Deficiency of the BKCa potassium channel displayed significant implications for the physiology of the human bronchial epithelium.

Plasma membrane large-conductance calcium-activated potassium (BKCa) channels are important players in various physiological processes, including those mediated by epithelia. Like other cell types, human bronchial epithelial (HBE) cells also express BKCa in the inner mitochondrial membrane (mitoBKCa). The genetic relationships between these mitochondrial and plasma membrane channels and the precise role of mitoBKCa in epithelium physiology are still unclear. Here, we tested the hypothesis that the mitoBKCa channel is encoded by the same gene as the plasma membrane BKCa channel in HBE cells. We also examined the impact of channel loss on the basic function of HBE cells, which is to create a tight barrier. For this purpose, we used CRISPR/Cas9 technology in 16HBE14o- cells to disrupt the KCNMA1 gene, which encodes the α-subunit responsible for forming the pore of the plasma membrane BKCa channel. Electrophysiological experiments demonstrated that the disruption of the KCNMA1 gene resulted in the loss of BKCa-type channels in the plasma membrane and mitochondria. We have also shown that HBE ΔαBKCa cells exhibited a significant decrease in transepithelial electrical resistance which indicates a loss of tightness of the barrier created by these cells. We have also observed a decrease in mitochondrial respiration, which indicates a significant impairment of these organelles. In conclusion, our findings indicate that a single gene encodes both populations of the channel in HBE cells. Furthermore, this channel is critical for maintaining the proper function of epithelial cells as a cellular barrier.

The renal vasodilatation from ß-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels.

The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.

Cryo-EM structure of the Slo1 potassium channel with the auxiliary γ1 subunit suggests a mechanism for depolarization-independent activation.

Mammalian Ca2+-dependent Slo K+ channels can stably associate with auxiliary γ subunits which fundamentally alter their behavior. By a so far unknown mechanism, the four γ subunits reduce the need for voltage-dependent activation and, thereby, allow Slo to open independently of an action potential. Here, using cryo-EM, we reveal how the transmembrane helix of γ1/LRRC26 binds and presumably stabilizes the activated voltage-sensor domain of Slo1. The activation is further enhanced by an intracellular polybasic stretch which locally changes the charge gradient across the membrane. Our data provide a possible explanation for Slo1 regulation by the four γ subunits and also their different activation efficiencies. This suggests a novel activation mechanism of voltage-gated ion channels by auxiliary subunits.

Thyrotropin induces atherosclerosis by upregulating large conductance Ca2+-activated K+ channel subunits.

Hypothyroidism is associated with elevated levels of serum thyrotropin (TSH), which have been shown to promote abnormal proliferation of vascular smooth muscle cells and contribute to the development of atherosclerosis. However, the specific mechanisms underlying the TSH-induced abnormal proliferation of vascular smooth muscle cells remain unclear. The objective of this study was to investigate the role of TSH in the progression of atherosclerosis. Our research findings revealed that hypothyroidism can trigger early atherosclerotic changes in the aorta of Wistar rats. In alignment with our in vitro experiments, we observed that TSH induces abnormal proliferation of aortic smooth muscle cells by modulating the expression of α and ß1 subunits of large conductance Ca2+-activated K+ (BKCa) channels within these cells via the cAMP/PKA signaling pathway. These results collectively indicate that TSH acts through the cAMP/PKA signaling pathway to upregulate the expression of α and ß1 subunits of BKCa channels, thereby promoting abnormal proliferation of arterial smooth muscle cells. These findings may provide a basis for the clinical prevention and treatment of atherosclerosis caused by elevated TSH levels.

The lncRNA lnc-TSI antagonizes sorafenib resistance in hepatocellular carcinoma via downregulating miR-4726-5p expression and upregulating KCNMA1 expression.

Acquired drug resistance is a main reason for limiting the application of sorafenib in HCC treatment. This study aimed to explore the role and mechanisms of a novel long non-coding RNA (lncRNA), lnc-TSI, in sorafenib resistance of HCC. The interaction between lnc-TSI and miR-4726-5p, and miR-4726-5p and KCNMA1 were predicted using bioinformatic tools. Expression of the molecules in the lnc-TSI/miR-4726-5p/KCNMA1 axis in clinical samples and cell lines, as well as the sorafenib resistant HCC cell lines, was determined using qRT-PCR or western blotting. Expressions of lnc-TSI, miR-4726-5p, and KCNMA1 were manipulated in HepG2 and Huh7 cells through plasmid transfection or lentivirus infection. The CCK-8, flow cytometry, and Tunel assays were employed to determine the role of this axis on sorafenib resistance of HCC. A xenograft model was established using sorafenib-resistant HepG2 and Huh7 cells followed by in vivo sorafenib treatments to confirm the in vitro findings. Lnc-TSI and KCNMA1 expressions were significantly downregulated in HCC clinical samples and cell lines, especially in sorafenib resistance ones, while mi-4726-5p presented a reversed expression pattern. Lnc-TSI interacted with miR-4726-5p, and Lnc-TSI acts as a ceRNA via sponging miR-4726-5p in HCC cells. Overexpression of lnc-TSI and KCNMA1 promoted apoptosis and decreased cell viability of sorafenib-treated HCC cells, thus alleviated sorafenib resistance. miR-4726-5p mimic reversed the KCNMA1-mediated sorafenib sensitivity-promoting effect, while additional overexpression of lnc-TSI reversed the effect of miR-4726-5p. In vivo analysis also showed that overexpression of ln-TSI diminished sorafenib resistance in mice inoculated with sorafenib-resistant HCC cells via increasing KCNMA1 expression and decreasing miR-4726-5p expression. The lnc-TSI/miR-4726-5p/KCNMA1 axis plays a critical role in regulating the resistance of HCC to sorafenib, and might serve as a therapeutic target to manage sorafenib resistance of HCC in clinic.

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6 J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

[Overcoming chemoresistance by Ca2＋-activated K＋ channel inhibition in cancer spheroid models].

Ca2＋-activated K＋ channels play a critical role in the proliferation, apoptosis, migration, adhesion, and metastasis of various types of cancer cells by controlling Ca2＋ signaling and cell volumes. Their amplification correlated with a high tumor stage and poor prognosis and has the potential as tumor grade-associated markers. The amplification of the large-conductance Ca2＋-activated K＋ channel, KCa1.1 is observed in many types of cancers such as breast, colon, ovarian, prostate, pancreatic cancers and gliomas. The hypoxic tumor microenvironment (TME) promotes the anti-cancer drug resistance and stemness of solid tumors. Three-dimensional (3D) in vitro cancer spheroid models mimic the TME of human solid tumors, and are efficient tools for investigating chemoresistance and stemness. We here introduce the mechanisms underlying the post-translational modification of KCa1.1 and the overcome of chemo- and antiandrogen-resistance by KCa1.1 inhibition in 3D cancer spheroid models. KCa1.1 is a key modulator of chemoresistance in KCa1.1-positive cancer cells, indicating that targeting KCa1.1 is a promising therapeutic strategy for overcoming chemoresistance.

Long noncoding RNA KCNMA1-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the SMAD9 signaling pathway.

The human bone marrow mesenchymal stem cells (hBMSCs) undergo intense osteogenic differentiation, a crucial bone formation mechanism. Evidence from prior studies suggested an association between long noncoding RNAs (lncRNAs) and the osteogenic differentiation of hBMSCs. However, precise roles and molecular mechanisms are still largely unknown. In this work, we report for the first time that lncRNA KCNMA1 antisense RNA 1 (KCNMA1-AS1) plays a vital role in regulating hBMSCs' osteogenic differentiation. Here, it was observed that the KCNMA1-AS1 expression levels were significantly upregulated during osteogenic differentiation. In addition, KCNMA1-AS1 overexpression enhanced in vitro osteogenic differentiation of hBMSCs and in vivo bone formation, whereas knockdown of KCNMA1-AS1 resulted in the opposite result. Additionally, the interaction between KCNMA1-AS1 and mothers against decapentaplegic homolog 9 (SMAD9) was confirmed by an RNA pull-down experiment, mass spectrometry, and RIP assay. This interaction regulated the activation of the SMAD9 signaling pathway. Moreover, rescue assays demonstrated that the inhibitor of the SMAD9 signaling pathway reversed the stimulative effects on osteogenic differentiation of hBMSCs by KCNMA1-AS1 overexpression. Altogether, our results stipulate that KCNMA1-AS1 promotes osteogenic differentiation of hBMSCs via activating the SMAD9 signaling pathway and can serve as a biomarker and therapeutic target in treating bone defects.

BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site.

The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory ß and γ subunits. The γ1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BKα is indispensable in BK channel modulation by the γ1 subunit. Notably, synthetic peptide mimics of the γ1-3 subunits' C-terminal positively charged regions caused 30-50 mV shifts in BKα channel voltage-gating toward the hyperpolarization direction. The cationic cell-penetrating HIV-1 Tat peptide exerted a similar BK channel-activating effect. The BK channel-activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the γ subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the γ1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the γ1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary γ subunits.

Cholesterol and PIP2 Modulation of BKCa Channels.

Ca2+/voltage-gated, large conductance K+ channels (BKCa) are formed by homotetrameric association of α (slo1) subunits. Their activity, however, is suited to tissue-specific physiology largely due to their association with regulatory subunits (ß and γ types), chaperone proteins, localized signaling, and the channel's lipid microenvironment. PIP2 and cholesterol can modulate BKCa activity independently of downstream signaling, yet activating Ca2+i levels and regulatory subunits control ligand action. At physiological Ca2+i and voltages, cholesterol and PIP2 reduce and increase slo1 channel activity, respectively. Moreover, slo1 proteins provide sites that seem to recognize cholesterol and PIP2: seven CRAC motifs in the slo1 cytosolic tail and a string of positively charged residues (Arg329, Lys330, Lys331) immediately after S6, respectively. A model that could explain the modulation of BKCa activity by cholesterol and/or PIP2 is hypothesized. The roles of additional sites, whether in slo1 or BKCa regulatory subunits, for PIP2 and/or cholesterol to modulate BKCa function are also discussed.

Co-dependent regulation of p-BRAF and potassium channel KCNMA1 levels drives glioma progression.

BRAF mutations have been found in gliomas which exhibit abnormal electrophysiological activities, implying their potential links with the ion channel functions. In this study, we identified the Drosophila potassium channel, Slowpoke (Slo), the ortholog of human KCNMA1, as a critical factor involved in dRafGOF glioma progression. Slo was upregulated in dRafGOF glioma. Knockdown of slo led to decreases in dRafGOF levels, glioma cell proliferation, and tumor-related phenotypes. Overexpression of slo in glial cells elevated dRaf expression and promoted cell proliferation. Similar mutual regulations of p-BRAF and KCNMA1 levels were then recapitulated in human glioma cells with the BRAF mutation. Elevated p-BRAF and KCNMA1 were also observed in HEK293T cells upon the treatment of 20 mM KCl, which causes membrane depolarization. Knockdown KCNMA1 in these cells led to a further decrease in cell viability. Based on these results, we conclude that the levels of p-BRAF and KCNMA1 are co-dependent and mutually regulated. We propose that, in depolarized glioma cells with BRAF mutations, high KCNMA1 levels act to repolarize membrane potential and facilitate cell growth. Our study provides a new strategy to antagonize the progression of gliomas as induced by BRAF mutations.

Deletion of large-conductance calcium-activated potassium channels promotes vascular remodelling through the CTRP7-mediated PI3K/Akt signaling pathway.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture, and abnormal expression or dysfunction of this channel is linked to many vascular diseases. Vascular remodelling is the early pathological basis of severe vascular diseases. Delaying the progression of vascular remodelling can reduce cardiovascular events, but the pathogenesis remains unclear. To clarify the role of BK channels in vascular remodelling, we use rats with BK channel α subunit knockout (BK α ‒/‒). The results show that BK α ‒/‒ rats have smaller inner and outer diameters, thickened aortic walls, increased fibrosis, and disordered elastic fibers of the aortas compared with WT rats. When the expression and function of BK α are inhibited in human umbilical arterial smooth muscle cells (HUASMCs), the expressions of matrix metalloproteinase 2 (MMP2), MMP9, and interleukin-6 are enhanced, while the expressions of smooth muscle cell contractile phenotype proteins are reduced. RNA sequencing, bioinformatics analysis and qPCR verification show that C1q/tumor necrosis factor-related protein 7 ( CTRP7) is the downstream target gene. Furthermore, except for that of MMPs, a similar pattern of IL-6, smooth muscle cell contractile phenotype proteins expression trend is observed after CTRP7 knockdown. Moreover, knockdown of both BK α and CTRP7 in HUASMCs activates PI3K/Akt signaling. Additionally, CTRP7 is expressed in vascular smooth muscle cells (VSMCs), and BK α deficiency activates the PI3K/Akt pathway by reducing CTRP7 level. Therefore, we first show that BK channel deficiency leads to vascular remodelling. The BK channel and CTRP7 may serve as potential targets for the treatment of cardiovascular diseases.

The function of BK channels extracted and purified within SMALPs.

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of ß and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and ß1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of ß1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified ß1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

TRPC6 interacted with KCa1.1 channels to regulate the proliferation and apoptosis of glioma cells.

Malignant glioma is the most aggressive and deadliest brain malignancy. TRPC6 and KCa1.1, two ion channels, have been considered as potential therapeutic targets for malignant glioma treatment. TRPC6, a Ca2+-permeable channel, plays a vital role in promoting tumorigenesis and the progression of glioma. KCa1.1, a large-conductance Ca2+-activated channel, is also involved in growth and migration of glioma. However, the underlying mechanism by which these two ion channels promote glioma progression was unclear. In our study, we found that TRPC6 upregulated the expression of KCa1.1, while the immunoprecipitation analysis also showed that TRPC6 interacts with KCa1.1 channels in glioma cells. The currents of KCa1.1 recorded by the whole-cell patch clamp technique were increased by TRPC6 in glioma cells, suggesting that TRPC6 can provide a Ca2+ source for the activation of KCa1.1 channels. It was also suggested that TRPC6 regulates the proliferation and apoptosis of glioma cells through KCa1.1 channels in vitro. Therefore, C6-bearing glioma rats were established to validate the results in vitro. After the administration of paxilline (a specific inhibitor of KCa1.1 channels), TRPC6-dependent growth of glioma was inhibited in vivo. We also found that TRPC6 enhanced co-expression with KCa1.1 in glioma. These all suggested that TRPC6/KCa1.1 signal plays a role in promoting the growth of glioma. Our results provided new evidence for TRPC6 and KCa1.1 as potential targets for glioma treatment.

Moderate-intensity exercise training reduces vasorelaxation of mesenteric arteries: role of BKCa channels and nitric oxide.

Exercise training (ET) is well established to induce vascular adaptations on the metabolically active muscles. These adaptations include increased function of vascular potassium channels and enhanced endothelium-dependent relaxations. However, the available data on the effect of ET on vasculatures that normally constrict during exercise, such as mesenteric arteries (MA), are scarce and not conclusive. Therefore, this study hypothesized that 10 weeks of moderate-intensity ET would result in adaptations towards more vasoconstriction or/and less vasodilatation of MA. Young Fischer 344 rats were randomly assigned to a sedentary group (SED; n=24) or exercise training group (EXE; n=28). The EXE rats underwent a progressive treadmill ET program for 10 weeks. Isometric tensions of small (SED; 252.9+/-29.5 microm, EXE; 248.6+/-34.4 microm) and large (SED; 397.7+/-85.3 microm, EXE; 414.0+/-86.95 microm) MA were recorded in response to cumulative phenylephrine concentrations (PE; 0-30 microM) in the presence and absence of the BKCa channel blocker, Iberiotoxin (100 nM). In another set of experiments, tensions in response to cumulative concentration-response curves of acetylcholine (ACh) or sodium nitroprusside (SNP) were obtained, and pEC50s were compared. Immunoblotting was performed to measure protein expression levels of the BKCa channel subunits and eNOS. ET did not alter the basal tension of small and large MA but significantly increased their responses to PE, and reduced the effect of BKCa channels in opposing the contractile responses to PE without changes in the protein expression level of BKCa subunits. ET also elicited a size-dependent functional adaptations that involved reduced endothelium-independent and endothelium-dependent relaxations. In large MA the sensitivity to SNP was decreased more than in small MA suggesting impaired nitric oxide (NO)-dependent mechanisms within the vascular smooth muscle cells of ET group. Whereas the shift in pEC50 of ACh-induced relaxation of small MA would suggest more effect on the production of NO within the endothelium, which is not changed in large MA of ET group. However, the eNOS protein expression level was not significantly changed between the ET and SED groups. In conclusion, our results indicate an increase in contraction and reduced relaxation of MA after 10 weeks of ET, an adaptation that may help shunt blood flow to metabolically active tissues during acute exercise.

The LRRC family of BK channel regulatory subunits: potential roles in health and disease.

Large conductance K+ channels, termed BK channels, regulate a variety of cellular and physiological functions. Although universally activated by changes in voltage or [Ca2+ ]i , the threshold for BK channel activation varies among loci of expression, often arising from cell-specific regulatory subunits including a family of leucine rich repeat-containing (LRRC) γ subunits (LRRC26, LRRC52, LRRC55 and LRRC38). The 'founding' member of this family, LRRC26, was originally identified as a tumour suppressor in various cancers. An LRRC26 knockout (KO) mouse model recently revealed that LRRC26 is also highly expressed in secretory epithelial cells and partners with BK channels in the salivary gland and colonic goblet cells to promote sustained K+ fluxes likely essential for normal secretory function. To accomplish this, LRRC26 negatively shifts the range of BK channel activation such that channels contribute to K+ flux near typical epithelial cell resting conditions. In colon, the absence of LRRC26 increases vulnerability to colitis. LRRC26-containing BK channels are also likely important regulators of epithelial function in other loci, including airways, female reproductive tract and mammary gland. Based on an LRRC52 KO mouse model, LRRC52 regulation of large conductance K+ channels plays a role both in sperm function and in cochlear inner hair cells. Although our understanding of LRRC-containing BK channels remains rudimentary, KO mouse models may help define other organs in which LRRC-containing channels support normal function. A key topic for future work concerns identification of endogenous mechanisms, whether post-translational or via gene regulation, that may impact LRRC-dependent pathologies.

Cholesterol Inhibition of Slo1 Channels Is Calcium-Dependent and Can Be Mediated by Either High-Affinity Calcium-Sensing Site in the Slo1 Cytosolic Tail.

Calcium- and voltage-gated K+ channels of large conductance (BKs) are expressed in the cell membranes of all excitable tissues. Currents mediated by BK channel-forming slo1 homotetramers are consistently inhibited by increases in membrane cholesterol (CLR). The molecular mechanisms leading to this CLR action, however, remain unknown. Slo1 channels are activated by increases in calcium (Ca2+) nearby Ca2+-recognition sites in the slo1 cytosolic tail: one high-affinity and one low-affinity site locate to the regulator of conductance for K+ (RCK) 1 domain, whereas another high-affinity site locates within the RCK2 domain. Here, we first evaluated the crosstalking between Ca2+ and CLR on the function of slo1 (cbv1 isoform) channels reconstituted into planar lipid bilayers. CLR robustly reduced channel open probability while barely decreasing unitary current amplitude, with CLR maximal effects being observed at 10-30 µM internal Ca2+ CLR actions were not only modulated by internal Ca2+ levels but also disappeared in absence of this divalent. Moreover, in absence of Ca2+, BK channel-activating concentrations of magnesium (10 mM) did not support CLR action. Next, we evaluated CLR actions on channels where the different Ca2+-sensing sites present in the slo1 cytosolic domain became nonfunctional via mutagenesis. CLR still reduced the activity of low-affinity Ca2+ (RCK1:E379A, E404A) mutants. In contrast, CLR became inefficacious when both high-affinity Ca2+ sites were mutated (RCK1:D367A,D372A and RCK2:D899N,D900N,D901N,D902N,D903N), yet still was able to decrease the activity of each high-affinity site mutant. Therefore, BK channel inhibition by CLR selectively requires optimal levels of Ca2+ being recognized by either of the slo1 high-affinity Ca2+-sensing sites. SIGNIFICANCE STATEMENT: Results reveal that inhibition of calcium/voltage-gated K+ channel of large conductance (BK) (slo1) channels by membrane cholesterol requires a physiologically range of internal calcium (Ca2+) and is selectively linked to the two high-affinity Ca2+-sensing sites located in the cytosolic tail domain, which underscores that Ca2+ and cholesterol actions are allosterically coupled to the channel gate. Cholesterol modification of BK channel activity likely contributes to disruption of normal physiology by common health conditions that are triggered by disruption of cholesterol homeostasis.

Electromagnetic energy (670 nm) stimulates vasodilation through activation of the large conductance potassium channel (BKCa).

INTRODUCTION: Peripheral artery disease (PAD) is a highly morbid condition in which impaired blood flow to the limbs leads to pain and tissue loss. Previously we identified 670 nm electromagnetic energy (R/NIR) to increase nitric oxide levels in cells and tissue. NO elicits relaxation of smooth muscle (SMC) by stimulating potassium efflux and membrane hyperpolarization. The actions of energy on ion channel activity have yet to be explored. Here we hypothesized R/NIR stimulates vasodilation through activation of potassium channels in SMC. METHODS: Femoral arteries or facial arteries from C57Bl/6 and Slo1-/- mice were isolated, pressurized to 60 mmHg, pre-constricted with U46619, and irradiated twice with energy R/NIR (10 mW/cm2 for 5 min) with a 10 min dark period between irradiations. Single-channel K+ currents were recorded at room temperature from cell-attached and excised inside-out membrane patches of freshly isolated mouse femoral arterial muscle cells using the patch-clamp technique. RESULTS: R/NIR stimulated vasodilation requires functional activation of the large conductance potassium channels. There is a voltage dependent outward current in SMC with light stimulation, which is due to increases in the open state probability of channel opening. R/NIR modulation of channel opening is eliminated pharmacologically (paxilline) and genetically (BKca α subunit knockout). There is no direct action of light to modulate channel activity as excised patches did not increase the open state probability of channel opening. CONCLUSION: R/NIR vasodilation requires indirect activation of the BKca channel.

Neonatal Diabetes in Patients Affected by Liang-Wang Syndrome Carrying KCNMA1 Variant p.(Gly375Arg) Suggest a Potential Role of Ca2+ and Voltage-Activated K+ Channel Activity in Human Insulin Secretion.

Liang-Wang syndrome (LIWAS) is a polymalformative syndrome first described in 2019 caused by heterozygous mutation of the KCNMA1 gene encoding the Ca2+ and voltage-activated K+ channel (BKC). The KCNMA1 variant p.(Gly356Arg) abolishes the function of BKC and blocks the generation of K+ current. The phenotype of this variant includes developmental delay, and visceral and connective tissue malformations. So far, only three cases of LWAS have been described, one of which also had neonatal diabetes (ND). We present the case of a newborn affected by LIWAS carrying the p.(Gly375Arg) variant who manifested diabetes in the first week of life. The description of our case strongly increases the frequency of ND in LIWAS patients and suggests a role of BK inactivation in human insulin secretion. The knowledge on the role of BKC in insulin secretion is very poor. Analyzing the possible mechanisms that could explain the association of LIWAS with ND, we speculate that BK inactivation might impair insulin secretion through the alteration of ion-dependent membrane activities and mitochondrial functions in ß-cells, as well as the impaired intra-islet vessel reactivity.