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 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 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 Olivocochlear suppression of outer hair cells in vivo: evidence for combined action of BK and SK2 channels throughout the cochlea

2013 ◽  
Vol 109 (6) ◽  
pp. 1525-1534 ◽  
Author(s):  
Stéphane F. Maison ◽  
Sonja J. Pyott ◽  
Andrea L. Meredith ◽  
M. Charles Liberman
Keyword(s):  
Hair Cells ◽  
In Vitro Study ◽  
Bk Channels ◽  
Outer Hair Cells ◽  
Cochlear Hair Cells ◽  
Α Subunit ◽  
Combined Action ◽  
Cholinergic Effects

Cholinergic inhibition of cochlear hair cells via olivocochlear (OC)-efferent feedback is mediated by Ca2+ entry through α9-/α10-nicotinic receptors, but the nature of the K+ channels activated by this Ca2+ entry has been debated (Yoshida N, Hequembourg SJ, Atencio CA, Rosowski JJ, Liberman MC. J Neurophysiol 85: 84–88, 2001). A recent in vitro study (Wersinger E, McLean WJ, Fuchs PA, Pyott SJ. PLoS One 5: e13836, 2010) suggests that small-conductance (SK2) channels mediate cholinergic effects in the apical turn, whereas large-conductance (BK) channels mediate basal turn effects. Here, we measure, as a function of cochlear frequency, the magnitude of BK and SK2 expression in outer hair cells and the strength of in vivo OC suppression in BK+/+ mice vs. BK−/− lacking the obligatory α-subunit (Meredith AL, Thorneloe KS, Werner ME, Nelson MT, Aldrich RW. J Biol Chem 279: 36746–36752, 2004). Except at the extreme apical tip, we see immunostaining for both BK and SK2 in BK+/+. Correspondingly, at all testable frequencies (8–45 kHz), we see evidence for both SK2 and BK contributions to OC effects evoked by electrically stimulating the OC bundle: OC-mediated suppression was reduced, but not eliminated, at all frequencies in the BK−/− ears. The suppression remaining in BK nulls was blocked by strychnine, suggesting involvement of α9-/α10-cholinergic receptors, coupled to activation of the remaining SK2 channels.

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 Calcium induced calcium release in proximity to hair cell BK channels revealed by PKA activation

2019 ◽  
Author(s):  
Jun-ping Bai ◽  
Na Xue ◽  
Omolara Lawal ◽  
Anda Nyati ◽  
Joseph Santos-Sacchi ◽  
...  
Keyword(s):  
Calcium Release ◽  
Critical Role ◽  
Bk Channels ◽  
Ip3 Receptors ◽  
Spatial Spread ◽  
Synaptic Release ◽  
Close Proximity ◽  
Electrical Resonance ◽  
High Concentrations ◽  
Calcium Induced Calcium Release

AbstractLarge conductance calcium-activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are dependent on inward flow of Ca2+, and intracellular buffering of Ca2+. Entry of Ca2+ is further amplified locally by Ca2+ induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. PKA activation increases BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.

scholarly journals Generation of Functional Fluorescent BK Channels by Random Insertion of GFP Variants

2005 ◽  
Vol 126 (5) ◽  
pp. 429-438 ◽  
Author(s):  
Teresa Giraldez ◽  
Thomas E. Hughes ◽  
Fred J. Sigworth
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Surface Expression ◽  
Bk Channels ◽  
Cell Surface Expression ◽  
Α Subunit ◽  
Tn5 Transposon ◽  
Strong Shift ◽  
Random Insertion

The yellow and cyan variants of green fluorescent protein (GFP) constitute an excellent pair for fluorescence resonance energy transfer (FRET) and can be used to study conformational rearrangements of proteins. Our aim was to develop a library of fluorescent large conductance voltage- and Ca2+-gated channels (BK or slo channels) for future use in FRET studies. We report the results of a random insertion of YFP and CFP into multiple sites of the α subunit of the hslo channel using a Tn5 transposon-based technique. 55 unique fluorescent fusion proteins were obtained and tested for cell surface expression and channel function. 19 constructs are expressed at the plasma membrane and show voltage and Ca2+-dependent currents. In 16 of them the voltage and Ca2+ dependence is very similar to the wild-type channel. Two insertions in the Ca2+ bowl and one in the RCK2 domain showed a strong shift in the G-V curve. The remaining 36 constructs were retained intracellularly; a solubility assay suggests that these proteins are not forming intracellular aggregates. The “success rate” of 19 out of 55 hslo insertion constructs compares very favorably with other studies of random GFP fusions.

scholarly journals Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels

2015 ◽  
Vol 145 (4) ◽  
pp. 331-343 ◽  
Author(s):  
Yutao Tian ◽  
Florian Ullrich ◽  
Rong Xu ◽  
Stefan H. Heinemann ◽  
Shangwei Hou ◽  
...  
Keyword(s):  
Divalent Cation ◽  
Critical Role ◽  
Cell Types ◽  
Bk Channels ◽  
Bk Channel ◽  
Positive Direction ◽  
Human Embryonic Kidney Cells ◽  
Auxiliary Subunits ◽  
Voltage Dependent ◽  
Negative Surface

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca2+- and voltage-dependent K+ (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (β and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + β1 and Slo1 + β4 channel complexes and failed to alter macroscopic currents through Slo1 + β2 and Slo1 + β2 Δ2–19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of β subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When β1 or β4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation–dependent modulatory influences.

scholarly journals Hearing at threshold intensities: by slow mechanical traveling waves or by fast cochlear fluid pressure waves

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Haim Sohmer
Keyword(s):  
Soft Tissue ◽  
Hair Cells ◽  
Traveling Wave ◽  
Basilar Membrane ◽  
Fluid Pressure ◽  
Low Frequency ◽  
Frequency Discrimination ◽  
Outer Hair Cells ◽  
Common Mechanism ◽  
Frequency Stimulation

The three modes of auditory stimulation (air, bone and soft tissue conduction) at threshold intensities are thought to share a common excitation mechanism: the stimuli induce passive displacements of the basilar membrane propagating from the base to the apex (slow mechanical traveling wave), which activate the outer hair cells, producing active displacements, which sum with the passive displacements. However, theoretical analyses and modeling of cochlear mechanics provide indications that the slow mechanical basilar membrane traveling wave may not be able to excite the cochlea at threshold intensities with the frequency discrimination observed. These analyses are complemented by several independent lines of research results supporting the notion that cochlear excitation at threshold may not involve a passive traveling wave, and the fast cochlear fluid pressures may directly activate the outer hair cells: opening of the sealed inner ear in patients undergoing cochlear implantation is not accompanied by threshold elevations to low frequency stimulation which would be expected to result from opening the cochlea, reducing cochlear impedance, altering hydrodynamics. The magnitude of the passive displacements at threshold is negligible. Isolated outer hair cells in fluid display tuned mechanical motility to fluid pressures which likely act on stretch sensitive ion channels in the walls of the cells. Vibrations delivered to soft tissue body sites elicit hearing. Thus, based on theoretical and experimental evidence, the common mechanism eliciting hearing during threshold stimulation by air, bone and soft tissue conduction may involve the fast-cochlear fluid pressures which directly activate the outer hair cells.

scholarly journals Isolated domains of recombinant human apo-metallothionein 1A are folded at neutral pH: a denaturant and heat-induced unfolding study using ESI-MS

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Gordon W. Irvine ◽  
Natalie Korkola ◽  
Martin J. Stillman
Keyword(s):  
Critical Role ◽  
Cysteine Residues ◽  
Small Molecular Weight ◽  
Esi Ms ◽  
Neutral Ph ◽  
Induced Folding ◽  
Starting Point ◽  
High Metal ◽  
High Concentrations ◽  
Product Species

Metallothioneins (MTs) are characterized by their high metal loading capacity, small molecular weight, and abundant cysteine residues. It has long been thought that metal-free, or apo-MT peptides were unstructured and only adopted as a distinct conformation upon forming the metal clusters, described as metal-induced folding. More recent studies have suggested that the presence of a globular, yet loosely defined structure actually exists that can be disrupted or unfolded. Residue modification and ion-mobility ESI (IM-ESI)-MS have been used to examine this unusual unfolding process. The structure of apo-MT plays a critical role as the starting point in the flexible metalation pathways that can accommodate numerous soft metals. ESI-MS measurements of the product species formed following the cysteine alkylation of the isolated domain fragments of recombinant human apo-MT 1A with n-ethylmaleimide (NEM) were used in the present study to monitor the denaturant- and heat-induced unfolding at physiological pH. The results indicate that these apo-MT fragments adopt distinct structures at neutral pH that react co-operatively with NEM when folded and non-cooperatively when heated or exposed to high concentrations of the denaturant guanidinium chloride (GdmCl). From these studies, we can conclude that at neutral pH, the domain fragments are folded into globular structures where some of the free cysteine residues are buried within the core and are stabilized by hydrogen bonds. Metalation therefore, must take place from the folded conformation.

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