Kv2.1 membrane corrals: Novel regulators of K+ channel function and trafficking

Michael Tamkun

Program in Molecular, Cellular and

Developmental Neuroscience

Department of Biomedical Sciences

Colorado State University

Requirements for live cell imaging

Fast and sensitive microscopes

Olympus FV1000 laser scanning confocal with PMT-based spectral detectors

Wide-field systems with deconvolutionSpinning disk confocal with CCD camera detection

Useful tags

Fluorescent proteins fused onto the protein to be imagedQuantum dots

Cell system with normal trafficking and regulation

Cultured HEK cells, neurons and cardiac myocytes

Four imaging techniques to be illustrated are

Single molecule tracking using quantum dots.

Analysis of diffusion via fluorescence recovery after photobleach (FRAP)

Analysis of stability using photoactivation

Imaging to direct analysis of single molecule function(location/function studies)

Epitope insertion

BADCFPGFPPA-GFPYFPmRFP

HA peptideMyc peptideBiotin acceptor peptideGFP

Useful tags for Kv channel trafficking in live cells

Long SB, Campbell EB, Mackinnon R. (2005) Science 309, 897-903

Kv1.2/Kvbeta2.1 structure

Single channel tracking

• Express Kv2.1 with the loopBAD extracellular tag(GGGAGGLV GLNDIFEAQKIEWHEAR GGGAGG),

• Biotinylate with biotin ligase

• Label Kv2.1 with streptavidin tagged Qdots (QD605 or 655)

• Image as fast as possible1-10 frames/sec

Quantum dots

CdSe in the core and ZnS in the shell

Colloidal semiconductor nanocrystals, 10 to 50 atoms in diameter and a total of 100 to 100,000 atoms within the quantum dot volume. Typically between 10 and 50 nm in size.

QuickTime™ and a decompressor

are needed to see this picture.

Quantum dot based tracking of individual Kv2.1 channels (HEK cells)

Streptavidin 605 Qdots bound after biotinylation of the extracellular loopBAD site

Mean Square Displacement Analysis of Diffusion

MSD(nt) 1N n {[x( jt nt) x( jt)]2

j1

N n

[y( jt nt) y( jt)]2}

t is the time interval at which images were taken, x(t) and y(t) are the coordinates of a Qdot at time t, and N is the total number of images in a recording. n and j are positive integers with n=1, 2, … ,(N-1). The apparent diffusion coefficient can be calculated as one fourth of the slope of the linear regression line fitted to the n = 2 to 10 values of the MSD(nt).

Unrestricted Directed Restricted

Time TimeTime

MS

D

MS

D

MS

D

D= Slope/4

MSD analysis of Kv2.1 diffusion

FRAP experiments indicate Kv1.4 channels also ignore the Kv2.1 cluster-forming perimeter fence

CFP-Kv2.1 co-expressedwith YFP-Kv1.4

Use of photoactivatable GFP to monitor Kv2.1 stability

405 nm laser based activation within rectangle

Channels are composed of two dsRED-Kv2.1 and two PA-GFP-Kv2.1 subunits

Single channel analysis of Kv2.1 clusters

GFP fluorescencesuggests 250-300 channels/ m2

Macroscopic currents are seen outside clusters

On cluster single channel events

9 pSPo=0.2 at -25 mV

Kv2.1 clusters are likely to perform two basic physiological roles. They are platforms that

1. Organize and regulate the efficient insertion and retrieval of Kv2.1 molecules at the cell surface.

2. Co-localize Kv2.1 near signaling molecules which regulate its function. Clustered Kv2.1 represent a reserve pool of inactive channels stored on the cell surface.

Microscope systems used

Olympus FV1000 owned by the Tamkun lab- two PMT-based spectral detectors, one standard PMT, two scan heads for simultaneous dual wavelength excitation and imaging

Zeiss 510 Meta system housed in A/Z- single PMT-based spectral detector, two standard PMTs

Olympus based wide-field (left side) and Yokogawa spinning disk (right side) with Mosaic system for simultaneous, multi-point photoactivation and photobleach

Olympus based TIRF system to be installed in BMS next month

CO2 Induces Redistribution of Kv2.1 in the Intact Rat Brain

Misonou, H., Mohapatra, D. P., Menegola, M., and Trimmer, J. S. (2005) J Neurosci 25(48), 11184-11193