Movements of individual BKCa channels in live cell membrane monitored by site-specific labeling using quantum dots.
The movements of BK(Ca) channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 μm(2), with an initial diffusion coefficient of 0.033 μm(2)/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal β4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BK(Ca) channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells.
School of Life Sciences, Gwangju Institute of Science Technology, Gwangju, Korea.
Sehoon
S
Won
Hae-Deun
HD
Kim
Ji-Yeon
JY
Kim
Byoung-Cheol
BC
Lee
Sunghoe
S
Chang
Chul-Seung
CS
Park
D000818:Animals; D001693:Biological Transport, Active; D055592:Biophysical Phenomena; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits; D045663:Quantum Dots; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008164:Luminescent Proteins; D015221:Potassium Channels; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C494842:Kcnma1 protein, rat