AJP - Heart Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H229-H237, 2008. First published October 26, 2007; doi:10.1152/ajpheart.01038.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Movies
Right arrow All Versions of this Article:
294/1/H229    most recent
01038.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O'Connell, K. M. S.
Right arrow Articles by Tamkun, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O'Connell, K. M. S.
Right arrow Articles by Tamkun, M. M.

Localization and mobility of the delayed-rectifer K+ channel Kv2.1 in adult cardiomyocytes

Kristen M. S. O'Connell,1 Jennifer D. Whitesell,1 and Michael M. Tamkun1,2

1Department of Biomedical Sciences and 2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado

Submitted 7 September 2007 ; accepted in final form 21 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
The delayed-rectifier voltage-gated K+ channel (Kv) 2.1 underlies the cardiac slow K+ current in the rodent heart and is particularly interesting in that both its function and localization are regulated by many stimuli in neuronal systems. However, standard immunolocalization approaches do not detect cardiac Kv2.1; therefore, little is known regarding its localization in the heart. In the present study, we used recombinant adenovirus to determine the subcellular localization and lateral mobility of green fluorescent protein (GFP)-Kv2.1 and yellow fluorescent protein-Kv1.4 in atrial and ventricular myocytes. In atrial myocytes, Kv2.1 formed large clusters on the cell surface similar to those observed in hippocampal neurons, whereas Kv1.4 was evenly distributed over both the peripheral sarcolemma and the transverse tubules. However, fluorescence recovery after photobleach (FRAP) experiments indicate that atrial Kv2.1 was immobile, whereas Kv1.4 was mobile ({tau} = 252 ± 42 s). In ventricular myocytes, Kv2.1 did not form clusters and was localized primarily in the transverse-axial tubules and sarcolemma. In contrast, Kv1.4 was found only in transverse tubules and sarcolemma. FRAP studies revealed that Kv2.1 has a higher mobility in ventricular myocytes ({tau} = 479 ± 178 s), although its mobility is slower than Kv1.4 ({tau}1 = 18.9 ± 2.3 s; {tau}2 = 305 ± 55 s). We also observed the movement of small, intracellular transport vesicles containing GFP-Kv2.1 within ventricular myocytes. These data are the first evidence of Kv2.1 localization in living myocytes and indicate that Kv2.1 may have distinct physiological roles in atrial and ventricular myocytes.

ion channel localization; lateral diffusion; adenovirus; voltage-gated potassium channels; live cell confocal microscopy

THE CARDIAC ACTION POTENTIAL is a complex balance of the activity of a variety of ion channels. Among these, voltage-gated K+ channels play a crucial role in cardiac repolarization. However, it is becoming increasingly clear that ion channel function is not the sole determinant of appropriate cellular excitability but that channels must also be correctly localized. Although the local distribution of ion channels can influence local membrane potential (19), channels are also often associated with modulatory signal transduction pathways critical for proper regulation of channel activity. For example, mutations within KCNQ1 that cause Long QT Syndrome disrupt the interaction of KCNQ1 with the AKAP yotiao, causing aberrant sympathetic regulation of the guinea pig cardiac slow K+ current (27). A form of Brugada syndrome is caused by a mutation in the cardiac voltage-gated Na+ channel (Nav) 1.5 channel that prevents its association with cardiac ankyrin-G, resulting in a failure of Nav1.5 to localize to the cell surface of cardiomyocytes (32).

Voltage-gated K+ channel (Kv) 2.1 is a slow delayed-rectifier K+ channel that underlies the slow K+ current (Ik,slow2) in rat cardiomyocytes (6, 10, 24, 26, 49, 50). Targeted elimination of Kv2 channels in mouse ventricular myocytes leads to prolongation of the action potential duration and the QT interval (24, 49, 50), demonstrating a role for this channel in repolarization and cardiac chronotropy. Although a role for Kv2.1 in human cardiac repolarization has yet to be confirmed, both Kv2.1 message (37) and Kv2.1 protein (48) have been detected in human atria and ventricles, suggesting the channel may have a similar role in human myocardium. In neurons, Kv2.1 has been shown to be sensitive to increased electrical activity (16, 29), intracellular Ca2+ (28, 33), O2, and hypoxia (22, 28, 38) and muscarinic stimulation (31). In response to these stimuli, the channel becomes dephosphorylated and undergoes a concomitant hyperpolarizing shift in the voltage dependence of activation, thus dampening neuronal excitability, particularly during periods of hyperexcitability (30). Kv2.1 also exhibits a high degree of localization: in hippocampal neurons, the channel is restricted to the somatodendritic region, where it forms large clusters on the cell surface (3, 35). In response to the aforementioned stimuli, Kv2.1 is dispersed from these clusters, becoming more evenly distributed over the cell surface (28, 29, 31), supporting a close relationship between channel function and localization.

Our previous studies demonstrated that the confinement of Kv2.1 to these cluster domains is likely to be via retention behind an actin fence, as opposed to binding to cytoskeletal-linked scaffolding proteins (35, 45). Single particle tracking of individual Kv2.1 channels on the cell surface revealed that, within the cluster domain, the channel is freely mobile (45). Kv2.1 is the first reported example of an ion channel whose localization is determined by such a cytoskeletal corral.

Although a considerable amount is known regarding the function of cardiac ion channels, including Kv2.1, surprisingly little is known about the subcellular localization of these channels in heart. In this study, we used recombinant adenovirus to express green fluorescent protein (GFP)-tagged Kv channels in isolated myocytes from both adult atria and ventricle to investigate the subcellular localization of the channels in these regions. This approach also allowed us to examine the dynamics of channel mobility in the surface membrane of expressing myocytes. In atrial myocytes, Kv2.1 exhibited a clustered distribution similar to that observed in HEK cells and neurons. However, live cell imaging and fluorescence recovery after photobleaching (FRAP) indicated that the channel's behavior is markedly different from that observed in other cell types. This difference may indicate that the channel is retained within the cluster domain by a mechanism unique to atrial myocytes. In ventricular myocytes, Kv2.1 does not form clusters but does have a cell surface mobility similar to that observed in neurons and HEK cells. In both cell types, the localization and mobility of Kv2.1 was distinct from that of Kv1.4, indicating that these two channels are handled in a subtype-specific fashion.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 
Construction of adenoviral vectors. Yellow fluorescent protein (YFP)-Kv1.4myc was constructed as previously described (36). Adenovirus carrying YFP-Kv1.4myc (Ad-YFP-Kv1.4) was made by digesting pYFP-Kv1.4myc with NheI and HincII. The YFP-Kv1.4 fragment was inserted into pShuttle-CMV transformed into the dam– strain Escherichia coli SCS110 and digested with XbaI and EcoRV. The pShuttle-YFP-Kv1.4myc plasmid was then linearized with PmeI, and the linearized vector cotransformed with the adenoviral backbone pAdEasy-1 into BJ5183 competent E. coli for recombination. Recombinants were linearized with PacI and transfected into HEK293 cells for adenoviral packaging. After 5–7 days, when most of the cells turned yellow and detached from the dish, the virus was harvested and used to infect T-75 flasks for further amplification (20).

The GFP-Kv2.1 adenovirus was a generous gift from Dr. Jim Trimmer at the University of California at Davis (3).

Isolation and adenoviral infection of adult rat cardiomyocytes. All protocols were reviewed and approved by the Colorado State University Animal Care and Use Committee.

Myocytes were isolated from the hearts of 6- to 12-wk-old male Sprague-Dawley rats. Rats were anesthetized using 60 mg/kg ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and 10 mg/kg xylazine, and the heart was removed. An aortic cannula was inserted, and the heart was mounted on a modified Langendorff apparatus for perfusion. The hearts were perfused with perfusion buffer containing (in mM): 135 NaCl, 5.0 KCl, 0.4 KH2PO4, 1.4 MgCl2, 10 HEPES, 10 glucose, 20 taurine, 10 creatine, and 0.125 K2EGTA, pH = 7.35, osmolality = 320 mmol/kg. After 5 min, the heart was perfused with digestion buffer, which was made by adding 0.25 mg/ml Liberase Blendzyme 4 (Roche Applied Science, Indianapolis, IN) and 12.5 µM CaCl2 in perfusion buffer. Hearts were digested for 20 min at 3 ml/min with digestion buffer and then removed from the Langendorff apparatus. The atria were removed, and both the atria and ventricles were minced into small (<1 cm) pieces. The ventricular tissue was digested by nutating a further 20 min in digestion buffer. Individual myocytes were dispersed from both the atrial and ventricular tissue by trituration and gentle centrifugation. After dispersion, Ca2+ was reintroduced stepwise to a final concentration of 1.2 mM. Both atrial and ventricular myocytes were resuspended in medium 199 supplemented with 2 mg/ml fraction V BSA (Sigma), 2 mM carnitine, 5 mM creatine, 5 mM taurine, 0.1 µM insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin and plated on enhanced chemiluminescence attachment matrix-coated (Millipore, Billerica, MA) 35-mm glass bottom dishes (MatTek, Ashland, MA).

Myocytes were infected with adenovirus carrying either GFP-Kv2.1 or YFP-Kv1.4myc 1 h after isolation. The volume of purified virus necessary for maximum expression was determined empirically. For expression of YFP-Kv1.4myc, myocytes were incubated with 20 µl of purified virus. For expression of GFP-Kv2.1, myocytes were incubated with 200 µl of purified virus overnight, and the media was changed the following day. Expression of YFP-Kv1.4 was typically observed 24–36 h after infection, whereas GFP-Kv2.1 expression was observed ~48 h after infection.

Live cell confocal imaging. Myocytes expressing GFP-Kv2.1 or YFP-Kv1.4 were imaged using a Fluoview1000 laser scanning confocal microscope (Olympus America, Center Valley, PA). A x60, 1.4 numerical aperture oil immersion objective with the pinhole diameter set for 1 Airy Unit was used for all images. GFP was excited by illumination with the 488-nm line of an Ar laser set at 0.5–1% transmission and emission collected using the variable bandpass filter set to 500–550 nm. Excitation of YFP was accomplished by illumination with the 514-nm line set at 1–2% transmission and emission collected using the variable bandpass filter set at 530–580 nm. For all images, the detector voltage was adjusted as necessary to use the full 12-bit dynamic range of the detector. All images were acquired at either 512 x 512 (time series) or 1024 x 1024 (z-stacks) resolution.

For FRAP experiments, GFP was photobleached using a 405-nm diode laser. A circular region of interest (ROI) was photobleached using the SIM scanner of the FV1000 set in tornado scan mode with the 405-nm diode laser set at 25% transmission for 1 s. Pre- and postbleach images were acquired as described above. Three to five prebleach images were acquired for each experiment, and recovery was monitored for ~20 min. The average fluorescence intensity within the bleach ROI was normalized to the prebleach intensity for each time point and plotted as a function of time. Recovery kinetics were analyzed by exponential fitting of the recovery time course:

Formula 1(1)
where Ai is the amplitude of each component, t is time, and {tau}i is the time constant of recovery of each component. The mobile fraction (Mf) was calculated as:

Formula 2(2)
where F{infty} is the fluorescence intensity at the end of the recovery period, F0 is the fluorescence intensity immediately postbleach, and Fi is the initial fluorescence intensity before photobleach. Cumulative photobleach outside of the FRAP ROI during the course of the imaging was typically 10–15% of the initial prebleach intensity and was not corrected for.

Vesicle tracking was done by manually drawing an ROI around the vesicle in each time point and then using the Quantitation Module of Volocity 4.1 (Improvision, Lexington, MA) to track the points using the shortest path model, with a minimum distance between objects of 0.2 µm.

All off-line image analysis was performed using Olympus Fluoview Software (version 1.6) and Volocity 4.1. Images were median filtered using a 3 x 3 filter and adjusted for brightness and contrast in Volocity. No other manipulation of the images was performed. All other data analysis was performed using SigmaPlot 8 (SPSS, Chicago, IL).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
GRANTS
 REFERENCES
 
Culture and infection of acutely dissociated cardiomyocytes. The standard approach for examining the subcellular localization of proteins is immunolabeling with specific antibodies. However, antibodies against Kv2.1 (which recognize an epitope in the COOH terminus) that efficiently label the channel in neurons (35) do not recognize Kv2.1 in cardiac myocytes (data not shown). Kv2.1 is abundant in adult rodent atrial and ventricular myocytes (4, 10, 37) so the inability of antibodies to recognize the channel may point to a modification of the channel's COOH terminus that is unique to the heart. Because cardiac myocytes are amenable to transduction by viral vectors, we turned to recombinant adenoviruses to express GFP-tagged Kv2.1 in adult myocytes, an approach that has been used previously to overexpress ion channel proteins and accessory proteins such as cardiac Ca2+ channel β-subunits (43) and calmodulin (1).

Because it typically requires 24–48 h for robust protein expression following adenoviral infection, it was first necessary to ascertain whether we could isolate cardiac myocytes and maintain them in culture long enough for sufficient expression of the recombinant protein while maintaining the structural integrity of the elaborate membrane system found in ventricular myocytes. One of the consequences of culturing adult cardiomyocytes is the dedifferentiation of the cells and eventual loss of the transverse tubule (T tubule) system (51). Such a breakdown in the plasma membrane system would clearly be detrimental to our study and cast doubt upon our results. Figure 1 shows a rat ventricular myocyte maintained in culture for 48 h. To determine whether the T tubule system was intact, we stained the plasma membrane with the lipophilic carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylinocarbocyanine perchlorate (DiIC18; Molecular Probes), which efficiently labels plasma membrane structures. As seen in Fig. 1, diI labeled both the peripheral sarcolemma and the T tubules. Adult ventricular myocytes also have axial tubules that branch off the transverse tubules; these were also labeled by diI (Fig. 1B), indicating that the complete T tubule system in 48-h-old ventricular myocytes is still present.


Figure 1
View larger version (70K):
[in this window]
[in a new window]

  		Fig. 1. The axial and transverse tubule (T tubule) system is intact in adult rat ventricular myocytes 48 h following isolation. A: acutely isolated ventricular myocyte 48 h after isolation labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylinocarbocyanine perchlorate (diI) to mark sarcolemmal structures. Left, diI fluorescence; right, DIC image of cell in right panel. B: enlarged image of region highlighted by the box in A. Arrow, T tubule; arrowhead, axial tubule.

 
Localization of Kv2.1 in atrial myocytes. Kv2.1 is highly expressed in the atria, where it underlies a significant component of the Ik,slow and rat cardiac steady-state noninactivating (Iss) currents (7); as such, it is an important repolarizing channel in the atria. To examine the subcellular localization of Kv2.1, we infected acutely isolated adult rat atrial myocytes with Ad-GFP-Kv2.1. Although atrial myocytes are not as robust as ventricular myocytes, we were able to maintain isolated atrial myocytes in culture for ~24 h, which was long enough for Ad-GFP-Kv2.1 expression while still maintaining contractility and the overall spindle-shaped morphology of atrial myocytes.

As shown in Fig. 2A and supplemental movie 1 (all supplemental material can be found with the online version of this article), GFP-Kv2.1 expression in atrial myocytes was localized to large surface clusters that closely resemble those typically seen in other cell types, such as hippocampal neurons (35). The Kv2.1 clusters in atrial myocytes are somewhat smaller that their neuronal counterparts, with an average area of 380 ± 40 nm2 vs. 890 ± 160 nm2 in neurons (35). Unlike neuronal Kv2.1 clusters, the atrial clusters appear to be regularly aligned along the long axis of the cell (Fig. 2A and Supplemental Movie 1). Such an alignment suggests an interaction of Kv2.1 with some intracellular component. We recently demonstrated in HEK cells and hippocampal neurons (35, 45) that the cortical actin cytoskeleton plays a major role in the maintenance of Kv2.1 clusters. Given the similar appearance of the atrial clusters, it seems likely that cortical actin plays a significant role in the localization of atrial Kv2.1 as well. Unfortunately, given the abundance of actin in the contractile machinery, detection of the relatively small pool of cortical actin is difficult, and we have been unable to identify actin structures similar to those we observed in HEK cells (45).


Figure 2
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 2. Adenoviral-mediated expression of fluorescent protein-tagged voltage-gated K+ (Kv) channels in adult atrial myocytes. Confocal images of live atrial myocytes expressing adenovirus carrying green fluorescent protein-Kv2.1 (Ad-GFP-Kv2.1, A) and adenovirus carrying yellow fluorescent protein-Kv1.4myc (Ad-YFP-Kv1.4, B). Atrial myocytes were imaged 24 h after adenoviral infection and were kept at 37°C in imaging saline during imaging. Scale bars = 10 µm.

 
We previously showed that, even in HEK cells, different Kv channels exhibit isoform-specific surface localization (36). Although Kv2.1 is clustered in HEK cells, the related channel Kv1.4, which underlies the slow transient outward current in the heart, is evenly distributed over the cell surface (36). Therefore, to determine whether the expression pattern of Kv2.1 observed in atrial myocytes is specific to Kv2.1, we used adenovirus to express YFP-tagged Kv1.4 in isolated atrial myocytes. As shown in Fig. 2B, YFP fluorescence was evenly distributed over the peripheral sarcolemma, with no obvious clustering of Kv1.4. Additionally, Kv1.4 was localized to striations within the myocyte. The spacing of these striations was ~2 µm, consistent with z-line spacing, supporting the idea that these structures are T tubules rather than an intracellular accumulation of mistrafficked channel. Because atrial myocytes do not typically have an elaborate T tubule system such as found in ventricular myocytes, this localization was unexpected. However, it has been reported that a subset of atrial myocytes do possess a rudimentary T tubule system (23); perhaps it is in these cells that Kv1.4 is preferentially expressed. It is unlikely that this pattern is due to contamination of the atrial myocyte culture with ventricular myocytes, since none of these cells exhibited the distinctive rod-shaped morphology typical of ventricular myocytes. These data indicate that the cell surface clusters are a phenotype specific to Kv2.1.

Mobility of Kv2.1 in atrial myocytes. A significant advantage to the use of adenovirus to express fluorescent protein-tagged channels in living myocytes is the ability to monitor the dynamics of protein localization in real time. In HEK cells and hippocampal neurons, Kv2.1-containing surface clusters are dynamic structures whose size and shape change over short periods of time. These clusters are observed to move in the membrane at a rate of ~2 µm2/min (35, 45). Furthermore, clusters are frequently observed to both fuse with adjacent clusters and break apart to form new clusters (35). Using live-cell confocal imaging, we investigated whether the Kv2.1-containing clusters in atrial myocytes exhibit a similar behavior. As shown in Fig. 3 and Supplemental Movie 2, these atrial clusters are much more static than their noncardiac counterparts. In Fig. 3, several clusters on the bottom surface of a GFP-Kv2.1-expressing atrial myocyte are outlined in red. Over 15 min, the shape of these clusters changes very little, such that, even at the end of the experiment, their overall shape and relative position are still distinctive enough to readily identify the original clusters. None of these clusters, even very closely apposed clusters, was ever seen to fuse with another cluster, nor do they ever break apart to form new clusters (see Supplemental Movie 2; compare with Ref. 35).


Figure 3
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 3. GFP-Kv2.1 containing clusters in atrial myocytes are highly stable structures. This sequence of images is taken from a time-lapse series of a GFP-Kv2.1-expressing atrial myocyte. Three clusters are outlined in red to show that neither the shape nor the position of these structures changes significantly even after 15 min of imaging. Scale bar = 2.5 µm.

 
Dynamics of Kv2.1 within the cluster domain. We previously demonstrated that, in both HEK cells and hippocampal neurons, Kv2.1 is highly mobile within the boundaries of the cluster (35, 45). Given the apparent similarity between atrial Kv2.1 clusters and neuronal clusters, we investigated the mobility of Kv2.1 using FRAP. As shown in Fig. 4A, part of a GFP-Kv2.1 cluster was photobleached by high-intensity illumination. In stark contrast to Kv2.1 clusters in other cell types, no diffusion of Kv2.1 from the unbleached region to the bleached region was observed. Virtually no fluorescence recovery into the bleached region was seen, even at the end of an ~40-min recovery period. This is dramatically different from the situation in HEK cells and hippocampal neurons where the channel diffuses rapidly within the cluster. It was not possible to quantitate the recovery of GFP fluorescence in these experiments by calculating a Mf or a {tau} of recovery because of the extremely low recovery.


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 4. Analysis of Kv channel mobility in atrial myocytes using fluorescence recovery after photobleach (FRAP). A: one-half of a GFP-Kv2.1-containing cluster on the bottom surface of an atrial myocyte was photobleached (red circle). Even nearly 20 min later, there is no appreciable recovery of fluorescence in the bleached region (middle). Because there was no fluorescence recovery, it was not possible to determine time constants ({tau}) or a mobile fraction (Mf) for these cells. B: FRAP of atrial myocyte expressing YFP-Kv1.4. Unlike GFP-Kv2.1, there was rapid recovery of YFP fluorescence within the atrial myocyte. For this cell, {tau} = 182 s and Mf = 0.55. Red line is an exponential fit to the data.

 
To determine if the low mobility of GFP-Kv2.1 in atrial myocytes is a generalized feature common to all cardiac K+ channels, we used FRAP to examine the dynamics of YFP-Kv1.4 in atrial myocytes. As before, we drew an ROI encompassing a small region of the myocyte containing Kv1.4 (Fig. 4B). Unlike what we observed for Kv2.1, there was significant recovery of YFP fluorescence after photobleach, with an average Mf of 0.42 ± 0.13 (n = 3). Additionally, as in HEK cells, the time course of recovery for Kv1.4 was well described by a single exponential with a {tau} of 252 ± 42 s (n = 3). These parameters differ slightly from what we reported for YFP-Kv1.4 in HEK cells, where the Mf was 0.78 and {tau} was 133 s. It seems probable that interaction of Kv1.4 with elements not present in HEK cells (e.g., {alpha}-actinin) underlies this difference.

We previously demonstrated that, in other cell types, cortical actin plays a major role in defining the cluster boundary and restricting the channel to these domains. However, our previous work suggests that, within the cluster, Kv2.1 is not tethered and is freely mobile. This does not appear to be the case in atrial myocytes based on the evidence presented here. First, Kv2.1 exhibits a high degree of organization as evidenced by the regular alignment of the Kv2.1 clusters along the long axis of the cell. These clusters are spaced ~2 µm apart (see Fig. 2), suggesting they may be interacting with proteins at or near the T tubules or z-line. In hippocampal neurons, Kv2.1 has been shown to colocalize with ryanodine receptors (RyRs) at the level of both light and electron microscopy (3, 17). Thus there is the intriguing possibility that Kv2.1 and RyR2 may share a similar relationship in atrial myocytes. Second, although we do not exclude a role for cortical actin, particularly with respect to the maintenance of the cluster boundary, the immobility of Kv2.1 on the atrial surface membrane points to protein-protein interactions not present in other cell types.

Localization of Kv2.1 in ventricular myocytes. To determine the localization of Kv2.1 in ventricular myocytes, we used Ad-GFP-Kv2.1 to infect dissociated adult rat ventricular myocytes. As mentioned previously, in almost every other cell type, Kv2.1 forms large surface clusters. Interestingly, these clusters are absent from Ad-GFP-Kv2.1-expressing ventricular myocytes (Fig. 5A). Rather, the channel appears to be distributed over both the transverse (Fig. 5A) and axial (Fig. 5A) tubules (Supplemental Movie 3). Interestingly, although the large clusters seen in hippocampal neurons are absent, Kv2.1 is not distributed evenly over the T tubules but instead appears concentrated over smaller regions of the T tubular membrane (Fig. 5A). It is unlikely that this is because of a breakdown on the T tubular system, since diI efficiently labeled these membranes (Fig. 1). Several areas within the myocyte appear to be punctate (Fig. 5A, inset); however, three-dimensional reconstruction of the myocyte (Supplemental Movie 3) suggests that these may be regions where the T tubules branch rather than clusters of Kv2.1. Other ion channels, such as Kv1.5 and Nav1.5, have been found to be concentrated at the intercalated disk; however, Kv2.1 displays no such concentration to this region.


Figure 5
View larger version (94K):
[in this window]
[in a new window]

  		Fig. 5. Localization of adenovirally expressed Kv channels in adult rat ventricular myocytes. Adenoviral-mediated expression of fluorescent protein-tagged Kv channels in adult rat ventricular myocytes. Confocal images of live ventricular myocytes expressing Ad-GFP-Kv2.1 (A) and Ad-YFP-Kv1.4 (B). Myocytes were imaged 48 h following adenoviral infection. For imaging, cells were kept at 37°C in imaging saline (see METHODS). Inset shows high-resolution view of region highlighted in box. In A, arrows indicate transverse tubules, and arrowheads indicate axial tubules. Scale bars = 10 µm (2.5 µm in inset).

 
Nonclustered surface localization of Kv2.1 has been reported in COS cells, where the channel exists primarily in a dephosphorylated state (31). However, work from Brunet et al. (10) indicates that Kv2.1 in the ventricular tissue may be in the phosphorylated state, since Western blot analysis in that paper shows that Kv2.1 runs at a higher molecular weight than predicted (110 vs. ~95 kDa) in tissue homogenates from the ventricle of adult rats. Work from the Trimmer group has demonstrated that, in HEK cell and brain homogenates, this molecular weight difference is attributable to phosphorylation (31, 33, 40). Because the phosphorylation state of Kv2.1 is always associated with the clustered phenotype, this raises the intriguing question of whether the same holds true for ventricular Kv2.1. It will be interesting to determine whether ventricular Kv2.1 is in fact as extensively phosphorylated as it is in neurons, yet does not cluster, or whether the additional mass is because of an as yet unknown posttranslational modification.

Localization of Kv1.4 in ventricular myocytes. We again used Kv1.4 to determine whether the axial and transverse tubule localization of Kv2.1 was specific to this channel. As shown in Fig. 5B, Kv1.4 is evenly distributed over the peripheral sarcolemma and the transverse tubules, as well as the intercalated disk. Unlike Kv2.1, Kv1.4 appears to be absent from the axial tubules (Fig. 5B, inset) and was more strongly expressed on the peripheral membrane. These results indicate that, as in atrial myocytes, the cell handles these distinct Kv channels differently. Kv1.4 has been reported to interact with {alpha}-actinin (13), a key component of the cardiac z-line. Such an interaction is consistent with the expression pattern seen here. YFP-Kv1.4 also appears to be localized to the intercalated disk, like the related channel Kv1.5 (18). The apparent concentration of Kv1.4 to this region is unlikely to be an artifact of membrane folding at the ends of the myocytes, since Kv2.1 is not particularly enriched at the intercalated disk (see Fig. 5A).

In all the cells we examined, there was a significant pool of channel (both Kv2.1 and Kv1.4) in a perinuclear compartment (see, for example, Supplemental Movie 3). In heterologous systems such as HEK cells and cultured hippocampal neurons, both of these channels are efficiently trafficked to the cell surface, and perinuclear accumulation is infrequent. Immunolocalization of the cardiac voltage-gated Na+ channel Nav1.5 has demonstrated a similar nuclear localization (52), suggesting this could be a possible intracellular reserve pool of channel, although the possibility that it is merely an artifact of overexpression cannot be ruled out.

Mobility of Kv2.1 in ventricular myocytes. Given the distinct subcellular localization of Kv2.1 in ventricular myocytes (i.e., the lack of large surface clusters), we were interested to see if there was also a change in channel mobility. As shown in Fig. 6A, we drew an ROI encompassing GFP-Kv2.1 localized within T tubules and photobleached GFP using high-intensity 405-nm laser light and then monitored the recovery of fluorescence in the ROI by scanning once every 5 s for ~30 min. Fluorescence recovery within the ROI was normalized to its prebleach intensity, plotted as a function of time, and then fit according to Eq. 1 (see METHODS). As we previously observed for Kv2.1 expressed in HEK cells and hippocampal neurons, GFP-Kv2.1 exhibited little recovery following photobleach in ventricular myocytes, with an average Mf of 0.30 ± 0.06 (n = 7). Even taking into account cumulative photobleach during continuous imaging, <50% recovery is observed during the 30-min recovery period. Unlike FRAP of Kv2.1 in HEK cells, the time course of recovery in ventricular myocytes was well fit by a single exponential, with a {tau} of 497 ± 178 s (n = 7; Fig. 3A, right).


Figure 6
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 6. Analysis of Kv channel mobility in ventricular myocytes using FRAP. A: FRAP of a ventricular myocyte expressing GFP-Kv2.1. GFP fluorescence within the T tubules (red circle) was photobleached, and recovery was monitored within the bleach region. Even after ~25 min of recovery, only ~40% of the fluorescence has recovered (right). For this cell, {tau} = 136.3 s and Mf = 0.39. Scale bar = 3.4 µm. B: FRAP of a ventricular myocyte expressing YFP-Kv1.4. A region in the center of the cell containing T tubules was bleached, and recovery was monitored within the bleach region. For this cell, {tau}1 = 28.7 s, {tau}2 = 590 s, and Mf = 0.67. Scale bar = 6.1 µm. For both cells, the imaging interval was 5 s, and analysis was performed on the region described by the red circle. Red lines are exponential fits to the data.

 
Mobility of Kv1.4 in ventricular myocytes. In HEK cells, Kv1.4 is highly mobile and is diffusely localized over the cell surface. In a previous study, Burke et al. (11) showed that coexpression of Kv1.4 with PSD-95 resulted in immobilization of the channel. In ventricular myocytes, Kv1.4 appears to be localized to the T tubules and the z-line. Given the abundance of specialized scaffolding proteins and other cytoskeletal elements at this region (21), in conjunction with a previous report that Kv1.4 binds {alpha}-actinin, a key component of the cardiac z-line, it seemed likely that Kv1.4 at the T tubule would exhibit a reduced mobility. However, as shown in Fig. 6B, recovery of YFP fluorescence following photobleach was more rapid and more complete than what we observed for Kv2.1, with an Mf of 0.60 ± 0.04 (n = 7).

The kinetics of Kv1.4 mobility, however, were quite different from what we previously reported in HEK cells (36). Whereas the time course of recovery in that study could be well fit by a single exponential, Kv1.4 recovery was best fit by two exponentials, with {tau} of 18.9 ± 2.3 s and 305 ± 55 s, respectively. The relative contribution of each of these components was roughly equal, indicating that the Mf is made up of both pools of channel. From these data, it appears that a pool of Kv1.4 exhibits a mobility even higher than what we measured in HEK cells and even the slow component of Kv1.4 displays a higher mobility than Kv2.1 in these cells. It therefore seems unlikely that Kv1.4 interacts with {alpha}-actinin or any other scaffolding protein, since such an interaction would be expected to significantly lower the mobility of the channel as is observed for tethered neurotransmitter receptors (14, 46).

The high mobility of Kv1.4 in ventricular myocytes demonstrates that the immobilization of Kv2.1 is not a generalized feature of cardiac ion channels. Therefore, although Kv2.1 does not display the large clusters typical of neurons, it is nevertheless either tethered to cytoskeletal structures or part of a large macromolecular complex, either of which would be expected to lower its mobility. Given the distinct pattern of localization for cardiac Kv2.1, the exact nature of this restriction is likely to be distinct from the mechanism underlying Kv2.1 restriction in HEK cells or hippocampal neurons.

Vesicular transport of Kv2.1 in ventricular myocytes. In GFP-Kv2.1-transfected HEK cells and hippocampal neurons, we have observed what appear to be small, intracellular trafficking vesicles containing GFP-Kv2.1 (35, 45). Similar structures exist in Ad-GFP-Kv2.1-expressing ventricular myocytes. Figure 7 and Supplemental Movie 4 show the path of one such vesicle within a ventricular myocyte. The vesicle appears to travel in the space between T tubules, moving a total of 16.4 µm before disappearing from the focal plane. Interestingly, the vesicle pauses two times during its journey. The first pause is brief, lasting only ~25 s, but the second pause is much longer, at 2 min and 15 s. As seen in Fig. 7 and Supplemental Movie 4, there appears to be an existing concentration of Kv2.1 in the vicinity of this long pause in the vesicle's movement; perhaps the vesicle becomes transiently associated with some component of the cytoskeleton or other macromolecular complex involved in the localization of Kv2.1. However, the vesicle does not deliver its cargo at this location, rather it continues on and leaves the focal plane. We have observed similar long pauses without fusion in the movement of Kv2.1-containing vesicles in HEK cells, suggesting that such transient interactions may be part of the targeting mechanism directing Kv2.1 to its ultimate destination. Between pauses, the average velocity of the vesicle is ~0.24 µm/s, consistent with the velocity we observed in transfected HEK cells. Similarly, the cardiac GFP-Kv2.1 vesicle is ~0.5 µm in diameter, nearly identical to these vesicles in other cell types.


Figure 7
View larger version (53K):
[in this window]
[in a new window]

  		Fig. 7. GFP-Kv2.1 vesicles transport channel within ventricular myocytes. The vesicle was tracked manually by drawing a region of interest (ROI) around the vesicle at each time point (red circles). The Quantitation Module of Volocity was then used to generate a track describing the movement of the vesicle through the myocyte. Images were taken every 3 s. The average velocity of the vesicle was 0.24 µm/s; this does not include time when the vesicle movement appeared to stop. Scale bar is 2.9 µm.

 
It should be noted that observation of these vesicles was a rare event, with a vesicle detected only one time during the imaging of any one myocyte, although we did observe them in three of seven GFP-Kv2.1-expressing myocytes imaged, suggesting they may be a common mechanism for the intracellular trafficking of Kv2.1.

Conclusions. To our knowledge, this is the first study using adenoviral-mediated overexpression of fluorescent protein-tagged Kv channels to visualize channel localization and mobility in live cardiomyocytes from both the atria and ventricle. Although in virtually every other cell type thus far examined Kv2.1 exhibits a clustered distribution on the cell surface, in ventricular myocytes, the channel displayed a relatively even distribution throughout the transverse-axial tubules. However, its mobility is similar to what we previously measured in HEK cells and hippocampal neurons. On the other hand, in atrial myocytes, although Kv2.1 does form cell surface clusters, FRAP experiments demonstrated that the channel's mobility in the membrane is significantly different from anything previously observed for Kv2.1.

What are the physiological implications of Kv channel localization to the T tubules? The T tubular space is being increasingly recognized as a specialized domain rather than simply invaginations of the peripheral membrane, and many ion channels, such as Kv4.2 (44), KCNQ1, and ERG1 (39), as well as those involved in excitation-contraction coupling, are localized there (8, 9). Kv2.1, which underlies the rodent Ik,slow2 current (6, 10, 24, 50) and may also contribute to the Iss current (6, 24, 49), is now also shown to be predominantly localized to the T tubular domain. Interestingly, ~71% of the total Iss current and 27% of the delayed-rectifier current in ventricular myocytes are distributed in the T tubules as determined by formamide-induced detubulation (8, 25), consistent with the localization we report here for Kv2.1.

The T tubules are a diffusion-restricted space, and the diffusion rate of K+ in the T tubules is slow, ~85 µm2/s (42); therefore, K+ accumulation within the lumen is likely. This is significant because Kv2.1 has been demonstrated to undergo a conductance switch in the presence of high extracellular K+ (2, 12, 47) such as might be found in the cardiac T tubule. However, what role, if any, this change in Kv2.1 conductance might play in the modulation of the action potential is unknown. The most intriguing possibility is that many of the Kv2.1 channels in the sarcolemma function primarily as voltage sensors rather than as K+-fluxing channels, in a manner analogous to the role of the skeletal muscle L-type Ca2+ channel in excitation-contraction coupling (15, 34). There is support for this idea in the literature: Benndorf et al. (5) compared the total gating charge associated with activation of Kv2.1 in Xenopus oocytes with the total amount of K+ current and found the delayed-rectifier current could be entirely accounted for by the opening of only ~1% of the channels on the surface. More recently, a nonconducting role for Kv2.1 in secretion was described in PC-12 cells, further supporting this idea (41). The possibility exists, therefore, that the majority of Kv2.1 channels on the surface membrane do not principally function as K+ channels but rather serve as voltage sensors to couple changes in membrane potential to intracellular pathways or, alternatively, are cell surface channels that act as a reserve pool, modified to be silent until they are required.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-49330 to M. M. Tamkun and K99-HL-087591 to K. M. S. O'Connell.


   ACKNOWLEDGMENTS
 
We thank Dr. James Trimmer for the generous gift of Ad-GFP-Kv2.1 and Cecile Weigle for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. S. O'Connell, Dept. of Biomedical Sciences, Campus Delivery 1617, Colorado State Univ., Fort Collins, CO 80523 (e-mail: koconnel{at}lamar.colostate.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS AND DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alseikhan BA, DeMaria CD, Colecraft HM, Yue DT. Engineered calmodulins reveal the unexpected eminence of Ca2+ channel inactivation in controlling heart excitation. Proc Natl Acad Sci USA 99: 17185–17190, 2002.[Abstract/Free Full Text]
  2. Andalib P, Wood MJ, Korn SJ. Control of outer vestibule dynamics and current magnitude in the Kv2.1 potassium channel. J Gen Physiol 120: 739–755, 2002.[Abstract/Free Full Text]
  3. Antonucci DE, Lim ST, Vassanelli S, Trimmer JS. Dynamic localization and clustering of dendritic Kv2.1 voltage-dependent potassium channels in developing hippocampal neurons. Neuroscience 108: 69–81, 2001.[CrossRef][Web of Science][Medline]
  4. Barry DM, Trimmer JS, Merlie JP, Nerbonne JM. Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels? Circ Res 77: 361–369, 1995.[Abstract/Free Full Text]
  5. Benndorf K, Koopmann R, Lorra C, Pongs O. Gating and conductance properties of a human delayed rectifier K+ channel expressed in frog oocytes. J Physiol 477: 1–14, 1994.[Abstract/Free Full Text]
  6. Bou-Abboud E, Li H, Nerbonne JM. Molecular diversity of the repolarizing voltage-gated K+ currents in mouse atrial cells. J Physiol 529: 345–358, 2000.[Abstract/Free Full Text]
  7. Bou-Abboud E, Nerbonne JM. Molecular correlates of the calcium-independent, depolarization-activated K+ currents in rat atrial myocytes. J Physiol (Lond) 517: 407–420, 1999.[Abstract/Free Full Text]
  8. Brette F, Orchard C. Resurgence of cardiac t-tubule research. Physiology (Bethesda) 22: 167–173, 2007.[CrossRef][Medline]
  9. Brette F, Orchard C. T-tubule function in mammalian cardiac myocytes. Circ Res 92: 1182–1192, 2003.[Abstract/Free Full Text]
  10. Brunet S, Aimond F, Li H, Guo W, Eldstrom J, Fedida D, Yamada KA, Nerbonne JM. Heterogeneous expression of repolarizing, voltage-gated K+ currents in adult mouse ventricles. J Physiol 559: 103–120, 2004.[Abstract/Free Full Text]
  11. Burke NA, Takimoto K, Li D, Han W, Watkins SC, Levitan ES. Distinct structural requirements for clustering and immobilization of K+ channels by PSD-95. J Gen Physiol 113: 71–80, 1999.[Abstract/Free Full Text]
  12. Consiglio JF, Andalib P, Korn SJ. Influence of pore residues on permeation properties in the Kv2.1 potassium channel Evidence for a selective functional interaction of K+ with the outer vestibule. J Gen Physiol 121: 111–124, 2003.[Abstract/Free Full Text]
  13. Cukovic D, Lu GW, Wible B, Steele DF, Fedida D. A discrete amino terminal domain of Kv1.5 and Kv14 potassium channels interacts with the spectrin repeats of alpha-actinin-2. FEBS Lett 498: 87–92, 2001.[CrossRef][Web of Science][Medline]
  14. Dahan M, Levi S, Luccardini C, Rostaing P, Riveau B, Triller A. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302: 442–445, 2003.[Abstract/Free Full Text]
  15. Dirksen RT. Bi-directional coupling between dihydropyridine receptors and ryanodine receptors. Front Biosci 7: d659–d670, 2002.[Web of Science][Medline]
  16. Du J, Haak LL, Phillips-Tansey E, Russell JT, McBain CJ. Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K+ channel subunit Kv2.1. J Physiol (Lond) 522: 19–31, 2000.[Abstract/Free Full Text]
  17. Du J, Tao-Cheng JH, Zerfas P, McBain CJ. The K+ channel Kv2.1 is apposed to astrocytic processes and is associated with inhibitory postsynaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons. Neuroscience 84: 37–48, 1998.[CrossRef][Web of Science][Medline]
  18. Eldstrom J, Van Wagoner DR, Moore ED, Fedida D. Localization of Kv1.5 channels in rat and canine myocyte sarcolemma. FEBS Lett 580: 6039–6046, 2006.[CrossRef][Web of Science][Medline]
  19. Frick A, Magee J, Johnston D. LTP is accompanied by an enhanced local excitability of pyramidal neuron dendrites. Nat Neurosci 7: 126–135, 2004.[CrossRef][Web of Science][Medline]
  20. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95: 2509–2514, 1998.[Abstract/Free Full Text]
  21. Hoshijima M. Mechanical stress-strain sensors embedded in cardiac cytoskeleton: Z disk, titin, and associated structures. Am J Physiol Heart Circ Physiol 290: H1313–H1325, 2006.[Abstract/Free Full Text]
  22. Hulme JT, Coppock EA, Felipe A, Martens JR, Tamkun MM. Oxygen sensitivity of cloned voltage-gated K+ channels expressed in the pulmonary vasculature. Circ Res 85: 489–497, 1999.[Abstract/Free Full Text]
  23. Kirk MM, Izu LT, Chen-Izu Y, McCulle SL, Wier WG, Balke CW, Shorofsky SR. Role of the transverse-axial tubule system in generating calcium sparks and calcium transients in rat atrial myocytes. J Physiol 547: 441–451, 2003.[Abstract/Free Full Text]
  24. Kodirov SA, Brunner M, Nerbonne JM, Buckett P, Mitchell GF, Koren G. Attenuation of I(K,slow1) and I(K,slow2) in Kv1/Kv2DN mice prolongs APD and QT intervals but does not suppress spontaneous or inducible arrhythmias. Am J Physiol Heart Circ Physiol 286: H368–H374, 2004.[Abstract/Free Full Text]
  25. Komukai K, Brette F, Yamanushi TT, Orchard CH. K(+) current distribution in rat sub-epicardial ventricular myocytes. Pflugers Arch 444: 532–538, 2002.[CrossRef][Web of Science][Medline]
  26. Li H, Guo W, Yamada KA, Nerbonne JM. Selective elimination of I(K,slow1) in mouse ventricular myocytes expressing a dominant negative Kv1.5{alpha} subunit. Am J Physiol Heart Circ Physiol 286: H319–H328, 2004.[Abstract/Free Full Text]
  27. Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR, Kass RS. Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science 295: 496–499, 2002.[Abstract/Free Full Text]
  28. Misonou H, Mohapatra DP, Menegola M, Trimmer JS. Calcium- and metabolic state-dependent modulation of the voltage-dependent Kv2.1 channel regulates neuronal excitability in response to ischemia. J Neurosci 25: 11184–11193, 2005.[Abstract/Free Full Text]
  29. Misonou H, Mohapatra DP, Park EW, Leung V, Zhen D, Misonou K, Anderson AE, Trimmer JS. Regulation of ion channel localization and phosphorylation by neuronal activity. Nat Neurosci 7: 711–718, 2004.[CrossRef][Web of Science][Medline]
  30. Misonou H, Mohapatra DP, Trimmer JS. Kv2.1: a voltage-gated K+ channel critical to dynamic control of neuronal excitability. Neurotoxicology 26: 743–752, 2005.[CrossRef][Web of Science][Medline]
  31. Mohapatra DP, Trimmer JS. The Kv2.1 C terminus can autonomously transfer Kv2.1-like phosphorylation-dependent localization, voltage-dependent gating, and muscarinic modulation to diverse Kv channels. J Neurosci 26: 685–695, 2006.[Abstract/Free Full Text]
  32. Mohler PJ, Rivolta I, Napolitano C, LeMaillet G, Lambert S, Priori SG, Bennett V. Nav1.5 E1053K mutation causing Brugada syndrome blocks binding to ankyrin-G and expression of Nav1.5 on the surface of cardiomyocytes. Proc Natl Acad Sci USA 101: 17533–17538, 2004.[Abstract/Free Full Text]
  33. Murakoshi H, Shi G, Scannevin RH, Trimmer JS. Phosphorylation of the Kv2.1 K+ channel alters voltage-dependent activation. Mol Pharmacol 52: 821–828, 1997.[Abstract/Free Full Text]
  34. Nakai J, Dirksen RT, Nguyen HT, Pessah IN, Beam KG, Allen PD. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature 380: 72–75, 1996.[CrossRef][Medline]
  35. O'Connell KM, Rolig AS, Whitesell JD, Tamkun MM. Kv2.1 potassium channels are retained within dynamic cell surface microdomains that are defined by a perimeter fence. J Neurosci 26: 9609–9618, 2006.[Abstract/Free Full Text]
  36. O'Connell KM, Tamkun MM. Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains. J Cell Sci 118: 2155–2166, 2005.[Abstract/Free Full Text]
  37. Ordog B, Brutyo E, Puskas LG, Papp JG, Varro A, Szabad J, Boldogkoi Z. Gene expression profiling of human cardiac potassium and sodium channels. Int J Cardiol 111: 386–393, 2006.[CrossRef][Web of Science][Medline]
  38. Patel AJ, Lazdunski M, Honore E. Kv2.1/Kv9.3, a novel ATP-dependent delayed-rectifier K+ channel in oxygen-sensitive pulmonary artery myocytes. EMBO J 16: 6615–6625, 1997.[CrossRef][Web of Science][Medline]
  39. Rasmussen HB, Moller M, Knaus HG, Jensen BS, Olesen SP, Jorgensen NK. Subcellular localization of the delayed rectifier K+ channels KCNQ1 and ERG1 in the rat heart. Am J Physiol Heart Circ Physiol 286: H1300–H1309, 2004.[Abstract/Free Full Text]
  40. Shi G, Kleinklaus AK, Marrion NV, Trimmer JS. Properties of Kv2.1 K+ channels expressed in transfected mammalian cells. J Biol Chem 269: 23204–23211, 1994.[Abstract/Free Full Text]
  41. Singer-Lahat D, Sheinin A, Chikvashvili D, Tsuk S, Greitzer D, Friedrich R, Feinshreiber L, Ashery U, Benveniste M, Levitan ES, Lotan I. K+ channel facilitation of exocytosis by dynamic interaction with syntaxin. J Neurosci 27: 1651–1658, 2007.[Abstract/Free Full Text]
  42. Swift F, Stromme TA, Amundsen B, Sejersted OM, Sjaastad I. Slow diffusion of K+ in the T tubules of rat cardiomyocytes. J Appl Physiol 101: 1170–1176, 2006.[Abstract/Free Full Text]
  43. Takahashi SX, Mittman S, Colecraft HM. Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating. Biophys J 84: 3007–3021, 2003.[Web of Science][Medline]
  44. Takeuchi S, Takagishi Y, Yasui K, Murata Y, Toyama J, Kodama I. Voltage-gated K+ channel I, Kv4.2, localizes predominantly to the transverse-axial tubular system of the rat myocyte. J Mol Cell Cardiol 32: 1361–1369, 2000.[CrossRef][Web of Science][Medline]
  45. Tamkun MM, O'Connell KM, Rolig AS. A cytoskeletal-based perimeter fence selectively corrals a sub-population of cell surface Kv2.1 channels. J Cell Sci 120: 2413–2423, 2007.[Abstract/Free Full Text]
  46. Tardin C, Cognet L, Bats C, Lounis B, Choquet D. Direct imaging of lateral movements of AMPA receptors inside synapses. Embo J 22: 4656–4665, 2003.[CrossRef][Web of Science][Medline]
  47. Trapani JG, Andalib P, Consiglio JF, Korn SJ. Control of single channel conductance in the outer vestibule of the Kv2.1 potassium channel. J Gen Physiol 128: 231–246, 2006.[Abstract/Free Full Text]
  48. Van Wagoner DR, Pond AL, McCarthy PM, Trimmer JS, Nerbonne JM. Outward K+ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ Res 80: 772–781, 1997.[Abstract/Free Full Text]
  49. Xu H, Barry DM, Li H, Brunet S, Guo W, Nerbonne JM. Attenuation of the slow component of delayed rectification, action potential prolongation, and triggered activity in mice expressing a dominant-negative Kv2 alpha subunit. Circ Res 85: 623–633, 1999.[Abstract/Free Full Text]
  50. Zhou J, Kodirov S, Murata M, Buckett PD, Nerbonne JM, Koren G. Regional upregulation of Kv2.1-encoded current, IK,slow2, in Kv1DN mice is abolished by crossbreeding with Kv2DN mice. Am J Physiol Heart Circ Physiol 284: H491–H500, 2003.[Abstract/Free Full Text]
  51. Zhou YY, Wang SQ, Zhu WZ, Chruscinski A, Kobilka BK, Ziman B, Wang S, Lakatta EG, Cheng H, Xiao RP. Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology. Am J Physiol Heart Circ Physiol 279: H429–H436, 2000.[Abstract/Free Full Text]
  52. Zimmer T, Biskup C, Dugarmaa S, Vogel F, Steinbis M, Bohle T, Wu YS, Dumaine R, Benndorf K. Functional expression of GFP-linked human heart sodium channel (hH1) and subcellular localization of the {alpha}-subunit in HEK293 cells and dog cardiac myocytes. J Membr Biol 186: 1–12, 2002.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
E. Balse, S. El-Haou, G. Dillanian, A. Dauphin, J. Eldstrom, D. Fedida, A. Coulombe, and S. N. Hatem
Cholesterol modulates the recruitment of Kv1.5 channels from Rab11-associated recycling endosome in native atrial myocytes
PNAS, August 25, 2009; 106(34): 14681 - 14686.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. Abi-Char, S. El-Haou, E. Balse, N. Neyroud, R. Vranckx, A. Coulombe, and S. N. Hatem
The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes
Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1851 - H1861.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Movies
Right arrow All Versions of this Article:
294/1/H229    most recent
01038.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by O'Connell, K. M. S.
Right arrow Articles by Tamkun, M. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by O'Connell, K. M. S.
Right arrow Articles by Tamkun, M. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.