L-WNK1 is required for BK channel activation in intercalated cells

AbstractBK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate control mice were placed on a high K+ (5% K+ as KCl) diet for at least 10 days. IC-L-WNK1-KO mice exhibited higher blood K+ concentrations ([K+]) than controls. BK channel-dependent whole-cell currents in ICs from cortical collecting ducts of high K+ fed IC-L-WNK1-KO mice were reduced compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice have higher blood [K+] and reduced IC BK channel currents are consistent with impaired urinary K+ secretion, and suggest that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

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L-WNK1 is required for BK channel activation in intercalated cells

AJP Renal Physiology ◽

10.1152/ajprenal.00472.2020 ◽

2021 ◽

Author(s):

Evan C. Ray ◽

Rolando Carrisoza-Gaytán ◽

Mohammad M Al-bataineh ◽

Allison L. Marciszyn ◽

Lubika J Nkashama ◽

...

Keyword(s):

Bk Channels ◽

Bk Channel ◽

Intercalated Cells ◽

Channel Activity ◽

Activity Increase ◽

Α Subunit ◽

High K ◽

Channel Expression ◽

Channel Dependent ◽

Expression And Activity

BK channels expressed in intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) mediate flow-induced K+ secretion. In the ASDN of mice and rabbits, IC BK channel expression and activity increase with a high K+ diet. In cell culture, the long isoform of the kinase WNK1 (L-WNK1) increases BK channel expression and activity. Apical L-WNK1 expression is selectively enhanced in ICs in the ASDN of rabbits on a high K+ diet, suggesting that L-WNK1 contributes to BK channel regulation by dietary K+. We examined the role of IC L-WNK1 expression in enhancing BK channel activity in response to a high K+ diet. Mice with an IC-selective deletion of L-WNK1 (IC-L-WNK1-KO) and littermate controls were placed on a high K+ (5% K+ as KCl) diet for 10 or more days. IC-L-WNK1-KO mice exhibited reduced IC apical/subapical BK α subunit expression and BK channel-dependent whole-cell currents compared to controls. Six-hour urinary K+ excretion in response a saline load was similar in IC-L-WNK1-KO mice and controls. The observations that IC-L-WNK1-KO mice on a high K+ diet have higher blood [K+] and reduced IC BK channel activity are consistent with impaired urinary K+ secretion, demonstrating that IC L-WNK1 has a role in the renal adaptation to a high K+ diet.

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Cell-specific regulation of L-WNK1 by dietary K+

AJP Renal Physiology ◽

10.1152/ajprenal.00226.2015 ◽

2016 ◽

Vol 310 (1) ◽

pp. F15-F26 ◽

Cited By ~ 8

Author(s):

Tennille N. Webb ◽

Rolando Carrisoza-Gaytan ◽

Nicolas Montalbetti ◽

Anna Rued ◽

Ankita Roy ◽

...

Keyword(s):

Bk Channels ◽

Bk Channel ◽

Intercalated Cells ◽

Distal Nephron ◽

Α Subunit ◽

High K ◽

Pseudohypoaldosteronism Type Ii ◽

Genes Encoding ◽

Channel Expression ◽

Specific Regulation

Flow-induced K+ secretion in the aldosterone-sensitive distal nephron is mediated by high-conductance Ca2+-activated K+ (BK) channels. Familial hyperkalemic hypertension (pseudohypoaldosteronism type II) is an inherited form of hypertension with decreased K+ secretion and increased Na+ reabsorption. This disorder is linked to mutations in genes encoding with-no-lysine kinase 1 (WNK1), WNK4, and Kelch-like 3/Cullin 3, two components of an E3 ubiquitin ligase complex that degrades WNKs. We examined whether the full-length (or “long”) form of WNK1 (L-WNK1) affected the expression of BK α-subunits in HEK cells. Overexpression of L-WNK1 promoted a significant increase in BK α-subunit whole cell abundance and functional channel expression. BK α-subunit abundance also increased with coexpression of a kinase dead L-WNK1 mutant (K233M) and with kidney-specific WNK1 (KS-WNK1), suggesting that the catalytic activity of L-WNK1 was not required to increase BK expression. We examined whether dietary K+ intake affected L-WNK1 expression in the aldosterone-sensitive distal nephron. We found a paucity of L-WNK1 labeling in cortical collecting ducts (CCDs) from rabbits on a low-K+ diet but observed robust staining for L-WNK1 primarily in intercalated cells when rabbits were fed a high-K+ diet. Our results and previous findings suggest that L-WNK1 exerts different effects on renal K+ secretory channels, inhibiting renal outer medullary K+ channels and activating BK channels. A high-K+ diet induced an increase in L-WNK1 expression selectively in intercalated cells and may contribute to enhanced BK channel expression and K+ secretion in CCDs.

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Disruption of TRPV1-mediated coupling of coronary blood flow to cardiac metabolism in diabetic mice: role of nitric oxide and BK channels

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00011.2012 ◽

2012 ◽

Vol 303 (2) ◽

pp. H216-H223 ◽

Cited By ~ 32

Author(s):

Giacinta Guarini ◽

Vahagn A. Ohanyan ◽

John G. Kmetz ◽

Daniel J. DelloStritto ◽

Roslin J. Thoppil ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Flow ◽

Coronary Blood Flow ◽

Cardiac Metabolism ◽

Bk Channels ◽

Bk Channel ◽

Transient Receptor Potential Vanilloid ◽

Trpv1 Channels ◽

Channel Dependent

We have previously shown transient receptor potential vanilloid subtype 1 (TRPV1) channel-dependent coronary function is compromised in pigs with metabolic syndrome (MetS). However, the mechanisms through which TRPV1 channels couple coronary blood flow to metabolism are not fully understood. We employed mice lacking TRPV1 [TRPV1(−/−)], db/db diabetic, and control C57BKS/J mice to determine the extent to which TRPV1 channels modulate coronary function and contribute to vascular dysfunction in diabetic cardiomyopathy. Animals were subjected to in vivo infusion of the TRPV1 agonist capsaicin to examine the hemodynamic actions of TRPV1 activation. Capsaicin (1–100 μg·kg−1·min−1) dose dependently increased coronary blood flow in control mice, which was inhibited by the TRPV1 antagonist capsazepine or the nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine methyl ester (l-NAME). In addition, the capsaicin-mediated increase in blood flow was attenuated in db/db mice. TRPV1(−/−) mice exhibited no changes in coronary blood flow in response to capsaicin. Vasoreactivity studies in isolated pressurized mouse coronary microvessels revealed a capsaicin-dependent relaxation that was inhibited by the TRPV1 inhibitor SB366791 l-NAME and to the large conductance calcium-sensitive potassium channel (BK) inhibitors iberiotoxin and Penetrim A. Similar to in vivo responses, capsaicin-mediated relaxation was impaired in db/db mice compared with controls. Changes in pH (pH 7.4–6.0) relaxed coronary vessels contracted to the thromboxane mimetic U46619 in all three groups of mice; however, pH-mediated relaxation was blunted in vessels obtained from TRPV1(−/−) and db/db mice compared with controls. Western blot analysis revealed decreased myocardial TRPV1 protein expression in db/db mice compared with controls. Our data reveal TRPV1 channels mediate coupling of myocardial blood flow to cardiac metabolism via a nitric oxide-dependent, BK channel-dependent pathway that is corrupted in diabetes.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00002.2008 ◽

2008 ◽

Vol 295 (3) ◽

pp. F780-F788 ◽

Cited By ~ 56

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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Detrusor overactivity is associated with downregulation of large-conductance calcium- and voltage-activated potassium channel protein

AJP Renal Physiology ◽

10.1152/ajprenal.00595.2009 ◽

2010 ◽

Vol 298 (6) ◽

pp. F1416-F1423 ◽

Cited By ~ 44

Author(s):

Shaohua Chang ◽

Cristiano Mendes Gomes ◽

Joseph A. Hypolite ◽

James Marx ◽

Jaber Alanzi ◽

...

Keyword(s):

Detrusor Overactivity ◽

Bladder Outlet Obstruction ◽

Rabbit Model ◽

Outlet Obstruction ◽

Bk Channels ◽

Bk Channel ◽

Β Subunit ◽

Α Subunit ◽

Translational Study ◽

Channel Expression

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet obstruction-induced DO is associated with downregulation of BK channels and whether BK channels affect myosin light chain 20 (MLC20) phosphorylation in detrusor smooth muscle (DSM). Partial bladder outlet obstruction (PBOO) was surgically induced in male New Zealand White rabbits. The rabbit PBOO model shows decreased voided volumes and increased voiding frequency. DSM from PBOO rabbits also show enhanced spontaneous contractions compared with control. Both BK channel α- and β-subunits were significantly decreased in DSM from PBOO rabbits. Immunostaining shows BKβ mainly expressed in DSM, and its expression is much less in PBOO DSM compared with control DSM. Furthermore, a translational study was performed to see whether the finding discovered in the animal model can be translated to human patients. The urodynamic study demonstrates several overactive DSM contractions during the urine-filling stage in benign prostatic hyperplasia (BPH) patients with DO, while DSM is very quiet in BPH patients without DO. DSM biopsies revealed significantly less BK channel expression at both mRNA and protein levels. The degree of downregulation of the BK β-subunit was greater than that of the BK α-subunit, and the downregulation of BK was only associated with DO, not BPH. Finally, the small interference (si) RNA-mediated downregulation of the BK β-subunit was employed to study the effect of BK depletion on MLC20 phosphorylation. siRNA-mediated BK channel reduction was associated with an increased MLC20 phosphorylation level in cultured DSM cells. In summary, PBOO-induced DO is associated with downregulation of BK channel expression in the rabbit model, and this finding can be translated to human BPH patients with DO. Furthermore, downregulation of the BK channel may contribute to DO by increasing the basal level of MLC20 phosphorylation.

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BK current contributions to action potentials across the first postnatal week reflect age dependent changes in BK current kinetics in rat hippocampal neurons

10.1101/839233 ◽

2019 ◽

Author(s):

Michael Hunsberger ◽

Michelle Mynlieff

Keyword(s):

Action Potential ◽

Action Potentials ◽

Hippocampal Neurons ◽

Expression Patterns ◽

Bk Channels ◽

Bk Channel ◽

Action Potential Firing ◽

Developmental Trends ◽

Channel Expression ◽

Old Rats

AbstractThe large conductance calcium-activated potassium (BK) channel is a critical regulator of neuronal action potential firing and follows two distinct trends in early postnatal development: an increase in total expression and a shift from the faster activating STREX isoform to the slower ZERO isoform. We analyzed the functional consequences of developmental trends in BK channel expression in hippocampal neurons isolated from neonatal rats aged one to seven days. Following overnight cultures, action potentials were recorded using whole-cell patch clamp electrophysiology. This population of neurons undergoes a steady increase in excitability during this time and the effect of blockade of BK channel activity with 100 nM iberiotoxin, changes as the neurons mature. BK currents contribute significantly more to single action potentials in neurons of one-day old rats (with BK blockade extending action potential duration by 0.46±0.12 ms) than in those of seven-day old rats (with BK blockade extending action potential duration by 0.17±0.05 ms). BK currents also contribute consistently to maintain firing rates in neurons of one-day old rats throughout extended action potential firing; BK blockade evenly depresses action potentials frequency across action potential trains. In neurons from seven-day old rats, BK blockade initially increases firing frequency and then progressively decreases frequency as firing continues, ultimately depressing neuronal firing rates to a greater extent than in the neurons from one day old animals. These results are consistent with a transition from low expression of a fast activating BK isoform (STREX) to high expression of a slower activating isoform (ZERO).New and NoteworthyThis work describes the early developmental trends of BK channel activity. Early developmental trends in expression of BK channels, both total expression and relative isoform expression, have been previously reported, but little work describes the effect of these changes in expression patterns on excitability. Here, we show that early changes in BK channel expression patterns lead to changes in the role of BK channels in determining the action potential waveform and neuronal excitability.

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Segmental differences in upregulated apical potassium channels in mammalian colon during potassium adaptation

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00181.2015 ◽

2016 ◽

Vol 311 (5) ◽

pp. G785-G793 ◽

Cited By ~ 5

Author(s):

Matthew D. Perry ◽

Vazhaikkurichi M. Rajendran ◽

Kenneth A. MacLennan ◽

Geoffrey I. Sandle

Keyword(s):

Distal Colon ◽

Proximal Colon ◽

Bk Channels ◽

Bk Channel ◽

Patch Clamp Recording ◽

Α Subunit ◽

Subunit Protein ◽

Clear Cut ◽

Potassium Adaptation ◽

Channel Expression

Rat proximal and distal colon are net K+secretory and net K+absorptive epithelia, respectively. Chronic dietary K+loading increases net K+secretion in the proximal colon and transforms net K+absorption to net K+secretion in the distal colon, but changes in apical K+channel expression are unclear. We evaluated expression/activity of apical K+(BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting ( P < 0.001). Thus apical BK channels are normally more abundant in K+secretory proximal colon than in K+absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+secretory response to dietary K+loading.

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Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

AJP Cell Physiology ◽

10.1152/ajpcell.00482.2004 ◽

2006 ◽

Vol 290 (1) ◽

pp. C77-C86 ◽

Cited By ~ 24

Author(s):

Hiroko Sade ◽

Katsuhiko Muraki ◽

Susumu Ohya ◽

Noriyuki Hatano ◽

Yuji Imaizumi

Keyword(s):

Cell Voltage ◽

Similar Efficacy ◽

Bk Channels ◽

Bk Channel ◽

Bathing Solution ◽

Channel Activity ◽

Α Subunit ◽

Hek 293 ◽

Intracellular Ca ◽

Channel Currents

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the α-subunit of the BK channel (BK-α), both α- and β1-subunits (BK-αβ1), or both α- and β4-subunits (BK-αβ4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-αβ1 within a similar concentration range. Because methAEA could potentiate BK-α, BK-αβ1, and BK-αβ4 with similar efficacy, the β-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-α and HEK-BK-αβ1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.

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Coronary Large Conductance Ca2+-Activated K+ Channel Dysfunction in Diabetes Mellitus

Frontiers in Physiology ◽

10.3389/fphys.2021.750618 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tong Lu ◽

Hon-Chi Lee

Keyword(s):

Diabetes Mellitus ◽

Vascular Tone ◽

Microvascular Complications ◽

Bk Channels ◽

Bk Channel ◽

K Channel ◽

Organ Perfusion ◽

Biophysical Properties ◽

Diabetic Vasculopathy ◽

Channel Expression

Diabetes mellitus (DM) is an independent risk of macrovascular and microvascular complications, while cardiovascular diseases remain a leading cause of death in both men and women with diabetes. Large conductance Ca2+-activated K+ (BK) channels are abundantly expressed in arteries and are the key ionic determinant of vascular tone and organ perfusion. It is well established that the downregulation of vascular BK channel function with reduced BK channel protein expression and altered intrinsic BK channel biophysical properties is associated with diabetic vasculopathy. Recent efforts also showed that diabetes-associated changes in signaling pathways and transcriptional factors contribute to the downregulation of BK channel expression. This manuscript will review our current understandings on the molecular, physiological, and biophysical mechanisms that underlie coronary BK channelopathy in diabetes mellitus.

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Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

AJP Cell Physiology ◽

10.1152/ajpcell.00450.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C45-C54 ◽

Cited By ~ 42

Author(s):

Kirill Essin ◽

Birgit Salanova ◽

Ralph Kettritz ◽

Matthias Sausbier ◽

Friedrich C. Luft ◽

...

Keyword(s):

Innate Immunity ◽

Patch Clamp ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Bk Channels ◽

Bk Channel ◽

Proton Channel ◽

Nadph Oxidase Activity ◽

Intracellular Ca ◽

Channel Currents

Large-conductance Ca2+-activated K+(BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK−/−) mice, we stimulated NADPH oxidase activity with 12- O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2−and H2O2production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca2+and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+by N-formyl-l-methionyl-l-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK−/−and BK+/+mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

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