Furosemide reduces BK-αß4-mediated K+ secretion in mice on an alkaline high-K+ diet.

Special high-K diets have cardioprotective effects and are often warranted in conjunction with diuretics such as furosemide for treating hypertension. However, it is not understood how a high-K diet (HK) influences the actions of diuretics on renal K+ handling. Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. We previously found that an alkaline urine is required for large conductance Ca2+-activated K+ (BK)-αß4-mediated K+ secretion in mice on HK. We therefore hypothesized that furosemide could reduce BK-αß4-mediated K+ secretion by acidifying the urine. Treating with furosemide (drinking water) for 11 days led to decreased urine pH in both wild-type (WT) and BK-ß4-knockout mice (BK-ß4-KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-ß4-KO. Western blotting and immunofluorescence staining showed that furosemide treatment decreased cortical expression of BK-ß4 and reduced apical localization of BK-α in connecting tubules. Addition of the carbonic anhydrase inhibitor, acetazolamide, to furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to control levels. Acetazolamide plus furosemide also restored the cortical expression of BK-ß4 and BK-α in connecting tubules. These results indicate that in mice adapted to HK, furosemide reduces BK-αß4-mediated K+ secretion by acidifying the urine.

High-fat diet-induced obesity alters nitric oxide-mediated neuromuscular transmission and smooth muscle excitability in the mouse distal colon.

We tested the hypothesis that colonic enteric neurotransmission and smooth muscle cell (SMC) function are altered in mice fed a high-fat diet (HFD). We used wild-type (WT) mice and mice lacking the ß1-subunit of the BK channel (BKß1 (-/-)). WT mice fed a HFD had increased myenteric plexus oxidative stress, a 28% decrease in nitrergic neurons, and a 20% decrease in basal nitric oxide (NO) levels. Circular muscle inhibitory junction potentials (IJPs) were reduced in HFD WT mice. The NO synthase inhibitor nitro-l-arginine (NLA) was less effective at inhibiting relaxations in HFD compared with control diet (CD) WT mice (11 vs. 37%, P < 0.05). SMCs from HFD WT mice had depolarized membrane potentials (-47 ± 2 mV) and continuous action potential firing compared with CD WT mice (-53 ± 2 mV, P < 0.05), which showed rhythmic firing. SMCs from HFD or CD fed BKß1 (-/-) mice fired action potentials continuously. NLA depolarized membrane potential and caused continuous firing only in SMCs from CD WT mice. Sodium nitroprusside (NO donor) hyperpolarized membrane potential and changed continuous to rhythmic action potential firing in SMCs from HFD WT and BKß1 (-/-) mice. Migrating motor complexes were disrupted in colons from BKß1 (-/-) mice and HFD WT mice. BK channel α-subunit protein and ß1-subunit mRNA expression were similar in CD and HFD WT mice. We conclude that HFD-induced obesity disrupts inhibitory neuromuscular transmission, SMC excitability, and colonic motility by promoting oxidative stress, loss of nitrergic neurons, and SMC BK channel dysfunction.

Knockout of the BK ß4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability.

BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK ß4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of ß4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the ß4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons.

Hyperaldosteronism after decreased renal K+ excretion in KCNMB2 knockout mice.

The kidney is the primary organ ensuring K(+) homeostasis. K(+) is secreted into the urine in the distal tubule by two mechanisms: by the renal outer medullary K(+) channel (Kir1.1) and by the Ca(2+)-activated K(+) channel (KCa1.1). Here, we report a novel knockout mouse of the ß2-subunit of the KCa1.1 channel (KCNMB2), which displays hyperaldosteronism after decreased renal K(+) excretion. KCNMB2(-/-) mice displayed hyperaldosteronism, normal plasma K(+) concentration, and produced dilute urine with decreased K(+) concentration. The normokalemia indicated that hyperaldosteronism did not result from primary aldosteronism. Activation of the renin-angiotensin-aldosterone system was also ruled out as renal renin mRNA expression was reduced in KCNMB2(-/-) mice. Renal K(+) excretion rates were similar in the two genotypes; however, KCNMB2(-/-) mice required elevated plasma aldosterone to achieve K(+) balance. Blockade of the mineralocorticoid receptor with eplerenone triggered mild hyperkalemia and unmasked reduced renal K(+) excretion in KCNMB2(-/-) mice. Knockout mice for the α-subunit of the KCa1.1 channel (KCNMA1(-/-) mice) have hyperaldosteronism, are hypertensive, and lack flow-induced K(+) secretion. KCNMB2(-/-) mice share the phenotypic traits of normokalemia and hyperaldosteronism with KCNMA1(-/-) mice but were normotensive and displayed intact flow-induced K(+) secretion. Despite elevated plasma aldosterone, KNCMB2(-/-) mice did not display salt-sensitive hypertension and were able to decrease plasma aldosterone on a high-Na(+) diet, although plasma aldosterone remained elevated in KCNMB2(-/-) mice. In summary, KCNMB2(-/-) mice have a reduced ability to excrete K(+) into the urine but achieve K(+) balance through an aldosterone-mediated, ß2-independent mechanism. The phenotype of KCNMB2 mice was similar but milder than the phenotype of KCNMA1(-/-) mice.

BK channel ß1-subunit deficiency exacerbates vascular fibrosis and remodelling but does not promote hypertension in high-fat fed obesity in mice.

OBJECTIVE: Reduced expression or increased degradation of BK (large conductance Ca-activated K) channel ß1-subunits has been associated with increased vascular tone and hypertension in some metabolic diseases. The contribution of BK channel function to control of blood pressure (BP), heart rate (HR) and vascular function/structure was determined in wild-type and BK channel ß1-subunit knockout mice fed a high-fat or control diet. METHODS AND RESULTS: After 24 weeks of high-fat diet, wild-type and BK ß1-knockout mice were obese, diabetic, but normotensive. High-fat-BK ß1-knockout mice had decreased HR, while high-fat-wild-type mice had increased HR compared with mice on the control diet. Ganglion blockade caused a greater fall in BP and HR in mice on a high-fat diet than in mice on the control diet. ß1-adrenergic receptor blockade reduced BP and HR equally in all groups. α1-adrenergic receptor blockade decreased BP in high-fat-BK ß1-knockout mice only. Echocardiographic evaluation revealed left ventricular hypertrophy in high-fat-BK ß1-knockout mice. Although under anaesthesia, mice on a high-fat diet had higher absolute stroke volume and cardiac output, these measures were similar to control mice when adjusted for body weight. Mesenteric arteries from high-fat-BK ß1-knockout mice had higher norepinephrine reactivity, greater wall thickness and collagen accumulation than high-fat-wild-type mesenteric arteries. Compared with control-wild-type mesenteric arteries, high-fat-wild-type mesenteric arteries had blunted contractile responses to a BK channel blocker, although BK α-subunit (protein) and ß1-subunit (mRNA) expression were unchanged. CONCLUSION: BK channel deficiency promotes increased sympathetic control of BP, and vascular dysfunction, remodelling and fibrosis, but does not cause hypertension in high-fat fed mice.

Knockout of the BK ß2 subunit abolishes inactivation of BK currents in mouse adrenal chromaffin cells and results in slow-wave burst activity.

Rat and mouse adrenal medullary chromaffin cells (CCs) express an inactivating BK current. This inactivation is thought to arise from the assembly of up to four ß2 auxiliary subunits (encoded by the kcnmb2 gene) with a tetramer of pore-forming Slo1 α subunits. Although the physiological consequences of inactivation remain unclear, differences in depolarization-evoked firing among CCs have been proposed to arise from the ability of ß2 subunits to shift the range of BK channel activation. To investigate the role of BK channels containing ß2 subunits, we generated mice in which the gene encoding ß2 was deleted (ß2 knockout [KO]). Comparison of proteins from wild-type (WT) and ß2 KO mice allowed unambiguous demonstration of the presence of ß2 subunit in various tissues and its coassembly with the Slo1 α subunit. We compared current properties and cell firing properties of WT and ß2 KO CCs in slices and found that ß2 KO abolished inactivation, slowed action potential (AP) repolarization, and, during constant current injection, decreased AP firing. These results support the idea that the ß2-mediated shift of the BK channel activation range affects repetitive firing and AP properties. Unexpectedly, CCs from ß2 KO mice show an increased tendency toward spontaneous burst firing, suggesting that the particular properties of BK channels in the absence of ß2 subunits may predispose to burst firing.

Altered L-type Ca2+ channel activity contributes to exacerbated hypoperfusion and mortality in smooth muscle cell BK channel-deficient septic mice.

We determined the contribution of vascular large conductance Ca2+-activated K+ (BK) and L-type Ca2+ channel dysregulation to exaggerated mortality in cecal ligation/puncture (CLP)-induced septic BK channel ß1-subunit knockout (BK ß1-KO, smooth muscle specific) mice. CLP-induced hemodynamic changes and mortality were assessed over 7 days in wild-type (WT) and BK ß1-KO mice that were either untreated, given volume resuscitation (saline), or saline + calcium channel blocker nicardipine. Some mice were euthanized 24 h post-CLP to measure tissue injury and vascular and immune responses. CLP-induced hypotension was similar in untreated WT and BK ß1-KO mice, but BK ß1-KO mice died sooner. At 24 h post-CLP (mortality latency in BK ß1-KO mice), untreated CLP-BK ß1-KO mice showed more severe hypothermia, lower tissue perfusion, polymorphonuclear neutrophil infiltration-independent severe intestinal necrosis, and higher serum cytokine levels than CLP-WT mice. Saline resuscitation improved survival in CLP-WT but not CLP-BK ß1-KO mice. Saline + nicardipine-treated CLP-BK ß1-KO mice exhibited longer survival times, higher tissue perfusion, less intestinal injury, and lower cytokines versus untreated CLP-BK ß1-KO mice. These improvements were absent in treated CLP-WT mice, although saline + nicardipine improved blood pressure similarly in both septic mice. At 24 h post-CLP, BK and L-type Ca2+ channel functions in vitro were maintained in mesenteric arteries from WT mice. Mesenteric arteries from BK ß1-KO mice had blunted BK/enhanced L-type Ca2+ channel function. We conclude that vascular BK channel deficiency exaggerates mortality in septic BK ß1-KO mice by activating L-type Ca2+ channels leading to blood pressure-independent tissue ischemia.

Relation between BK-α/ß4-mediated potassium secretion and ENaC-mediated sodium reabsorption.

The large-conductance, calcium-activated BK-α/ß4 potassium channel, localized to the intercalated cells of the distal nephron, mediates potassium secretion during high-potassium, alkaline diets. Here we determine whether BK-α/ß4-mediated potassium transport is dependent on epithelial sodium channel (ENaC)-mediated sodium reabsorption. We maximized sodium-potassium exchange in the distal nephron by feeding mice a low-sodium, high-potassium diet. Wild-type and BK-ß4 knockout mice were maintained on a low-sodium, high-potassium, alkaline diet or a low-sodium, high-potassium, acidic diet for 7-10 days. Wild-type mice maintained potassium homeostasis on the alkaline, but not acid, diet. BK-ß4 knockout mice could not maintain potassium homeostasis on either diet. During the last 12 h of diet, wild-type mice on either a regular, alkaline, or an acid diet, or knockout mice on an alkaline diet, were administered amiloride (an ENaC inhibitor). Amiloride enhanced sodium excretion in all wild-type and knockout groups to similar values; however, amiloride diminished potassium excretion by 59% in wild-type but only by 33% in knockout mice on an alkaline diet. Similarly, amiloride decreased the trans-tubular potassium gradient by 68% in wild-type but only by 42% in knockout mice on an alkaline diet. Amiloride treatment equally enhanced sodium excretion and diminished potassium secretion in knockout mice on an alkaline diet and wild-type mice on an acid diet. Thus, the enhanced effect of amiloride on potassium secretion in wild-type compared to knockout mice on the alkaline diet clarify a BK- α/ß4-mediated potassium secretory pathway in intercalated cells driven by ENaC-mediated sodium reabsorption linked to bicarbonate secretion.

Bicarbonate promotes BK-α/ß4-mediated K excretion in the renal distal nephron.

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-ß4 in acid/base-transporting intercalated cells (IC), we determined whether BK-ß4 knockout mice (ß4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-ß4 expression increased (Western blot). When WT and ß4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and ß4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with ß4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and ß4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in ß-IC, comparing WT and ß4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in ß4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of ß4KO. When WT on LNaHK were given NH(4)Cl in the drinking water, K excretion was reduced to the magnitude of ß4KO on LNaHK. These results show that WT, but not ß4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/ß4-mediated K secretion is promoted by bicarbonaturia.

Smooth muscle cholesterol enables BK ß1 subunit-mediated channel inhibition and subsequent vasoconstriction evoked by alcohol.

OBJECTIVE: Hypercholesterolemia and alcohol drinking constitute independent risk factors for cerebrovascular disease. Alcohol constricts cerebral arteries in several species, including humans. This action results from inhibition of voltage- and calcium-gated potassium channels (BK) in vascular smooth muscle cells (VSMC). BK activity is also modulated by membrane cholesterol. We investigated whether VSMC cholesterol regulates ethanol actions on BK and cerebral arteries. METHODS AND RESULTS: After myogenic tone development, cholesterol depletion of rat, resistance-size cerebral arteries ablated ethanol-induced constriction, a result that was identical in intact and endothelium-free vessels. Cholesterol depletion reduced ethanol inhibition of BK whether the channel was studied in VSMC or after rat cerebral artery myocyte subunit (cbv1+ß1) reconstitution into phospholipid bilayers. Homomeric cbv1 channels reconstituted into bilayers and VSMC BK from ß1 knockout mice were both resistant to ethanol-induced inhibition. Moreover, arteries from ß1 knockout mice failed to respond to ethanol even when VSMC cholesterol was kept unmodified. Remarkably, ethanol inhibition of cbv1+ß1 in bilayers and wt mouse VSMC BK were drastically blunted by cholesterol depletion. Consistently, cholesterol depletion suppressed ethanol constriction of wt mouse arteries. CONCLUSION: VSMC cholesterol and BK ß1 are both required for ethanol inhibition of BK and the resulting cerebral artery constriction, with health-related implications for manipulating cholesterol levels in alcohol-induced cerebrovascular disease.

BK channel ß1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitation­contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation­contraction coupling to more negative voltage ranges.

Hypertension of Kcnmb1-/- is linked to deficient K secretion and aldosteronism.

Mice lacking the beta1-subunit (gene, Kcnmb1; protein, BK-beta1) of the large Ca-activated K channel (BK) are hypertensive. This phenotype is thought to result from diminished BK currents in vascular smooth muscle where BK-beta1 is an ancillary subunit. However, the beta1-subunit is also expressed in the renal connecting tubule (CNT), a segment of the aldosterone-sensitive distal nephron, where it associates with BK and facilitates K secretion. Because of the correlation between certain forms of hypertension and renal defects, particularly in the distal nephron, it was determined whether the hypertension of Kcnmb1(-/-) has a renal origin. We found that Kcnmb1(-/-) are hypertensive, volume expanded, and have reduced urinary K and Na clearances. These conditions are exacerbated when the animals are fed a high K diet (5% K; HK). Supplementing HK-fed Kcnmb1(-/-) with eplerenone (mineralocorticoid receptor antagonist) corrected the fluid imbalance and more than 70% of the hypertension. Finally, plasma [aldo] was elevated in Kcnmb1(-/-) under basal conditions (control diet, 0.6% K) and increased significantly more than wild type when fed the HK diet. We conclude that the majority of the hypertension of Kcnmb1(-/-) is due to aldosteronism, resulting from renal potassium retention and hyperkalemia.