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.

BK K+ channels are critical regulators of neuron and muscle excitability, comprised of a tetramer of pore-forming αsubunits from the KCNMA1 gene and cell- and tissue-selective β subunits (KCNMB1-4). Mutations in KCNMA1 are associated with neurological disorders, including autism. However, little is known about the role of neuronal BK channel β subunits in human neuropathology. The β2 subunit is expressed in central neurons and imparts inactivation to BK channels, as well as altering activation and deactivation gating. In this study, we report the functional effect of G124R, a novel KCNMB2 mutation obtained from whole-exome sequencing of a patient diagnosed with autism spectrum disorder. Residue G124, located in the extracellular loop between TM1 and TM2, is conserved across species, and the G124R missense mutation is predicted deleterious with computational tools. To investigate the pathogenicity potential, BK channels were co-expressed with β2WT and β2G124R subunits in HEK293T cells. BK/β2 currents were assessed from inside-out patches under physiological K+ conditions (140/6 mM K+ and 10 μM Ca2+) during activation and inactivation (voltage-dependence and kinetics). Using β2 subunits lacking inactivation (β2IR) revealed that currents from BK/β2IRG124R channels activated 2-fold faster and deactivated 2-fold slower compared with currents from BK/β2IRWT channels, with no change in the voltage-dependence of activation (V1/2). Despite the changes in the BK channel opening and closing, BK/β2G124R inactivation rates (τinact and τrecovery), and the V1/2 of inactivation, were unaltered compared with BK/β2WT channels under standard steady-state voltage protocols. Action potential-evoked current was also unchanged. Thus, the mutant phenotype suggests the β2G124R TM1-TM2 extracellular loop could regulate BK channel activation and deactivation kinetics. However, additional evidence is needed to validate pathogenicity for this patient-associated variant in KCNMB2.

KCNMA1-linked channelopathy is an emerging neurological disorder characterized by heterogeneous and overlapping combinations of movement disorder, seizure, developmental delay, and intellectual disability. KCNMA1 encodes the BK K+ channel, which contributes to both excitatory and inhibitory neuronal and muscle activity. Understanding the basis of the disorder is an important area of active investigation; however, the rare prevalence has hampered the development of large patient cohorts necessary to establish genotype-phenotype correlations. In this review, we summarize 37 KCNMA1 alleles from 69 patients currently defining the channelopathy and assess key diagnostic and clinical hallmarks. At present, 3 variants are classified as gain-of-function with respect to BK channel activity, 14 loss-of-function, 15 variants of uncertain significance, and putative benign/VUS. Symptoms associated with these variants were curated from patient-provided information and prior publications to define the spectrum of clinical phenotypes. In this newly expanded cohort, seizures showed no differential distribution between patients harboring GOF and LOF variants, while movement disorders segregated by mutation type. Paroxysmal non-kinesigenic dyskinesia was predominantly observed among patients with GOF alleles of the BK channel, although not exclusively so, while additional movement disorders were observed in patients with LOF variants. Neurodevelopmental and structural brain abnormalities were prevalent in patients with LOF mutations. In contrast to mutations, disease-associated KCNMA1 single nucleotide polymorphisms were not predominantly related to neurological phenotypes but covered a wider set of peripheral physiological functions. Together, this review provides additional evidence exploring the genetic and biochemical basis for KCNMA1-linked channelopathy and summarizes the clinical repository of patient symptoms across multiple types of KCNMA1 gene variants.

Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (β1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 μM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca2+- and voltage-dependent K+ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary β1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with β1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca2+ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the β1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary β1 subunit.

BK Ca2+-activated K+ channels are important regulators of membrane excitability. Multiple regulatory mechanisms tailor BK current properties across tissues, such as alternative splicing, posttranslational modifications, and auxiliary subunits. Another potential mechanism for modulating BK channel activity is genetic variation due to single nucleotide polymorphisms (SNPs). The gene encoding the human BK α subunit, KCNMA1, contains hundreds of SNPs. However, the variation in BK channel activity due to SNPs is not well studied. Here, we screened the effects of four SNPs (A138V, C495G, N599D, and R800W) on BK currents in HEK293T cells, selected based on predicted protein pathogenicity or disease linkage. We found that the SNPs C495G and R800W had the largest effects on BK currents, affecting the conductance-voltage relationship across multiple Ca2+ conditions in the context of two BK channel splice variants. In symmetrical K+, C495G shifted the V1/2 to more hyperpolarized potentials (by -15 to -20 mV) and accelerated activation, indicating C495G confers some gain-of-function properties. R800W shifted the V1/2 to more depolarized potentials (+15 to +35 mV) and slowed activation, conferring loss-of-function properties. Moreover, the C495G and R800W effects on current properties were found to persist with posttranslational modifications. In contrast, A138V and N599D had smaller and more variable effects on current properties. Neither application of alkaline phosphatase to patches, which results in increased BK channel activity attributed to channel dephosphorylation, nor bidirectional redox modulations completely abrogated SNP effects on BK currents. Lastly, in physiological K+, C495G increased the amplitude of action potential (AP)-evoked BK currents, while R800W had a more limited effect. However, the introduction of R800W in parallel with the epilepsy-linked mutation D434G (D434G/R800W) decreased the amplitude of AP-evoked BK currents compared with D434G alone. These results suggest that in a physiological context, C495G could increase BK activation, while the effects of the loss-of-function SNP R800W could oppose the gain-of-function effects of an epilepsy-linked mutation. Together, these results implicate naturally occurring human genetic variation as a potential modifier of BK channel activity across a variety of conditions.

The differentiation of fibroblasts into myofibroblasts is critical for the development of fibrotic disorders, including idiopathic pulmonary fibrosis (IPF). Previously, we demonstrated that fibroblasts from patients with IPF exhibit changes in DNA methylation across the genome that contribute to a profibrotic phenotype. One of the top differentially methylated genes identified in our previous study was KCNMB1, which codes for the β subunit of the large-conductance potassium (BK, also known as MaxiK or KCa1.1) channel. Here, we examined how the expression of KCNMB1 differed between IPF fibroblasts and normal cells, and how BK channels affected myofibroblast differentiation. Fibroblasts from patients with IPF exhibited increased expression of KCNMB1, which corresponded to increased DNA methylation within the gene body. Patch-clamp experiments demonstrated that IPF fibroblasts had increased BK channel activity. Knockdown of KCNMB1 attenuated the ability of fibroblasts to contract collagen gels, and this was associated with a loss of α-smooth muscle actin (SMA) expression. Pharmacologic activation of BK channels stimulated α-SMA expression, whereas BK channel inhibitors blocked the upregulation of α-SMA. The ability of BK channels to enhance α-SMA expression was dependent on intracellular calcium, as activation of BK channels resulted in increased levels of intracellular calcium and the effects of BK agonists were abolished when calcium was removed. Together, our findings demonstrate that epigenetic upregulation of KCNMB1 contributes to increased BK channel activity in IPF fibroblasts, and identify a newfound role for BK channels in myofibroblast differentiation.

Primary fluid secretion in secretory epithelia relies on the unidirectional transport of ions and water across a single cell layer. This mechanism requires the asymmetric apico-basal distribution of ion transporters and intracellular Ca2+ signaling. The primary aim of the present study was to verify the localization and the identity of Ca2+-dependent ion channels in acinar cells of the mouse lacrimal gland.
Whole-cell patch-clamp-electrophysiology, spatially localized flash-photolysis of Ca2+ and temporally resolved digital Ca2+-imaging was combined. Immunostaining of enzymatically isolated mouse lacrimal acinar cells was performed.
We show that the Ca2+-dependent K+-conductance is paxilline-sensitive, abundant in the luminal, but negligible in the basal membrane; and co-localizes with Cl--conductance. These data suggest that both Cl- and K+ are secreted into the lumen and thus they account for the high luminal [Cl-] (∼141 mM), but not for the relatively low [K+] (<17 mM) of the primary fluid. Accordingly, these results also imply that K+ must be reabsorbed from the primary tear fluid by the acinar cells. We hypothesized that apically-localized Na+-K+ pumps are responsible for K+-reabsorption. To test this possibility, immunostaining of lacrimal acinar cells was performed using anti-Na+-K+ ATP-ase antibody. We found positive fluorescence signal not only in the basal, but in the apical membrane of acinar cells too.
Based on these results we propose a new primary fluid-secretion model in the lacrimal gland, in which the paracellular pathway of Na+ secretion is supplemented by a transcellular pathway driven by apical Na+-K+ pumps.

The big conductance Ca2+-dependent K+ channel, also known as BK, MaxiK, Slo1, or KCa1.1, is a ligand- and voltage-gated K+ channel. Although structure-function studies of the past decades, involving mutagenesis and electrophysiological measurements, revealed fine details of the mechanism of BK channel gating, the exact molecular details remained unknown until the quaternary structure of the protein has been solved at a resolution of 3.5 Å using cryo-electron microscopy. In this short review, we are going to summarize these results and interpret the gating model of the BK channel in the light of the recent structural results.

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl- efflux and the subsequent paracellular Na+ transport. In this model, the Na+-K+ pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl- transport via basolateral Na+-K+-2Cl- cotransport is generated by the Na+-K+ pump. In addition, the continuous electrochemical gradient for Cl- flow during acinar cell stimulation is maintained by the basolateral K+ efflux. However, using a combination of single-cell electrophysiology and Ca2+-imaging, we demonstrate that photolysis of Ca2+ close to the apical membrane of parotid acinar cells triggered significant K+ current, indicating that a substantial amount of K+ is secreted into the lumen during stimulation. Nevertheless, the K+ content of the primary saliva is relatively low, suggesting that K+ might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na+-K+ pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K+ from and secretes Na+ to the lumen, which can partially supplement the paracellular Na+ pathway.