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

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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
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Requirements for live cell imaging Fast and
sensitive microscopes Olympus FV1000 laser
scanning confocal with PMT-based spectral
detectors Wide-field systems with
deconvolution Spinning disk confocal with CCD
camera detection Useful tags Fluorescent
proteins fused onto the protein to be
imaged Quantum dots Cell system with normal
trafficking and regulation Cultured HEK cells,
neurons and cardiac myocytes
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• 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)

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Useful tags for Kv channel trafficking in live
cells
HA peptide Myc peptide Biotin acceptor peptide GFP
Epitope insertion
BAD CFP GFP PA-GFP YFP mRFP
Kv1.2/Kvbeta2.1 structure
Long SB, Campbell EB, Mackinnon R. (2005)
Science 309, 897-903
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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 possible
• 1-10 frames/sec

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Quantum dots
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.
CdSe in the core and ZnS in the shell
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• Quantum dot based tracking of individual Kv2.1
  channels (HEK cells)

Streptavidin 605 Qdots bound after biotinylation
of the extracellular loopBAD site
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Mean Square Displacement Analysis of Diffusion
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 n1, 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(n?t).
Restricted
Unrestricted
Directed
MSD
MSD
MSD
D Slope/4
Time
Time
Time
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MSD analysis of Kv2.1 diffusion
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FRAP experiments indicate Kv1.4 channels also
ignore the Kv2.1 cluster-forming perimeter fence
CFP-Kv2.1 co-expressed with YFP-Kv1.4
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Use of photoactivatable GFP to monitor Kv2.1
stability
Channels are composed of two dsRED-Kv2.1 and two
PA-GFP-Kv2.1 subunits
405 nm laser based activation within rectangle
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Single channel analysis of Kv2.1 clusters
GFP fluorescence suggests 250-300 channels/ ?m2
9 pS Po0.2 at -25 mV
Macroscopic currents are seen outside clusters
On cluster single channel events
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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.
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Microscope systems used
Zeiss 510 Meta system housed in A/Z- single
PMT-based spectral detector, two standard PMTs
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
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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
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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