The hypothalamic arcuate nucleus (ARH) is a brain region critical for regulation of food intake and a primary area for the action of leptin in the CNS. In lean mice, the adipokine leptin inhibits neuropeptide Y (NPY) and agouti-related peptide (AgRP) neuronal activity, resulting in decreased food intake. Here we show that diet-induced obesity in mice is associated with persistent activation of NPY neurons and a failure of leptin to reduce the firing rate or hyperpolarize the resting membrane potential. However, the molecular mechanism whereby diet uncouples leptin's effect on neuronal excitability remains to be fully elucidated. In NPY neurons from lean mice, the Kv channel blocker 4-aminopyridine inhibited leptin-induced changes in input resistance and spike rate. Consistent with this, we found that ARH NPY neurons have a large, leptin-sensitive delayed rectifier K ϩ current and that leptin sensitivity of this current is blunted in neurons from diet-induced obese mice. This current is primarily carried by Kv2-containing channels, as the Kv2 channel inhibitor stromatoxin-1 significantly increased the spontaneous firing rate in NPY neurons from lean mice. In HEK cells, leptin induced a significant hyperpolarizing shift in the voltage dependence of Kv2.1 but had no effect on the function of the closely related channel Kv2.2 when these channels were coexpressed with the long isoform of the leptin receptor LepRb. Our results suggest that dynamic modulation of somatic Kv2.1 channels regulates the intrinsic excitability of NPY neurons to modulate the spontaneous activity and the integration of synaptic input onto these neurons in the ARH.

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...

The Kv2.1 K + channel is highly expressed throughout the brain, where it regulates excitability during periods of high-frequency stimulation. Kv2.1 is unique among Kv channels in that it targets to large surface clusters on the neuronal soma and proximal dendrites. These clusters also form in transfected HEK cells. Following excessive excitatory stimulation, Kv2.1 declusters with an accompanying 20- to 30-mV hyperpolarizing shift in the activation threshold. Although most Kv2.1 channels are clustered, there is a pool of Kv2.1 resident outside of these domains. Using the cell-attached patch clamp technique, we investigated the hypothesis that Kv2.1 activity varies as a function of cell surface location. We found that clustered Kv2.1 channels do not efficiently conduct K + , whereas the nonclustered channels are responsible for the high threshold delayed rectifier K + current typical of Kv2.1. Comparison of gating and ionic currents indicates only 2% of the surface channels conduct, suggesting that the clustered channels still respond to membrane potential changes. Declustering induced via either actin depolymerization or alkaline phosphatase treatment did not increase whole-cell currents. Dephosphorylation resulted in a 25-mV hyperpolarizing shift, whereas actin depolymerization did not alter the activation midpoint. Taken together, these data demonstrate that clusters do not contain high threshold Kv2.1 channels whose voltage sensitivity shifts upon declustering; nor are they a reservoir of nonconducting channels that are activated upon release. On the basis of these findings, we propose unique roles for the clustered Kv2.1 that are independent of K + conductance.

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The Kv2.1 delayed-rectifier channel trafficks to 1-3 μm2 clusters on the surface of neurons and transfected HEK cells. Single quantum dot (Qdot) tracking and FRAP approaches were used to quantify the diffusion of GFP-labeled Kv2.1 channels on the cell surface and address the mechanisms underlying the formation of these unique membrane structures. Mean square displacement analysis of single Kv2.1 channel tracks inside or outside the surface clusters yielded mean diffusion coefficients of 0.03±0.02 μm2/second and 0.06±0.05 μm2/second, respectively. Kv2.1 channels outside the clusters effectively ignore the cluster boundary, readily diffusing through these microdomains. However, in 5% of the tracks analyzed, single, non-clustered channels were observed to cross into a cluster and become corralled within the cluster perimeter. Alexa Fluor 594-labelled phalloidin staining and mCherry-Kv2.1 co-expression with GFP-actin indicated that the Kv2.1 surface clusters form where the cortical actin cytoskeleton is reduced. Kv2.1 channels lacking the C-terminus do not form clusters, freely diffusing over the cell surface with a mean diffusion coefficient of 0.07±0.04 μm2/second. These data support a model whereby the Kv2.1 clusters are formed by sub-membrane cytoskeletal structures that limit the lateral diffusion of only the sub-population of Kv2.1 channels carrying the appropriate modifications on the Kv2.1 C-terminus.