scholarly journals Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation

2012 ◽  
Vol 303 (2) ◽  
pp. C143-C150 ◽  
Author(s):  
Alexei Surguchev ◽  
Jun-Ping Bai ◽  
Powrnima Joshi ◽  
Dhasakumar Navaratnam
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
High Frequency ◽  
Messenger Rna ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Α Subunit

Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

scholarly journals β4-Subunit increases Slo responsiveness to physiological Ca2+ concentrations and together with β1 reduces surface expression of Slo in hair cells

2011 ◽  
Vol 300 (3) ◽  
pp. C435-C446 ◽  
Author(s):  
Jun-Ping Bai ◽  
Alexei Surguchev ◽  
Dhasakumar Navaratnam
Keyword(s):  
Hair Cells ◽  
Critical Role ◽  
Relaxation Times ◽  
Low Frequency ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Operating Voltage ◽  
Α Subunit ◽  
Voltage Range

Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca2+ at a hair cell's operating voltage range (spanning −40 to −60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca2+ concentrations at −50 mV. We have cloned the chick β4- and β1-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The β4-subunit in particular is responsible for the BK channel's increased responsiveness to Ca2+ at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both β-subunits suggest additional mechanisms responsible for BK channel function in hair cells.

scholarly journals CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation

2012 ◽  
Vol 302 (5) ◽  
pp. C766-C780 ◽  
Author(s):  
Jun-Ping Bai ◽  
Alexei Surguchev ◽  
Powrnima Joshi ◽  
Liza Gross ◽  
Dhasakumar Navaratnam
Keyword(s):  
Hair Cells ◽  
Critical Role ◽  
Frequency Discrimination ◽  
Surface Expression ◽  
Bk Channels ◽  
Cyclin Dependent Kinase ◽  
Α Subunit ◽  
Human Embryonic Kidney Cells ◽  
Accessory Subunits ◽  
High Concentrations

Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.

scholarly journals Regulation of large conductance calcium- and voltage-activated potassium (BK) channels by S-palmitoylation

2013 ◽  
Vol 41 (1) ◽  
pp. 67-71 ◽  
Author(s):  
Michael J. Shipston
Keyword(s):  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Post Translational Modification ◽  
Physiological Control ◽  
Α Subunit ◽  
Channel Trafficking ◽  
C Terminus ◽  
Family Protein ◽  
Health And Disease

BK (large conductance calcium- and voltage-activated potassium) channels are important determinants of physiological control in the nervous, endocrine and vascular systems with channel dysfunction associated with major disorders ranging from epilepsy to hypertension and obesity. Thus the mechanisms that control channel surface expression and/or activity are important determinants of their (patho)physiological function. BK channels are S-acylated (palmitoylated) at two distinct sites within the N- and C-terminus of the pore-forming α-subunit. Palmitoylation of the N-terminus controls channel trafficking and surface expression whereas palmitoylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. Recent studies are beginning to reveal mechanistic insights into how palmitoylation controls channel trafficking and cross-talk with phosphorylation-dependent signalling pathways. Intriguingly, each site of palmitoylation is regulated by distinct zDHHCs (palmitoyl acyltransferases) and APTs (acyl thioesterases). This supports that different mechanisms may control substrate specificity by zDHHCs and APTs even within the same target protein. As palmitoylation is dynamically regulated, this fundamental post-translational modification represents an important determinant of BK channel physiology in health and disease.

scholarly journals GENERATION OF MOSAIC STEM CELL DERIVED INNER EAR ORGANOIDS TO DETERMINE PARACRINE EFFECTS OF TMPRSS3

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Radoslaw Nabrzyski, BS ◽  
Pei-Ciao Tang, PhD ◽  
Alpha Alex ◽  
Rick F. Nelson, MD, PhD
Keyword(s):  
Stem Cell ◽  
Hair Cell ◽  
Cell Survival ◽  
Inner Ear ◽  
Hair Cells ◽  
Embryonic Stem ◽  
Premature Stop Codon ◽  
Bk Channels ◽  
Bk Channel ◽  
Exon 2

Background and Hypothesis: The carefully timed treatment of mouse embryonic stem cell (mESC) cultures with small molecules results in mESC differentiation into 3D organoids containing all components of the inner ear such as hair cells (HCs), support cells and neurons. Loss of TMPRSS3 function, which is a transmembrane extracellular protease, has been previously shown to lead to rapid HC degeneration between culture days 36 (D36) and D38 in 3D organoids. Mosaic organoids would allow for analysis of developmental dynamics, cell-cell interactions and even therapeutic rescue efficiency. We hypothesized that we could develop an inner ear mosaic organoid containing cells with and without TMPRSS3 and that the Tmprsss3KO mosaic organoids would have greater hair cell survival than Tmprss3KO-only organoids due to the compensatory effect of intact TMPRSS3 on control hair cells within the same vesicle. Experimental Design or Project Methods: Two mESC cell lines (R1E background) were previously generated using CRISPR-Cas9n. Control reporter line (tdTomato) expresses a tdTomato reporter gene in all cells under the CAG promoter at the ROSA26 locus. Tmprss3 knockout line (Tmprss3KO) contains a 2A-nGFP cassette with a premature stop codon into exon 2 of the Tmprss3 gene. We generated Tmprss3KO mosaic (Tmprss3KO:tdTomato) and control mosaic (R1E:tdTomato) organoids. The aggregates were analyzed on days 25, 33 and 38 after being fixed, sectioned and stained for tdTomato, SOX2, MYO7A, cleaved CASPASE3, and BK channels using immunohistochemistry. Results: We have successfully used mESCs to generate Tmprss3KO mosaic inner ear organoids with similar efficiency to that of control mosaic organoids. Preliminary data suggests that the hair cell survival rates were similar across all vesicle types in the Tmprss3KO mosaic organoids. Additionally, the Tmprss3KO mosaic organoids had a significantly decreased overall BK channel expression by D38. Conclusion and Potential Impact: The successful generation of mosaic organoids achieved here under both conditions sets the stage for future studies of intercellular interactions and therapeutics in this domain. More replicates are necessary to make a definitive conclusion about the effectiveness of control cells on Tmprss3KO rescue.

scholarly journals LRRC52 regulates BK channel function and localization in mouse cochlear inner hair cells

2019 ◽  
Vol 116 (37) ◽  
pp. 18397-18403 ◽  
Author(s):  
Christopher J. Lingle ◽  
Pedro L. Martinez-Espinosa ◽  
Aizhen Yang-Hood ◽  
Luis E. Boero ◽  
Shelby Payne ◽  
...  
Keyword(s):  
Hair Cells ◽  
Glutamate Release ◽  
Nerve Fibers ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Macromolecular Complex ◽  
Inner Hair Cells ◽  
Channel Function ◽  
Voltage Dependent

The perception of sound relies on sensory hair cells in the cochlea that convert the mechanical energy of sound into release of glutamate onto postsynaptic auditory nerve fibers. The hair cell receptor potential regulates the strength of synaptic transmission and is shaped by a variety of voltage-dependent conductances. Among these conductances, the Ca2+- and voltage-activated large conductance Ca2+-activated K+channel (BK) current is prominent, and in mammalian inner hair cells (IHCs) displays unusual properties. First, BK currents activate at unprecedentedly negative membrane potentials (−60 mV) even in the absence of intracellular Ca2+elevations. Second, BK channels are positioned in clusters away from the voltage-dependent Ca2+channels that mediate glutamate release from IHCs. Here, we test the contributions of two recently identified leucine-rich-repeat–containing (LRRC) regulatory γ subunits, LRRC26 and LRRC52, to BK channel function and localization in mouse IHCs. Whereas BK currents and channel localization were unaltered in IHCs fromLrrc26knockout (KO) mice, BK current activation was shifted more than +200 mV in IHCs fromLrrc52KO mice. Furthermore, the absence of LRRC52 disrupted BK channel localization in the IHCs. Given that heterologous coexpression of LRRC52 with BK α subunits shifts BK current gating about −90 mV, to account for the profound change in BK activation range caused by removal of LRRC52, we suggest that additional factors may help define the IHC BK gating range. LRRC52, through stabilization of a macromolecular complex, may help retain some other components essential both for activation of BK currents at negative membrane potentials and for appropriate BK channel positioning.

β-Subunit–Dependent Modulation of hSlo BK Current by Arachidonic Acid

2007 ◽  
Vol 97 (1) ◽  
pp. 62-69 ◽  
Author(s):  
X. Sun ◽  
D. Zhou ◽  
P. Zhang ◽  
E. G. Moczydlowski ◽  
G. G. Haddad
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Conformational Changes ◽  
Unsaturated Fatty Acids ◽  
Saturated Fatty Acids ◽  
Nordihydroguaiaretic Acid ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Α Subunit

In this study, we examined the effect of arachidonic acid (AA) on the BK α-subunit with or without β-subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo-α current and slowed inactivation only when β2/3 subunit was co-expressed. The β2-subunit–dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites. Other cis unsaturated fatty acids (docosahexaenoic and oleic acid) also enhanced hSlo-α + β2 currents and slowed inactivation, whereas saturated fatty acids (palmitic, stearic, and caprylic acid) were without effect. Pretreatment with trypsin to remove the cytosolic inactivation domain largely occluded AA action. Intracellularly applied free synthetic β2-ball peptide induced inactivation of the hSlo-α current, and AA failed to enhance this current and slow the inactivation. These results suggest that AA removes inactivation by interacting, possibly through conformational changes, with β2 to prevent the inactivation ball from reaching its receptor. Our data reveal a novel mechanism of β-subunit–dependent modulation of BK channels by AA. In freshly dissociated mouse neocortical neurons, AA eliminated a transient component of whole cell K+ currents. BK channel inactivation may be a specific mechanism by which AA and other unsaturated fatty acids influence neuronal death/survival in neuropathological conditions.

scholarly journals Site-specific deacylation by ABHD17a controls BK channel splice variant activity

2020 ◽  
Vol 295 (49) ◽  
pp. 16487-16496 ◽  
Author(s):  
Heather McClafferty ◽  
Hamish Runciman ◽  
Michael J. Shipston
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Membrane Trafficking ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Site Specific ◽  
And Function ◽  
A Site

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

scholarly journals Chronic Prenatal Hypoxia Down-Regulated BK Channel Β1 Subunits in Mesenteric Artery Smooth Muscle Cells of the Offspring

2018 ◽  
Vol 45 (4) ◽  
pp. 1603-1616 ◽  
Author(s):  
Bailin Liu ◽  
Yanping Liu ◽  
Ruixiu Shi ◽  
Xueqin Feng ◽  
Xiang Li ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Bk Channels ◽  
Bk Channel ◽  
Mesenteric Arteries ◽  
Prenatal Hypoxia ◽  
Pregnant Rats ◽  
Α Subunit

Background/Aims: Chronic hypoxia in utero could impair vascular functions in the offspring, underlying mechanisms are unclear. This study investigated functional alteration in large-conductance Ca2+-activated K+ (BK) channels in offspring mesenteric arteries following prenatal hypoxia. Methods: Pregnant rats were exposed to normoxic control (21% O2, Con) or hypoxic (10.5% O2, Hy) conditions from gestational day 5 to 21, their 7-month-old adult male offspring were tested for blood pressure, vascular BK channel functions and expression using patch clamp and wire myograh technique, western blotting, and qRT-PCR. Results: Prenatal hypoxia increased pressor responses and vasoconstrictions to phenylephrine in the offspring. Whole-cell currents density of BK channels and amplitude of spontaneous transient outward currents (STOCs), not the frequency, were significantly reduced in Hy vascular myocytes. The sensitivity of BK channels to voltage, Ca2+, and tamoxifen were reduced in Hy myocytes, whereas the number of channels per patch and the single-channel conductance were unchanged. Prenatal hypoxia impaired NS1102- and tamoxifen-mediated relaxation in mesenteric arteries precontracted with phenylephrine in the presence of Nω-nitro-L-arginine methyl ester. The mRNA and protein expression of BK channel β1, not the α-subunit, was decreased in Hy mesenteric arteries. Conclusions: Impaired BK channel β1-subunits in vascular smooth muscle cells contributed to vascular dysfunction in the offspring exposed to prenatal hypoxia.

scholarly journals BK Channels Mediate Cholinergic Inhibition of High Frequency Cochlear Hair Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (11) ◽  
pp. e13836 ◽  
Author(s):  
Eric Wersinger ◽  
Will J. McLean ◽  
Paul A. Fuchs ◽  
Sonja J. Pyott
Keyword(s):  
Hair Cells ◽  
High Frequency ◽  
Bk Channels ◽  
Cochlear Hair Cells

scholarly journals Effect of aldosterone on BK channel expression in mammalian cortical collecting duct

2008 ◽  
Vol 295 (3) ◽  
pp. F780-F788 ◽  
Author(s):  
Genevieve Estilo ◽  
Wen Liu ◽  
Nuria Pastor-Soler ◽  
Phillip Mitchell ◽  
Marcelo D. Carattino ◽  
...  
Keyword(s):  
Splice Variants ◽  
Collecting Duct ◽  
Bk Channels ◽  
Bk Channel ◽  
Mrna Abundance ◽  
Cortical Collecting Duct ◽  
Α Subunit ◽  
Channel Expression ◽  
Tubular Flow ◽  
Circulating Levels

Apical large-conductance Ca2+-activated K+ (BK) channels in the cortical collecting duct (CCD) mediate flow-stimulated K+ secretion. Dietary K+ loading for 10–14 days leads to an increase in BK channel mRNA abundance, enhanced flow-stimulated K+ secretion in microperfused CCDs, and a redistribution of immunodetectable channels from an intracellular pool to the apical membrane (Najjar F, Zhou H, Morimoto T, Bruns JB, Li HS, Liu W, Kleyman TR, Satlin LM. Am J Physiol Renal Physiol 289: F922–F932, 2005). To test whether this adaptation was mediated by a K+-induced increase in aldosterone, New Zealand White rabbits were fed a low-Na+ (LS) or high-Na+ (HS) diet for 7–10 days to alter circulating levels of aldosterone but not serum K+ concentration. Single CCDs were isolated for quantitation of BK channel subunit (total, α-splice variants, β-isoforms) mRNA abundance by real-time PCR and measurement of net transepithelial Na+ (JNa) and K+ (JK) transport by microperfusion; kidneys were processed for immunolocalization of BK α-subunit by immunofluorescence microscopy. At the time of death, LS rabbits excreted no urinary Na+ and had higher circulating levels of aldosterone than HS animals. The relative abundance of BK α-, β2-, and β4-subunit mRNA and localization of immunodetectable α-subunit were similar in CCDs from LS and HS animals. In response to an increase in tubular flow rate from ∼1 to 5 nl·min−1·mm−1, the increase in JNa was greater in LS vs. HS rabbits, yet the flow-stimulated increase in JK was similar in both groups. These data suggest that aldosterone does not contribute to the regulation of BK channel expression/activity in response to dietary K+ loading.

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