The mechanosensitive BKα/β1 channel localizes to cilia of principal cells in rabbit cortical collecting duct (CCD)

Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca2+- and/or stretch-dependent flow-induced K+ secretion (FIKS) and contributes to K+ adaptation in response to dietary K+ loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K+ secretory processes. In support of a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore-forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high-K+ diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca2+ concentration, albeit of nonciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K+-fed rabbits with channel subunit-specific antibodies revealed colocalization of immunodetectable BKα- and β1-subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low-K+ vs. high-K+ diet. Single-channel recordings of cilia revealed K+ channels with conductance and kinetics typical of the BK channel. The observations that 1) FIKS was preserved but 2) the high-amplitude Ca2+ peak elicited by flow was reduced in microperfused CCDs subject to pharmacological deciliation suggest that cilia BK channels do not contribute to K+ secretion in this segment, but that cilia serve as modulators of cell signaling.

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SGK1-dependent ENaC processing and trafficking in mice with high dietary K intake and elevated aldosterone

AJP Renal Physiology ◽

10.1152/ajprenal.00257.2016 ◽

2017 ◽

Vol 312 (1) ◽

pp. F65-F76 ◽

Cited By ~ 19

Author(s):

Lei Yang ◽

Gustavo Frindt ◽

Florian Lang ◽

Dietmar Kuhl ◽

Volker Vallon ◽

...

Keyword(s):

Knockout Mice ◽

Collecting Duct ◽

Surface Expression ◽

Bk Channels ◽

Na Channel ◽

Cortical Collecting Duct ◽

Wild Type ◽

Altered Expression ◽

Principal Cells ◽

High K

We examined renal Na and K transporters in mice with deletions in the gene encoding the aldosterone-induced protein SGK1. The knockout mice were hyperkalemic, and had altered expression of the subunits of the epithelial Na channel (ENaC). The kidneys showed decreased expression of the cleaved forms of the γENaC subunit, and the fully glycosylated form of the βENaC subunits when animals were fed a high-K diet. Knockout animals treated with exogenous aldosterone also had reduced subunit processing and diminished surface expression of βENaC and γENaC. Expression of the three upstream Na transporters NHE3, NKCC2, and NCC was reduced in both wild-type and knockout mice in response to K loading. The activity of ENaC measured as whole cell amiloride-sensitive current ( INa) in principal cells of the cortical collecting duct (CCD) was minimal under control conditions but was increased by a high-K diet to a similar extent in knockout and wild-type animals. INa in the connecting tubule also increased similarly in the two genotypes in response to exogenous aldosterone administration. The activities of both ROMK channels in principal cells and BK channels in intercalated cells of the CCD were unaffected by the deletion of SGK1. Acute treatment of animals with amiloride produced similar increases in Na excretion and decreases in K excretion in the two genotypes. The absence of changes in ENaC activity suggests compensation for decreased surface expression. Altered K balance in animals lacking SGK1 may reflect defects in ENaC-independent K excretion.

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Role of NKCC in BK channel-mediated net K+ secretion in the CCD

AJP Renal Physiology ◽

10.1152/ajprenal.00347.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. F1088-F1097 ◽

Cited By ~ 37

Author(s):

Wen Liu ◽

Carlos Schreck ◽

Richard A. Coleman ◽

James B. Wade ◽

Yubelka Hernandez ◽

...

Keyword(s):

Collecting Duct ◽

Bk Channels ◽

Bk Channel ◽

Taqman Assay ◽

Specific Cell ◽

Cortical Collecting Duct ◽

Ph Indicator ◽

Transport Pathway ◽

K Secretion

Apical SK/ROMK and BK channels mediate baseline and flow-induced K secretion (FIKS), respectively, in the cortical collecting duct (CCD). BK channels are detected in acid-base transporting intercalated (IC) and Na-absorbing principal (PC) cells. Although the density of BK channels is greater in IC than PC, Na-K-ATPase activity in IC is considered inadequate to sustain high rates of urinary K secretion. To test the hypothesis that basolateral NKCC in the CCD contributes to BK channel-mediated FIKS, we measured net K secretion ( JK) and Na absorption ( JNa) at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in vitro in the absence and presence of bumetanide, an inhibitor of NKCC, added to the bath. Bumetanide inhibited FIKS but not basal JK, JNa, or the flow-induced [Ca2+]i transient necessary for BK channel activation. Addition of luminal iberiotoxin, a BK channel inhibitor, to bumetanide-treated CCDs did not further reduce JK. Basolateral Cl removal reversibly inhibited FIKS but not basal JK or JNa. Quantitative PCR performed on single CCD samples using NKCC1- and 18S-specific primers and probes and the TaqMan assay confirmed the presence of the transcript in this nephron segment. To identify the specific cell type to which basolateral NKCC is localized, we exploited the ability of NKCC to accept NH4+ at its K-binding site to monitor the rate of bumetanide-sensitive cytosolic acidification after NH4+ addition to the bath in CCDs loaded with the pH indicator dye BCECF. Both IC and PC were found to have a basolateral bumetanide-sensitive NH4+ entry step and NKCC1-specific antibodies labeled the basolateral surfaces of both cell types in CCDs. These results suggest that BK channel-mediated FIKS is dependent on a basolateral bumetanide-sensitive, Cl-dependent transport pathway, proposed to be NKCC1, in both IC and PC in the CCD.

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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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Angiotensin II type 2 receptor regulates ROMK-like K+ channel activity in the renal cortical collecting duct during high dietary K+ adaptation

AJP Renal Physiology ◽

10.1152/ajprenal.00141.2014 ◽

2014 ◽

Vol 307 (7) ◽

pp. F833-F843 ◽

Cited By ~ 13

Author(s):

Yuan Wei ◽

Yi Liao ◽

Beth Zavilowitz ◽

Jin Ren ◽

Wen Liu ◽

...

Keyword(s):

Collecting Duct ◽

No Synthase ◽

K Channel ◽

Ang Ii ◽

Cortical Collecting Duct ◽

Channel Activity ◽

High K ◽

Pka Pathway

The kidney adjusts K+ excretion to match intake in part by regulation of the activity of apical K+ secretory channels, including renal outer medullary K+ (ROMK)-like K+ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K+ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K+ (HK)-fed but not normal K+ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K+-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.

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Insulin and IGF-1 activate Kir4.1/5.1 channels in cortical collecting duct principal cells to control basolateral membrane voltage

AJP Renal Physiology ◽

10.1152/ajprenal.00436.2015 ◽

2016 ◽

Vol 310 (4) ◽

pp. F311-F321 ◽

Cited By ~ 20

Author(s):

Oleg Zaika ◽

Oleg Palygin ◽

Viktor Tomilin ◽

Mykola Mamenko ◽

Alexander Staruschenko ◽

...

Keyword(s):

Single Channel ◽

Collecting Duct ◽

Basolateral Membrane ◽

Sodium Reabsorption ◽

Cortical Collecting Duct ◽

Open Probability ◽

Whole Cell ◽

Principal Cells ◽

Basolateral Side ◽

Kinase Cascade

Potassium Kir4.1/5.1 channels are abundantly expressed at the basolateral membrane of principal cells in the cortical collecting duct (CCD), where they are thought to modulate transport rates by controlling transepithelial voltage. Insulin and insulin-like growth factor-1 (IGF-1) stimulate apically localized epithelial sodium channels (ENaC) to augment sodium reabsorption in the CCD. However, little is known about their actions on potassium channels localized at the basolateral membrane. In this study, we implemented patch-clamp analysis in freshly isolated murine CCD to assess the effect of these hormones on Kir4.1/5.1 at both single channel and cellular levels. We demonstrated that K+-selective conductance via Kir4.1/5.1 is the major contributor to the macroscopic current recorded from the basolateral side in principal cells. Acute treatment with 10 μM amiloride (ENaC blocker), 100 nM tertiapin-Q (TPNQ; ROMK inhibitor), and 100 μM ouabain (Na+-K+-ATPase blocker) failed to produce a measurable effect on the macroscopic current. In contrast, Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM), virtually abolished whole cell K+-selective conductance. Insulin (100 nM) markedly increased the open probability of Kir4.1/5.1 and nortriptyline-sensitive whole cell current, leading to significant hyperpolarization of the basolateral membrane. Inhibition of the phosphatidylinositol 3-kinase cascade with LY294002 (20 μM) abolished action of insulin on Kir4.1/5.1. IGF-1 had similar stimulatory actions on Kir4.1/5.1-mediated conductance only when applied at a higher (500 nM) concentration and was ineffective at 100 nM. We concluded that both insulin and, to a lesser extent, IGF-1 activate Kir4.1/5.1 channel activity and open probability to hyperpolarize the basolateral membrane, thereby facilitating Na+ reabsorption in the CCD.

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Ca2+ dependence of flow-stimulated K secretion in the mammalian cortical collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00057.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. F227-F235 ◽

Cited By ~ 71

Author(s):

Wen Liu ◽

Tetsuji Morimoto ◽

Craig Woda ◽

Thomas R. Kleyman ◽

Lisa M. Satlin

Keyword(s):

Apical Membrane ◽

Collecting Duct ◽

Brefeldin A ◽

Bk Channels ◽

Bk Channel ◽

Sk Channels ◽

Cortical Collecting Duct ◽

Intracellular Ca ◽

K Secretion ◽

High Conductance

Apical low-conductance SK and high-conductance Ca2+-activated BK channels are present in distal nephron, including the cortical collecting duct (CCD). Flow-stimulated net K secretion ( JK) in the CCD is 1) blocked by iberiotoxin, an inhibitor of BK but not SK channels, and 2) associated with an increase in [Ca2+]i, leading us to conclude that BK channels mediate flow-stimulated JK. To examine the Ca2+ dependence and sources of Ca2+ contributing to flow-stimulated JK, JK and net Na absorption ( JNa) were measured at slow (∼1) and fast (∼5 nl·min−1·mm−1) flow rates in rabbit CCDs microperfused in the absence of luminal Ca2+ or after pretreatment with BAPTA-AM to chelate intracellular Ca2+, 2-aminoethoxydiphenyl borate (2-APB), to inhibit the inositol 1,4,5-trisphosphate (IP3) receptor or thapsigargin to deplete internal stores. These treatments, which do not affect flow-stimulated JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), inhibited flow-stimulated JK. Increases in [Ca2+]i stimulate exocytosis. To test whether flow induces exocytic insertion of preformed BK channels into the apical membrane, CCDs were pretreated with 10 μM colchicine (COL) to disrupt microtubule function or 5 μg/ml brefeldin-A (BFA) to inhibit delivery of channels from the intracellular pool to the plasma membrane. Both agents inhibited flow-stimulated JK but not JNa (Morimoto et al. Am J Physiol Renal Physiol 291: F663–F669, 2006), although COL but not BFA also blocked the flow-induced [Ca2+]i transient. We thus speculate that BK channel-mediated, flow-stimulated JK requires an increase in [Ca2+]i due, in part, to luminal Ca2+ entry and ER Ca2+ release, microtubule integrity, and exocytic insertion of preformed channels into the apical membrane.

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An unexpected journey: conceptual evolution of mechanoregulated potassium transport in the distal nephron

AJP Cell Physiology ◽

10.1152/ajpcell.00328.2015 ◽

2016 ◽

Vol 310 (4) ◽

pp. C243-C259 ◽

Cited By ~ 23

Author(s):

Rolando Carrisoza-Gaytan ◽

Marcelo D. Carattino ◽

Thomas R. Kleyman ◽

Lisa M. Satlin

Keyword(s):

Flow Rate ◽

Fluid Shear Stress ◽

Collecting Duct ◽

Critical Role ◽

Bk Channels ◽

Bk Channel ◽

Macromolecular Complex ◽

Urinary Flow ◽

Cortical Collecting Duct ◽

Distal Nephron

Flow-induced K secretion (FIKS) in the aldosterone-sensitive distal nephron (ASDN) is mediated by large-conductance, Ca2+/stretch-activated BK channels composed of pore-forming α-subunits (BKα) and accessory β-subunits. This channel also plays a critical role in the renal adaptation to dietary K loading. Within the ASDN, the cortical collecting duct (CCD) is a major site for the final renal regulation of K homeostasis. Principal cells in the ASDN possess a single apical cilium whereas the surfaces of adjacent intercalated cells, devoid of cilia, are decorated with abundant microvilli and microplicae. Increases in tubular (urinary) flow rate, induced by volume expansion, diuretics, or a high K diet, subject CCD cells to hydrodynamic forces (fluid shear stress, circumferential stretch, and drag/torque on apical cilia and presumably microvilli/microplicae) that are transduced into increases in principal (PC) and intercalated (IC) cell cytoplasmic Ca2+ concentration that activate apical voltage-, stretch- and Ca2+-activated BK channels, which mediate FIKS. This review summarizes studies by ourselves and others that have led to the evolving picture that the BK channel is localized in a macromolecular complex at the apical membrane, composed of mechanosensitive apical Ca2+ channels and a variety of kinases/phosphatases as well as other signaling molecules anchored to the cytoskeleton, and that an increase in tubular fluid flow rate leads to IC- and PC-specific responses determined, in large part, by the cell-specific composition of the BK channels.

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Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells

AJP Renal Physiology ◽

10.1152/ajprenal.00439.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. F1360-F1368 ◽

Cited By ~ 2

Author(s):

Ming-Ming Wu ◽

Yu-Jia Zhai ◽

Yu-Xia Li ◽

Qing-Qing Hu ◽

Zhi-Rui Wang ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Transient Receptor Potential ◽

Single Channel ◽

Apical Membrane ◽

Intact Cell ◽

Collecting Duct ◽

Physiological Role ◽

Cell Types ◽

Cortical Collecting Duct ◽

Principal Cells

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

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High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf02181471 ◽

1987 ◽

Vol 408 (3) ◽

pp. 282-290 ◽

Cited By ~ 43

Author(s):

Alfred H. Gitter ◽

Klaus W. Beyenbach ◽

Chadwick W. Christine ◽

Peter Gross ◽

Will W. Minuth ◽

...

Keyword(s):

Collecting Duct ◽

K Channel ◽

Cortical Collecting Duct ◽

Principal Cells ◽

Apical Membranes ◽

Cells Cultured ◽

High Conductance

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Vasopressin increases density of apical low-conductance K+ channels in rat CCD

AJP Renal Physiology ◽

10.1152/ajprenal.1993.264.3.f502 ◽

1993 ◽

Vol 264 (3) ◽

pp. F502-F509 ◽

Cited By ~ 19

Author(s):

A. C. Cassola ◽

G. Giebisch ◽

W. Wang

Keyword(s):

Collecting Duct ◽

Phosphodiesterase Inhibitor ◽

K Channel ◽

Channel Activity ◽

Dibutyryl Camp ◽

Open Probability ◽

Simultaneous Application ◽

K Channels ◽

Principal Cells ◽

High K

The effect of arginine vasopressin (AVP) on the low-conductance K+ channel in the apical membrane of rat cortical collecting duct (CCD) principal cells from animals on a control and high-K+ diet was studied using patch-clamp techniques. AVP stimulated apical low-conductance K+ channel activity in both control and high-K+ animals: application of 110-220 pM AVP induced a significant increase in the density of low-conductance K+ channels. In the presence of phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), administration of 22 pM AVP also increased channel activity. The action of AVP on low-conductance K+ channel activity was mimicked by simultaneous application of forskolin and 3-isobutyl-1-methylxanthine. Exogenously applied N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl-cAMP, 0.4-0.8 mM) also increased apical low-conductance K+ channel activity. Since channel open probability (Po) was almost saturated in the absence of AVP, the increase of channel activity induced by AVP, forskolin, and dibutyryl-cAMP resulted predominantly from stimulating previously silent K+ channels. We conclude that AVP induces an increase of low-conductance K+ channel activity of principal cells in rat CCD by the stimulation of cAMP-dependent protein kinase. The AVP-induced increase of low-conductance K+ channel activity can thus significantly contribute to the hormone-induced K+ secretion in the rat CCD.

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