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Cell migration is an important physiological process among others controlled by ion channel activity. Calcium-activated potassium channels ([K.sub.Ca]3.1) are required for optimal cell migration. Previously, we identified single human (h) [K.sub.Ca]3.1 channel proteins in the plasma membrane by means of quantum dot (QD) labeling. In the present study, we tracked single-channel proteins during migration to classify their dynamics in the plasma membrane of MDCK-F cells. Single [hK.sub.Ca]3.1 channels were visualized with QD- or Alexa488-conjugated antibodies and tracked at the basal cell membrane using time-lapse total internal reflection fluorescence (TIRF) microscopy. Analysis of the trajectories allowed the classification of channel dynamics. Channel tracks were compared with those of free QD-conjugated antibodies. The size of the label has a pronounced effect on [hK.sub.Ca]3.1 channel diffusion. QD-labeled channels have a (sub)diffusion coefficient [D.sub.QDbound] = 0.067 [micro][m.sup.2]/[s.sup.alpha], whereas that of Alexa488-1abeled channels is [D.sub.Alexa] = 0.139 [micro][m.sup.2]/s. Free QD-conjugated antibodies move much faster: [D.sub.QDfree] = 2.163 [micro][m.sup.2]/[s.sup.alpha] Plotting the mean squared distances (msd) covered by [hK.sub.Ca]3.1 channels as a function of time points to the mode of diffusion. Alexa488-1abeled channels diffuse normally, whereas the QD-label renders [hK.sub.Ca]3.1 channel diffusion anomalous. Free QDlabeled antibodies also diffuse anomalously. Hence, QDs slow down diffusion of [hK.sub.Ca]3.1 channels and change the mode of diffusion. These results, referring to the role of label size and properties of the extracellular environment, suggest that the pericellular glycocalyx has an important impact on labels used for single molecule tracking. Thus tracking fluorescent particles within the glycocalyx opens up a possibility to characterize the pericellular nanoenvironment. calcium-activated potassium channel 3.1, quantum dot; single molecule tracking; cell migration

[K.sup.+] channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many [K.sup.+] channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian [K.sup.+] channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on [K.sup.+] channel structure contrasts with the difficulties to visualize single [K.sup.+] channel proteins in their physiological environment. We describe a method to identify single [Ca.sup.2+]-activated [K.sup.+] channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single [K.sup.+] channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level. [Ca.sup.2+]-activated [K.sup.+] channel; migration doi:10.1152/ajpcell.00633.2005

Calcium-sensitive potassium channels (KCa3.1) are expressed in virtually all migrating cells. Their activity is required for optimal cell migration so that their blockade leads to slowing down. KCa3.1 channels must be inserted into the plasma membrane in order to elicit their physiological function. However, the plasma membrane of migrating cells is subject to rapid recycling by means of endo- and exocytosis. Here, we focussed on the endocytic internalization and the intracellular transport of the human isoform hKCa3.1. A hKCa3.1 channel construct with an HA-tag in the extracellularly located S3-S4 linker was transfected into migrating transformed renal epithelial MDCK-F cells. Channel internalization was visualized and quantified with immunofluorescence and a cell-based ELISA. Movement of hKCa3.1 channel containing vesicles as well as migration of MDCK-F cells were monitored by means of time lapse video microscopy. hKCa3.1 channels are endocytosed during migration. Most of the hKCa3.1 channel containing vesicles are moving at a speed of up to 2 µm/sec in a microtubule-dependent manner towards the front of MDCK-F cells. Our experiments indicate that endocytosis of hKCa3.1 channels is clathrin-dependent since they colocalize with clathrin adaptor proteins and since it is impaired when a C-terminal dileucine motif is mutated. The C-terminal dileucine motif is also important for the subcellular localization of hKCa3.1 channels in migrating cells. Mutated channels are no longer concentrated at the leading edge. We therefore propose that recycling of hKCa3.1 channels contributes to their characteristic subcellular distribution in migrating cells. J. Cell. Physiol. 227: 686-696, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

K+ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K+ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K+ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K+ channel structure contrasts with the difficulties to visualize single K+ channel proteins in their physiological environment. We describe a method to identify single Ca2+-activated K+ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K+ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level. [ABSTRACT FROM AUTHOR]

Cell migration is crucial for wound healing, immune defense, or formation of tumor metastases. In addition to the cytoskeleton, Ca2+ sensitive K+ channels (IK1) are also part of the cellular “migration machinery.” We showed that Ca2+ sensitive K+ channels support the retraction of the rear part of migrating MDCK-F cells by inducing a localized shrinkage at this cell pole. So far the molecular nature and in particular the subcellular distribution of these channels in MDCK-F cells is unknown. We compared the effect of IK1 channel blockers and activators on the current of a cloned IK1 channel from MDCK-F cells (cIK1) and the migratory behavior of these cells. Using IK1 channels labeled with a HA-tag or the enhanced green fluorescent protein we studied the subcellular distribution of the canine (cIK1) and the human (hIK1) channel protein in different migrating cells. The functional impact of cIK1 channel activity at the front or rear part of MDCK-F cells was assessed with a local superfusion technique and a detailed morphometric analysis. We show that it is cIK1 whose activity is required for migration of MDCK-F cells. IK1 channels are found in the entire plasma membrane, but they are concentrated at the cell front. This is in part due to membrane ruffling at this cell pole. However, there appears to be only little cIK1 channel activity at the front of MDCK-F cells. In our view this apparent discrepancy can be explained by differential regulation of IK1 channels at the front and rear part of migrating cells. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]