Role of an S4-S5 Linker Lysine in the Trafficking of the Ca2+-activated K+ Channels IK1 and SK3

Jones, Heather M.; Hamilton, Kirk L.; Devor, Daniel C.

doi:10.1074/jbc.m508601200

Cited by 27 publications

(33 citation statements)

References 61 publications

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“…1A). The internal addition of Tram-34 (1 M) resulted in a near total inhibition of channel activity that was slowly reversible confirming that the Ca 2ϩ -sensitive currents measured under our experimental conditions correspond to KCa3.1 (24,25,44). A series of insideout patch-clamp experiments was next performed to determine whether the KCa3.1 activity could be regulated by AMP.…”

Section: Resultsmentioning

confidence: 99%

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

Klein

Garneau²,

Trinh³

et al. 2009

American Journal of Physiology-Cell Physiology

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The vectorial transport of ions and water across epithelial cells depends to a large extent on the coordination of the apical and basolateral ion fluxes with energy supply. In this work we provide the first evidence for a regulation by the 5′-AMP-activated protein kinase (AMPK) of the calcium-activated potassium channel KCa3.1 expressed at the basolateral membrane of a large variety of epithelial cells. Inside-out patch-clamp experiments performed on human embryonic kidney (HEK) cells stably transfected with KCa3.1 first revealed a decrease in KCa3.1 activity following the internal addition of AMP at a fixed ATP concentration. This effect was dose dependent with half inhibition at 140 μM AMP in 1 mM ATP. Evidence for an interaction between the COOH-terminal region of KCa3.1 and the γ1-subunit of AMPK was next obtained by two-hybrid screening and pull-down experiments. Our two-hybrid analysis confirmed in addition that the amino acids extending from Asp380 to Ala400 in COOH-terminal were essential for the interaction AMPK-γ1/KCa3.1. Inside-out experiments on cells coexpressing KCa3.1 with the dominant negative AMPK-γ1-R299G mutant showed a reduced sensitivity of KCa3.1 to AMP, arguing for a functional link between KCa3.1 and the γ1-subunit of AMPK. More importantly, coimmunoprecipitation experiments carried out on bronchial epithelial NuLi cells provided direct evidence for the formation of a KCa3.1/AMPK-γ1 complex at endogenous AMPK and KCa3.1 expression levels. Finally, treating NuLi monolayers with the membrane permeant AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) caused a significant decrease of the KCa3.1-mediated short-circuit currents, an effect reversible by coincubation with the AMPK inhibitor Compound C. These observations argue for a regulation of KCa3.1 by AMPK in a functional epithelium through protein/protein interactions involving the γ1-subunit of AMPK.

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Section: Resultsmentioning

confidence: 99%

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

Klein

Garneau²,

Trinh³

et al. 2009

American Journal of Physiology-Cell Physiology

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“…In the past, specific leucines of the leucine zipper motifs of the C- and N termini of KCa3.1 have been mutated to alanine resulting in altered assembly and trafficking of the channel to the plasma membrane of human epithelial kidney (HEK293) cells (Syme et al, 2003; Jones et al, 2004, 2005). Additionally, Balut et al (2010a), using a novel-tagged channel approach reported, in non-polarized HEK293 cells, that KCa3.1 was endocytosed from the plasma membrane in <90 min and targeted, via a Rab7-ESCRT (endosomal sorting complexes required for transport)-dependent mechanism, for degradation by the lysosome.…”

Section: Introductionmentioning

confidence: 99%

Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium

2017

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In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

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“…Our immunoprecipitations (IP) and immunoblot (IB) protocols have been previously described (28,29,40,41). Briefly, cells were lysed and equivalent amounts of total protein were precleared with protein G-agarose beads (Invitrogen) and incubated with the indicated antibody.…”

Section: Methodsmentioning

confidence: 99%

“…The number of channels in the membrane (N) is regulated by the balance between the anterograde and retrograde trafficking pathways of the channels. Considerable progress has been made in understanding the mechanisms involved in the assembly/trafficking of KCa2.3 and KCa3.1 toward the plasma membrane (18,40,41,51,63); however, the mechanisms of endocytosis and downstream sorting of these channels are virtually unknown. Deciphering the molecular machinery that governs the (post)endocytic trafficking of KCa2.3 and KCa3.1 is the first step in advancing our understanding of how the number of channels is regulated at the plasma membrane, and whether this will result in an altered EDHF response.…”

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confidence: 99%

ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

Balut

Gao

Murray

et al. 2010

American Journal of Physiology-Cell Physiology

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The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

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Role of an S4-S5 Linker Lysine in the Trafficking of the Ca2+-activated K+ Channels IK1 and SK3

Cited by 27 publications

References 61 publications

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

Inhibition of the KCa3.1 channels by AMP-activated protein kinase in human airway epithelial cells

Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium

ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

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