A Novel Actin-Binding Domain on Slo1 Calcium-Activated Potassium Channels Is Necessary for Their Expression in the Plasma Membrane

Zou, Shengwei; Jha, Smita; Kim, Eun Young; Dryer, Stuart E.

doi:10.1124/mol.107.039743

Cited by 31 publications

(40 citation statements)

References 37 publications

(65 reference statements)

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“…Electrophysiological data were digitized and stored for offline analysis using PClamp software (Molecular Devices, Sunnyvale, CA). Whole-cell and inside-out patch recordings were performed in podocytes and HEK293T cells transiently expressing different Slo1 constructs using standard methods described in detail previously (Kim et al, 2007bZou et al, 2008). For whole-cell recordings from HEK293T cells, the bath solution contained 150 mM NaCl, 0.08 mM KCl, 0.8 mM MaCl 2 , 5.4 mM CaCl 2 , 10 mM glucose, and 10 mM HEPES, and the pH was adjusted to 7.4 with NaOH.…”

Section: Methodsmentioning

confidence: 99%

“…Currents were evoked by a series of 150-ms depolarizing steps from a holding potential of Ϫ80 mV. Activation curves were fitted to the Boltzmann function as described previously (Zou et al, 2008).…”

Section: Methodsmentioning

confidence: 99%

“…In this regard, we do not know the nature of the intracellular compartments that harbor Slo1 VEDEC subunits, and it may well depend on what cell type is considered. In chick ciliary ganglion neurons, some of it may be associated with cortical actin in post-Golgi pools, at least in some cell types (Chae and Dryer, 2005;Chae et al, 2005b;Zou et al, 2008), whereas other pools are likely to be retained in the endoplasmic reticulum (Lhuillier and Dryer, 2002) and Golgi apparatus (Chae et al, 2005b).…”

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confidence: 99%

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Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

Chiu¹,

Alvarez-Baron²,

Kim³

et al. 2010

Mol Pharmacol

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Large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels regulate the physiology of many cell types. A single vertebrate gene variously known as Slo1, KCa1.1, or KCNMA1 encodes the pore-forming subunits of BK Ca channel but is expressed in a potentially very large number of alternative splice variants. Two splice variants of Slo1, Slo1 VEDEC and Slo1 QEERL , which differ at the extreme COOH terminus, show markedly different steadystate expression levels on the cell surface. Here we show that Slo1 VEDEC and Slo1 QEERL can reciprocally coimmunoprecipitate, indicating that they form heteromeric complexes. Moreover, coexpression of even small amounts of Slo1 VEDEC markedly reduces surface expression of Slo1 QEERL and total Slo1 as indicated by cell-surface biotinylation assays. The effects of Slo1 VEDEC on steady-state surface expression can be attributed primarily to the last five residues of the protein based on surface expression of motif-swapped constructs of Slo1 in human embryonic kidney (HEK) 293T cells. In addition, the presence of the VEDEC motif at the COOH terminus of Slo1 channels is sufficient to confer a dominant-negative effect on cell surface expression of itself or other types of Slo1 subunits. Treating cells with short peptides containing the VEDEC motif increased surface expression of Slo1 VEDEC channels transiently expressed in HEK293T cells and increased current through endogenous BK Ca channels in mouse podocytes. Slo1 VEDEC and Slo1 QEERL channels are removed from the HEK293T cell surface with similar kinetics and to a similar extent, which suggests that the inhibitory effect of the VEDEC motif is exerted primarily on forward trafficking into the plasma membrane.The pore-forming subunits of large-conductance Ca 2ϩ -activated potassium (BK Ca ) channels are encoded by a conserved vertebrate gene called Slo1 (also known as KCNMA1 and KCa1

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

Chiu¹,

Alvarez-Baron²,

Kim³

et al. 2010

Mol Pharmacol

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“…A conserved sequence motif, L/I-X-D/E-X-X-L/I, establishes as a putative actin-binding domain (12). The sequence data of murine TMEM16A (GenBank accession number NM_178642) revealed that the open reading frame contained two actin-binding domain candidates, 192 LLEAGL at the N-terminus and 786 IIEIRL at the third intracellular loop (between S6 and S7 transmembrane domains) of TMEM16A channel.…”

Section: Short Communicationmentioning

confidence: 99%

Modulation of TMEM16A-Channel Activity as Ca2+ Activated Cl− Conductance via the Interaction With Actin Cytoskeleton in Murine Portal Vein

et al. 2014

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Abstract. TMEM16A is a major component of Ca 2+ -activated Cl − channel (CaCC) conductance in murine portal vein smooth muscle cells (mPVSMCs). Here, the regulation of CaCC activity by the actin cytoskeleton was examined in mPVSMCs. Actin disruption by cytochalasin D did not affect the current density, but increased the deactivation time constant in mPVSMCs. The elongated deactivation was recovered by jasplakinolide. When murine TMEM16A was transfected into HEK293 cells that have a poorly developed actin cytoskeleton, electrophysiological properties of CaCC currents were not changed by cytochalasin D. In conclusion, the CaCC activity in mPVSMCs is modified by the interaction of TMEM16A with abundant actin cytoskeleton.

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“…Gamma-actin is a major cytoskeletal protein in cochlear sensory cells and missense mutations in its gene are associated with DFNA20/26, an autosomal dominant, nonsyndromic, sensorineural, hearing loss (73). Thus, it is likely this mutation would affect the expression of the BK channel as demonstrated by F-actin, which plays a role in the organization and reorganization of BK via an actin-binding domain at the C terminus of BK (74). Moreover, depolymerization of F-actin via cofilin leads to a reduction in Ca 2ϩ currents regulated by L-type channels (75).…”

Section: Bk Protein Folding and Intracellular Ca 2ϩ Stores-ap-mentioning

confidence: 99%

A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea

Kathiresan

Harvey

Orchard

et al. 2009

Molecular & Cellular Proteomics

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The large conductance Ca 2؉ -activated K ؉ or BK channel has a role in sensory/neuronal excitation, intracellular signaling, and metabolism. In the non-mammalian cochlea, the onset of BK during development correlates with increased hearing sensitivity and underlies frequency tuning in non-mammals, whereas its role is less clear in mammalian hearing. To gain insights into BK function in mammals, coimmunoprecipitation and two-dimensional PAGE, combined with mass spectrometry, were used to reveal 174 putative BKAPs from cytoplasmic and membrane/cytoskeletal fractions of mouse cochlea. Eleven BKAPs were verified using reciprocal coimmunoprecipitation, including annexin, apolipoprotein, calmodulin, hippocalcin, and myelin P0, among others. These proteins were immunocolocalized with BK in sensory and neuronal cells. A bioinformatics approach was used to mine databases to reveal binary partners and the resultant protein network, as well as to determine previous ion channel affiliations, subcellular localization, and cellular processes. The search for binary partners using the IntAct molecular interaction database produced a putative global network of 160 nodes connected with 188 edges that contained 12 major hubs. Additional mining of databases revealed that more than 50% of primary BKAPs had prior affiliations with K ؉ and Ca 2؉ channels. Although a majority of BKAPs are found in either the cytoplasm or membrane and contribute to cellular processes that primarily involve metabolism (30.5%) and trafficking/scaffolding (23.6%), at least 20% are mitochondrial-related. Among the BKAPs are chaperonins such as calreticulin, GRP78, and HSP60 that, when reduced with siRNAs, alter BK␣ expression in CHO cells. Studies of BK␣ in mitochondria revealed compartmentalization in sensory cells, whereas heterologous expression of a BK-DEC splice variant cloned from cochlea revealed a BK mitochondrial candidate. The studies described herein provide insights into BK-related functions that include not only cell excitation, but also cell signaling and apoptosis, and involve proteins concerned with Ca 2؉ regulation, structure, and hearing loss. BK 1 channels act as sensors for membrane voltage and intracellular Ca 2ϩ , thereby linking cell excitability, metabolism, and signaling. BK channels, also known as Slo, are large conductance channels (100 -300 pS) (1) composed of four ␣-subunits that are regulated by four auxiliary ␤-subunits. The ␣-subunit of the BK channel has six to seven transmembranespanning regions (S0 -S6) where the S0 domain places the N terminus extracellularly as a binding site for the beta subunit. The transmembrane domains S1-S4 are responsible for sensing voltage changes, whereas the pore forming region, between S5-S6, conducts ions. BK has a large C-terminal region that contains target sequences for channel modulation such as a Ca 2ϩ bowl, two domains that regulate the conductance of K ϩ (RCK1 and RCK2), a tetramerization domain, leucine zipper motifs, a hemebinding motif, two phosphorylation sites, and a caveolin-targ...

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A Novel Actin-Binding Domain on Slo1 Calcium-Activated Potassium Channels Is Necessary for Their Expression in the Plasma Membrane

Cited by 31 publications

References 37 publications

Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

Modulation of TMEM16A-Channel Activity as Ca2+ Activated Cl− Conductance via the Interaction With Actin Cytoskeleton in Murine Portal Vein

A Protein Interaction Network for the Large Conductance Ca2+-activated K+ Channel in the Mouse Cochlea

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