1. 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

2. 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

3. 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)

4. Epitope insertion BAD CFP GFP PA-GFP YFP mRFP HA peptide Myc peptide Biotin acceptor peptide GFP 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

5. 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

6. 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.

7. Quantum dot based tracking of individual Kv2.1 channels (HEK cells) Streptavidin 605 Qdots bound after biotinylation of the extracellular loopBAD site

8. 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 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(n  t). Unrestricted Directed Restricted Time MSD D= Slope/4

9. MSD analysis of Kv2.1 diffusion

10. FRAP experiments indicate Kv1.4 channels also ignore the Kv2.1 cluster-forming perimeter fence CFP-Kv2.1 co-expressed with YFP-Kv1.4

11. 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

12. Single channel analysis of Kv2.1 clusters GFP fluorescence suggests 250-300 channels/  m 2 Macroscopic currents are seen outside clusters On cluster single channel events 9 pS P o =0.2 at -25 mV

13. 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.

14. 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

15. 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

16. CO 2 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