Rapid Cell Surface Kv2.1 Recycling Observed by Single Molecule Tracking

Abstract

We study the insertion and retrieval of voltage-gated potassium channels, Kv2.1, at the single molecule level. Kv2.1 channels are labeled with quantum dots (QDs) at an extracellular domain. We observe QDs being internalized by the cell and new QD-tagged channels being inserted into the membrane. Because labeling occurs solely on the cell surface, only recycled channels that were previously in the plasma membrane can carry emerging QDs. Controls with both GFP and QD labels indicate that newly arriving QDs are indeed Kv2.1 channels. Channels that are in the plasma membrane from the beginning of the experiment can be either recycled or newly synthesized channels, as we cannot separate between these two in this measurement. The residence time distribution of channels that are on the cell surface from the beginning of our measurements has a median of 119 s, whereas for recycled channels the median is only 81 s, a 32% reduction (n = 334). In both instances it is surprising how short the residence time is on the cell surface of these channels. We propose that rapid channel turnover, via recycling pathways, helps the cell to maintain specialized regions in the membrane, which are entropically unfavorable. We investigate the role of actin in Kv2.1 trafficking using actin polymerization inhibitors. Upon the application of 5 μM cytochalasin D and 80 μM swinholide A, we observe that the residence times of both newly synthesized and recycled proteins are significantly reduced. In cells treated with actin inhibitors, channels are no longer sequestered into specific microdomains. Thus, channel recycling may function as an important factor in membrane compartmentalization and may be enhanced by stimuli that disrupt this organization.