Kv2.1 Potassium Channels Are Retained within Dynamic Cell Surface Microdomains That Are Defined by a Perimeter Fence

Abstract

Ion channel localization to specific cell surface regions is essential for proper neuronal function. The Kv2.1 K ϩ channel forms large clusters on the plasma membrane of hippocampal neurons and transfected human embryonic kidney (HEK) cells. Using live cell imaging, we address mechanisms underlying this Kv2.1 clustering in both HEK cells and cultured hippocampal neurons. The Kv2.1-containing surface clusters have properties unlike those expected for a scaffolding protein bound channel. After channel is delivered to the plasma membrane via intracellular transport vesicles, it remains localized at the insertion site. Fluorescence recovery after photobleaching (FRAP) and quantum dot tracking experiments indicate that channel within the surface cluster is mobile (FRAP, ϭ 14.1 Ϯ 1.5 and 11.5 Ϯ 6.1 s in HEK cells and neurons, respectively). The cluster perimeter is not static, because after fusion of adjacent clusters, green fluorescent protein (GFP)-Kv2.1 completely exchanged between the two domains within 60 s. Treatment of hippocampal neurons expressing GFP-Kv2.1 with 5 M latrunculin A resulted in a significant increase in average cluster size from 0.89 Ϯ 0.16 m 2 to 12.15 Ϯ 1.4 m 2 with a concomitant decrease in cluster number. Additionally, Kv2.1 was no longer restricted to the cell body, suggesting a role for cortical actin in both cluster maintenance and localization. Thus, Kv2.1 surface domains likely trap mobile Kv2.1 channels within a well defined, but fluid, perimeter rather than being tightly bound to a scaffolding protein-containing complex. Channel moves directly into these clusters via trafficking vesicles. Such domains allow for efficient trafficking to the cell surface while sequestering channel with signaling proteins.Key words: Kv channel; restricted diffusion; membrane insertion; quantum dot tracking; fluorescence microscopy; hippocampal neurons IntroductionVoltage-gated ion channels are often highly localized in electrically excitable cells such as nerve and muscle. Voltage-gated Na ϩ channels form high-density arrays within the axon node of Ranvier, and voltage-gated K ϩ channels localize in the paranodal region (Rasband and Trimmer, 2001). In smooth muscle, Ca 2ϩ -dependent K ϩ channels are found adjacent to the ryanodine receptors of the sarcoplasmic reticulum (Wellman and Nelson, 2003). L-type voltage-gated Ca 2ϩ channels are exclusively localized to the T-tubule/sarcoplasmic reticulum triad junction in skeletal muscle (Dirksen, 2002). Whereas scaffolding proteins are known to cluster some neurotransmitter receptors (Kim and Sheng, 2004), the exact mechanisms underlying most ion channel localization remain unknown.The delayed rectifier Kv2.1 regulates somato-dendritic excitability in the mammalian CNS where it forms unique cell surface clusters on the soma and proximal dendrites of hippocampal neurons both in situ and in culture (Misonou et al., , 2005. Kv2.1 represents the predominant delayed rectifier current in these cells (Du et al., 2000). This clustering has been proposed to be attributa...

Localization-dependent activity of the Kv2.1 delayed-rectifier K + channel

“…As previously described, Kv2.1 clusters are high-density structures containing most of the Kv2.1 expressed on the plasma membrane (8,13,14). Binding of various reagents to extracellular epitopes has confirmed that these structures are indeed on the cell surface (13).…”

“…It is noteworthy that the Kv2.1 expression levels and localization and the regulation of localization and function are nearly identical between transfected HEK cells and the endogenous Kv2.1 in cultured hippocampal neurons (8,9,(13)(14)(15). We report here that the clustered Kv2.1 is nonconducting and that the majority of the whole-cell current is derived from the nonclustered channels.…”

“…Binding of various reagents to extracellular epitopes has confirmed that these structures are indeed on the cell surface (13). We hypothesized that the channels associated with these structures underlie the large high-threshold K + current attributed to Kv2.1 under steady-state conditions.…”

“…Because low passage HEK293 cells express few endogenous ion channels (we recorded no endogenous channel activity in nontransfected controls), we chose this system for the experiments in this study. We previously generated a tagged Kv2.1 construct, GFP-Kv2.1loopBAD (13,14) that carries green fluorescent protein (GFP) on the N terminus of Kv2.1 and a biotin acceptor domain (BAD) within the extracellular S1-S2 loop, enabling specific labeling of cell surface channels with streptavidin-conjugated quantum dots as illustrated in Fig. S1.…”

“…One of the hallmarks of Kv2.1 expression in mammalian neurons is specific targeting to large, high-density cell surface clusters restricted to the soma and proximal dendrites (8,12,13). Interestingly, not only is channel voltage dependence altered by the aforementioned stimuli, but also channel localization.…”