one minute, and in some instances are recaptured at the same location. The impact of transient confinement on the measured diffusion using the three fluorescence techniques were assessed using computer simulations of the biological experiments. Finally, the impact of endosomal CFTR on mobility measurements was assessed by fluorescence correlation spectroscopy (FCS). These results reveal unexpected features of CFTR dynamics which may influence its ion channel activity.

Key Words: FRAP, PDZ domains, chloride channel, cystic fibrosis, image correlation spectroscopy, single particle tracking

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