Topography of native SK channels revealed by force nanoscopy in living neurons.
The spatial distribution of ion channels is an important determinant of neuronal excitability. However, there are currently no quantitative techniques to map endogenous ion channels with single-channel resolution in living cells. Here, we demonstrate that integration of pharmacology with single-molecule atomic force microscopy (AFM) allows for the high-resolution mapping of native potassium channels in living neurons. We focus on calcium-activated small conductance (SK) potassium channels, which play a critical role in brain physiology. By linking apamin, a toxin that specifically binds to SK channels, to the tip of an AFM cantilever, we are able to detect binding events between the apamin and SK channels. We find that native SK channels from rat hippocampal neurons reside primarily in dendrites as single entities and in pairs. We also show that SK channel dendritic distribution is dynamic and under the control of protein kinase A. Our study demonstrates that integration of toxin pharmacology with single-molecule AFM can be used to quantitatively map individual native ion channels in living cells, and thus provides a new tool for the study of ion channels in cellular physiology.
Department of Mechanical Engineering, University of Connecticut, Storrs, Connecticut 06269, USA.
Jamie L
JL
Maciaszek
Heun
H
Soh
Randall S
RS
Walikonis
Anastasios V
AV
Tzingounis
George
G
Lykotrafitis
D000818:Animals; D000831:Animals, Newborn; D001030:Apamin / Q000494:pharmacology; D001703:Biophysics; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003712:Dendrites / Q000187:drug effects / Q000378:metabolism; D004558:Electric Stimulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D018625:Microscopy, Atomic Force; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D020033:Protein Isoforms; D051640:Small-Conductance Calcium-Activated Potassium Channels; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005576:Colforsin; D001030:Apamin