Research Article 2155 Targeting of voltage-gated potassium channel isoforms to distinct cell surface microdomains Kristen M. S. O’Connell1 and Michael M. Tamkun1,2,* 1Department of Biomedical Sciences and 2Department of Biochemistry and Molecular Biology, Colorado State University, Ft Collins, CO 80523, USA *Author for correspondence (e-mail: [email protected]) Accepted 23 February 2005 Journal of Cell Science 118, 2155-2166 Published by The Company of Biologists 2005 doi:10.1242/jcs.02348 Summary Voltage-gated potassium (Kv) channels regulate action diffused throughout the cell surface. Additionally, PA- potential duration in nerve and muscle; therefore changes GFP-Kv2.1 moved into regions of the cell membrane not in the number and location of surface channels can adjacent to the original photoactivation ROI. Sucrose profoundly influence electrical excitability. To investigate density gradient analysis indicated that half of Kv2.1 is trafficking of Kv2.1, 1.4 and 1.3 within the plasma part of a large, macromolecular complex while Kv1.4 membrane, we combined the expression of fluorescent sediments as predicted for the tetrameric channel complex. protein-tagged Kv channels with live cell confocal imaging. Disruption of membrane cholesterol by cyclodextrin Kv2.1 exhibited a clustered distribution in HEK cells minimally altered Kv2.1 mobility (Mf=0.32±0.03), but similar to that seen in hippocampal neurons, whereas significantly increased surface cluster size by at least Kv1.4 and Kv1.3 were evenly distributed over the plasma fourfold. By comparison, the mobility of Kv1.4 decreased membrane. Using FRAP, surface Kv2.1 displayed limited following cholesterol depletion with no change in surface mobility; approximately 40% of the fluorescence recovered distribution. The mobility of Kv1.3 was slightly increased within 20 minutes of photobleach (Mf=0.41±0.04). following cyclodextrin treatment. These results indicate Recovery occurred not by diffusion from adjacent that (1) Kv2.1, Kv1.4 and Kv1.3 exist in distinct membrane but probably by transport of nascent channel compartments that exhibit different trafficking properties, from within the cell. By contrast, the Kv1 family members (2) membrane cholesterol levels differentially modulate the Kv1.4 and Kv1.3 were highly mobile, both showing trafficking and localization of Kv channels and (3) Kv2.1 approximately 80% recovery (Kv 1.4 Mf=0.78±0.07; Kv1.3 expressed in HEK cells exhibits a surface distribution Mf=0.78±0.04; without correction for photobleach); unlike similar to that seen in native cells. Journal of Cell Science Kv2.1, recovery was consistent with diffusion of channel from membrane adjacent to the bleach region. Studies using PA-GFP-tagged channels were consistent with the Supplementary material available online at FRAP results. Following photoactivation of a small region http://jcs.biologists.org/cgi/content/full/118/10/2155/DC1 of plasma membrane PA-GFP-Kv2.1 remained restricted to the photoactivation ROI, while PA-GFP-Kv1.4 rapidly Key words: Kv channels, Membrane trafficking, Lipid rafts Introduction interact with PDZ proteins such as PSD-95 via binding Ion channel regulation is an important determinant of electrical domains within the C-terminus (Kim et al., 1995). However, excitability in the nervous and cardiovascular systems, skeletal such motifs are not present in all channels. For example, the muscle, gastrointestinal tract and uterus. Voltage-gated C-terminus of Kv2.1 plays a role in the clustering of Kv2.1 in potassium (Kv) channels play a key role in setting the resting hippocampal neurons (Lim et al., 2000), despite the absence of membrane potential and shaping action potential repolarization any known interaction motifs, such as PDZ binding domains in these functionally diverse systems. Kv channels comprise (Scannevin et al., 1996). A role for the N- and C-termini in the largest and most diverse class of voltage-gated ion channels channel trafficking has been shown for the differential and most cells express multiple channel types belonging to one trafficking of inward rectifier K (KIR) channel isoforms or more subfamilies. Structurally, Kv channel isoforms share a (Bendahhou et al., 2003; Ma et al., 2002). conserved core of six transmembrane domains, which includes Kv channels often show a highly specific subcellular the voltage sensor and the ion conducting pore. However, the localization; for example, the A-type channel Kv1.4 is found intracellular N- and C-termini are divergent, even in Kv only along axonal membranes (Sheng et al., 1992), whereas the channel isoforms with nearly identical functional properties, delayed rectifier Kv2.1 is found only on the soma and proximal giving rise to speculation that these domains regulate dendrites (Lim et al., 2000). Such compartmentalization of trafficking and localization in a manner unique to each channel. functionally distinct channels is significant, as it permits spatial Consistent with these differences, some Kv1 family channels regulation of electrical excitability as well as localizing signal bind α-actinin at the N-terminus (Maruoka et al., 2000) and transduction molecules near their ion channel substrates. We 2156 Journal of Cell Science 118 (10) previously reported that Kv channels differentially target to Live cell confocal imaging distinct lipid raft microdomains within the plasma membrane HEK cells expressing fluorescently tagged Kv channels were imaged (Martens et al., 2000; Martens et al., 2001), indicating that using a Zeiss LSM 510 Meta or Olympus FV1000 confocal protein-lipid interactions must also be considered as a potential microscope. For FRAP experiments on the Zeiss LSM510 Meta, YFP mechanism of Kv channel localization. Furthermore, many was excited with the 514 nm line of an Ar laser and emission collected signal transduction pathways, including those known to using a Long Pass 530 emission filter. A 63ϫ 1.4 NA oil immersion modulate Kv channels, are often found in lipid rafts, including objective was used for imaging with the pinhole diameter set for 1 kinases, nitric oxide synthase, GPI-linked proteins and others Airy Unit. YFP was photobleached by 40-50 bleach iterations over the bleach region of interest (ROI) with the 514 nm laser set to 100% (Martens et al., 2004; O’Connell et al., 2004). transmission. Pre- and postbleach images were acquired at 1% laser From a clinical standpoint, mutations resulting in power every 15 seconds for a total of 80 scans. The average mistrafficked ion channels are responsible for human disease. fluorescence intensity within the bleach ROI was normalized to the For example, the most common mutation in cystic fibrosis, prebleach intensity for each time point and recovery kinetics ∆F508 in the cystic fibrosis transmembrane conductance determined by exponential fitting of the average data: regulator (CFTR), results in a mistrafficked channel (Powell n − τ = − ( t/ i) and Zeitlin, 2002). Mutations in the human ether-a-go-go f(x) ∑ Ai(1 e ) , (1) related (HERG) K+ channel result in trafficking defects that are i=1 a common disease mechanism in the LQT2 form of Long QT where Ai is the amplitude of each component, t is time and τi is the syndrome (Delisle et al., 2004). Despite this clinical relevance, time constant of each component. The mobile fraction was calculated the mechanisms by which ion channels are trafficked remain as: poorly understood. Few studies have examined channel (Fϱ−F ) trafficking in live cells (see Burke et al., 1999), with most using M = 0 , (2) f − fixed preparations or purely biochemical approaches. Thus, it (Fi F0) is essential that we expand our basic understanding of Kv where Fϱ is the fluorescence intensity at the end of the FRAP channel localization in live cells. To this end, we examined the experiment, Fi is the initial fluorescence intensity prior to bleach and trafficking of three Kv channel isoforms. Kv2.1, Kv1.4 and F0 is the intensity immediately postbleach. Cumulative photobleach Kv1.3 exist in distinct membrane compartments that traffic via outside of the FRAP ROI was typically 10-15% of the prebleach distinct mechanisms in HEK 293 cells and are differentially intensity and did not exceed 15% for the experiments shown here. sensitive to the perturbation of raft lipid by depletion of This bleach was not corrected for in the data presented. We chose to membrane cholesterol. In addition, the behavior of Kv2.1 in analyze recovery in this manner since the Kv channels being these cells establishes HEK293 cells as a meaningful model examined here clearly localize to different compartments with system in which to explore the mechanisms underlying different mobilities and subcellular distribution. Calculating mobile trafficking and cell surface localization of this delayed rectifier fractions and using exponentials to approximate Kv channel photobleach recovery provides us with a relatively model-independent channel. means of comparing different Kv channel behaviors. Some FRAP studies were done using GFP-tagged channels on an Journal of Cell Science Olympus FluoView1000 confocal microscope. A circular region of Materials and Methods interest was photobleached in tornado scan mode with a 405 nm diode Preparation of cDNAs and cell transfection laser at 35% transmission for 1 second using the SIM scanner of the Standard PCR mutagenesis techniques were used to construct FV1000. The SIM scanner was synchronized with the main scanner Kv2.1HA and Kv1.4myc. To make Kv2.1HA, the HA epitope during bleach and acquisition. Following bleach, imaging was sequence YPYDVPDYA was inserted in tandem after Gly217 of the performed by raster scanning with the 488 nm line of a 40 mW Ar rat Kv2.1 primary sequence, which places the epitope in the laser at 1% transmission and the variable bandpass filter set at 505- extracellular S1-S2 loop. To make Kv1.4myc, the myc epitope 530 nm emission. A 60ϫ, 1.4 NA oil immersion objective was used sequence EQKLISEEDL was inserted in tandem after Gly430 of the with the pinhole set to 1 Airy Unit.