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

Abstract

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.

Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

“…Kv2.1 is distinct from other Kv channels in that it is efficiently trafficked to, and abundantly expressed on, the cell surface (Fox et al, 2013b). In addition, a majority of Kv2.1 in both transfected HEK cells and hippocampal neurons exists in a non-conducting state, and induction of this state is dependent on the cell surface density (Fox et al, 2013b;O'Connell et al, 2010). Our current findings -i.e.…”

“…Fluorescent-protein-, HA-epitope-and biotin acceptor domain (BAD)-tagged Kv channel constructs have been described previously (O'Connell et al, 2010;O'Connell et al, 2006;O'Connell and Tamkun, 2005;Tamkun et al, 2007) and are based on the Living Colors vector system (Clontech). The HA epitope or BAD sequence was contained within the extracellular domain between the first and second transmembrane domains of Kv2.1 (Tamkun et al, 2007).…”

“…We have previously demonstrated that the vast majority of clustered Kv2.1 channels are non-conducting with the majority of the K + flux coming from a subpopulation of channels that are diffusely localized over the cell surface (Fox et al, 2013b;O'Connell et al, 2010). Kv2.1 is expressed abundantly in the neuronal soma, suggesting that the channel takes on a non-traditional function that is not related to the regulation of action potentials, which requires a high level of expression -as expected of a structural or scaffolding protein.…”

“…To the best of our knowledge, Kv2.1 is the first plasma membrane protein that has been identified as governing the formation of ERplasma-membrane junctions under resting conditions. The presence of non-conducting Kv2.1 channels (Fox et al, 2013b;O'Connell et al, 2010) allows the clusters to achieve the high channel density that is required to induce junction formation without compromising electrical signaling. Given the well-accepted role of ER-plasmamembrane junctions in Ca 2+ homeostasis in immune cells (Hogan et al, 2010) and that Kv2.1 clusters serve as sites for endo-and exocytosis (Deutsch et al, 2012), the Kv2.1-induced ER-plasmamembrane junctions are proposed to represent a macromolecular complex that functions as a scaffolding site to couple membrane trafficking to Ca 2+ signaling.…”

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

“…In neurons, the precise trafficking of VGIC subtypes to distinct cell regions such as the axon hillock, presynaptic terminals, and distal dendrites allows specific subtypes to control voltage changes in subcellular compartments (6)(7)(8)(9). However, the presence of an ion channel subunit in a cell does not indicate whether it is activated under physiological conditions (10)(11)(12)(13). Determining which ion channels activate under particular physiological and pathophysiological conditions is a formidable challenge, yet this understanding is crucial for identification of channel subtypes as therapeutic targets to correct pathologies (4).…”