Subunit-specific inhibition of BK channels by piperine.

Piperine is the principal alkaloid present in black pepper and is well-known for its diverse pharmacological effects, including inhibition of different ion channels. Large conductance Ca2+-activated K+ channels (BK) are widely expressed across several tissues and play a vital role in many physiological functions. In this study, we investigated the pharmacological effects of piperine on various BK channel subunit compositions (BKα, BKαß1,4, BKαγ1,3) expressed in HEK293T cells. Piperine in zero Ca2+ reversibly inhibited currents from the pore-forming BKα channels in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) of 4.8 µM. Elevating the internal Ca2+ concentration from 0 to 100 µM significantly attenuated the inhibitory effects of piperine on BKα channels. The mutation G311S in the pore domain failed to alter the modulatory effects of piperine, whereas deletion of the entire cytoplasmic domain from BKα channels ablated its inhibitory effects. Addition of either BKß1 or ß4 regulatory subunits did not alter the efficacy of piperine on BKα channels. Interestingly, co-expression of either BKγ1 or BKγ3 subunits greatly diminished the ability of piperine to inhibit BKα channels. Our findings demonstrate that piperine is a potent natural modulator of BKα/BKαß1,4 subunits but not BKαγ1,3 subunits. The mechanism of piperine modulation appeared to be allosteric and differs from that of other BK pore blockers (paxilline, peptide toxins, and quaternary ammonium compounds). Together, our results unravel the potential of piperine to inhibit BK channels, providing a new tool to explore mechanisms underlying the effects of regulatory subunits.

Effects of Chronic Kidney Disease on Nanomechanics of the Endothelial Glycocalyx Are Mediated by the Mineralocorticoid Receptor.

Endothelial mechanics control vascular reactivity and are regulated by the mineralocorticoid receptor (MR) and its downstream target, the epithelial Na+ channel (ENaC). Endothelial dysfunction is a hallmark of chronic kidney disease (CKD), but its mechanisms are poorly understood. We hypothesized that CKD disrupts endothelial mechanics in an MR/ENaC-dependent process. METHODS: Primary human endothelial cells were cultured with uremic serum derived from children with stage 3-5 (predialysis) CKD or adult hemodialysis (HD) patients or healthy controls. The height and stiffness of the endothelial glycocalyx (eGC) and cortex were monitored by atomic force microscopy (AFM) using an ultrasensitive mechanical nanosensor. RESULTS: In a stage-dependent manner, sera from children with CKD induced a significant increase in eGC and cortex stiffness and an incremental reduction of the eGC height. AFM measurements were significantly associated with individual pulse wave velocity and serum concentrations of gut-derived uremic toxins. Serum from HD patients increased MR expression and mechanical stiffness of the endothelial cortex, an effect reversed by MR and ENaC antagonists, decreased eNOS expression and NO bioavailability, and augmented monocyte adhesion. CONCLUSION: These data indicate progressive structural damage of the endothelial surface with diminishing kidney function and identify the MR as a mediator of CKD-induced endothelial dysfunction.

SGK1.1 isoform is involved in nociceptive modulation, offering a protective effect against noxious cold stimulus in a sexually dimorphic manner.

The serum and glucocorticoid-regulated kinase 1 (SGK1) is a widely expressed protein in the Central Nervous System (CNS), involved in regulating the activity of a wide variety of ion channels and transporters and physiological functions, such as neuronal excitability. SGK1.1 is a neuronal splice isoform of SGK1, expressed exclusively in the CNS, distributed in brain and cerebellum, that decreases neuronal excitability via up-regulation of M-current, linked to Kv7.2/3 potassium channels. Strategies to maintain increased SGK1.1 activity could be helpful in decreasing neuronal hyperexcitability, as occurs in neuropathic pain. Transgenic mice overexpressing SGK1.1 (B6.Tg.sgk1) offer a particularly relevant opportunity to assess the physiological involvement of this protein in nociception. Behavior and physiological nociception were evaluated in male and female B6.Tg.sgk1 and wild-type mice (B6.WT), characterizing nociceptive thresholds to different nociceptive stimuli (thermal, chemical and mechanical), as well as the electrophysiological properties of cutaneous sensory Aδ-fibres isolated from the saphenous nerve. The acute antinociceptive effect of morphine was also evaluated. Compared with B6.WT animals, male and female B6.Tg.sgk1 mice showed increased spontaneous locomotor activity. Regarding nociception, there were no differences between transgenic and wild-type mice in heat, chemical and mechanical thresholds, but interestingly, male B6.Tg.sgk1 mice were less sensitive to cold stimulus; B6.Tg.sgk1 animals showed lower sensitivity to morphine. Electrophysiological properties of cutaneous primary afferent fibres were maintained. This is the first demonstration that the SGK1.1 isoform is involved in nociceptive modulation, offering a protective effect against noxious cold stimulus in a sexually dimorphic manner. B6.Tg.sgk1 mice offer a particularly relevant opportunity to further analyze the involvement of this protein in nociception, and studies in models of chronic, neuropathic pain are warranted.

Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations.

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1.

NMDA receptor-BK channel coupling regulates synaptic plasticity in the barrel cortex.

Postsynaptic N-methyl-D-aspartate receptors (NMDARs) are crucial mediators of synaptic plasticity due to their ability to act as coincidence detectors of presynaptic and postsynaptic neuronal activity. However, NMDARs exist within the molecular context of a variety of postsynaptic signaling proteins, which can fine-tune their function. Here, we describe a form of NMDAR suppression by large-conductance Ca2+- and voltage-gated K+ (BK) channels in the basal dendrites of a subset of barrel cortex layer 5 pyramidal neurons. We show that NMDAR activation increases intracellular Ca2+ in the vicinity of BK channels, thus activating K+ efflux and strong negative feedback inhibition. We further show that neurons exhibiting such NMDAR-BK coupling serve as high-pass filters for incoming synaptic inputs, precluding the induction of spike timing-dependent plasticity. Together, these data suggest that NMDAR-localized BK channels regulate synaptic integration and provide input-specific synaptic diversity to a thalamocortical circuit.

SGK1.1 limits brain damage after status epilepticus through M current-dependent and independent mechanisms.

Epilepsy is a neurological condition associated to significant brain damage produced by status epilepticus (SE) including neurodegeneration, gliosis and ectopic neurogenesis. Reduction of these processes constitutes a useful strategy to improve recovery and ameliorate negative outcomes after an initial insult. SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), has been shown to increase M-current density in neurons, leading to reduced excitability and protection against seizures. For this study, we used 4-5 months old male transgenic C57BL/6 J and FVB/NJ mice expressing near physiological levels of a constitutively active form of the kinase controlled by its endogenous promoter. Here we show that SGK1.1 activation potently reduces levels of neuronal death (assessed using Fluoro-Jade C staining) and reactive glial activation (reported by GFAP and Iba-1 markers) in limbic regions and cortex, 72 h after SE induced by kainate, even in the context of high seizure activity. This neuroprotective effect is not exclusively through M-current activation but is also directly linked to decreased apoptosis levels assessed by TUNEL assays and quantification of Bim and Bcl-xL by western blot of hippocampal protein extracts. Our results demonstrate that this newly described antiapoptotic role of SGK1.1 activation acts synergistically with the regulation of cellular excitability, resulting in a significant reduction of SE-induced brain damage in areas relevant to epileptogenesis.

Kv1.3 Channel Inhibition Limits Uremia-Induced Calcification in Mouse and Human Vascular Smooth Muscle.

Chronic kidney disease (CKD) significantly increases cardiovascular risk. In advanced CKD stages, accumulation of toxic circulating metabolites and mineral metabolism alterations triggers vascular calcification, characterized by vascular smooth muscle cell (VSMC) transdifferentiation and loss of the contractile phenotype. Phenotypic modulation of VSMC occurs with significant changes in gene expression. Even though ion channels are an integral component of VSMC function, the effects of uremia on ion channel remodeling has not been explored. We used an in vitro model of uremia-induced calcification of human aorta smooth muscle cells (HASMCs) to study the expression of 92 ion channel subunit genes. Uremic serum-induced extensive remodeling of ion channel expression consistent with loss of excitability but different from the one previously associated with transition from contractile to proliferative phenotypes. Among the ion channels tested, we found increased abundance and activity of voltage-dependent K+ channel Kv1.3. Enhanced Kv1.3 expression was also detected in aorta from a mouse model of CKD. Pharmacological inhibition or genetic ablation of Kv1.3 decreased the amount of calcium phosphate deposition induced by uremia, supporting an important role for this channel on uremia-induced VSMC calcification.

Differential regulation of BK channels by fragile X mental retardation protein.

Fragile X mental retardation protein (FMRP) is an RNA-binding protein prominently expressed in neurons. Missense mutations or complete loss of FMRP can potentially lead to fragile X syndrome, a common form of inherited intellectual disability. In addition to RNA regulation, FMRP was also proposed to modulate neuronal function by direct interaction with the large conductance Ca2+- and voltage-activated potassium channel (BK) ß4 regulatory subunits (BKß4). However, the molecular mechanisms underlying FMRP regulation of BK channels were not studied in detail. We have used electrophysiology and super-resolution stochastic optical reconstruction microscopy (STORM) to characterize the effects of FMRP on pore-forming BKα subunits, as well as the association with regulatory subunits BKß4. Our data indicate that, in the absence of coexpressed ß4, FMRP alters the steady-state properties of BKα channels by decreasing channel activation and deactivation rates. Analysis using the Horrigan-Aldrich model revealed alterations in the parameters associated with channel opening (L0) and voltage sensor activation (J0). Interestingly, FMRP also altered the biophysical properties of BKαß4 channels favoring channel opening, although not as dramatically as BKα. STORM experiments revealed clustered multi-protein complexes, consistent with FMRP interacting not only to BKαß4 but also to BKα. Lastly, we found that a partial loss-of-function mutation in FMRP (R138Q) counteracts many of its functional effects on BKα and BKαß4 channels. In summary, our data show that FMRP modulates the function of both BKα and BKαß4 channels.

SGK1.1 Reduces Kainic Acid-Induced Seizure Severity and Leads to Rapid Termination of Seizures.

Approaches to control epilepsy, one of the most important idiopathic brain disorders, are of great importance for public health. We have previously shown that in sympathetic neurons the neuronal isoform of the serum and glucocorticoid-regulated kinase (SGK1.1) increases the M-current, a well-known target for seizure control. The effect of SGK1.1 activation on kainate-induced seizures and neuronal excitability was studied in transgenic mice that express a permanently active form of the kinase, using electroencephalogram recordings and electrophysiological measurements in hippocampal brain slices. Our results demonstrate that SGK1.1 activation leads to reduced seizure severity and lower mortality rates following status epilepticus, in an M-current-dependent manner. EEG is characterized by reduced number, shorter duration, and early termination of kainate-induced seizures in the hippocampus and cortex. Hippocampal neurons show decreased excitability associated to increased M-current, without altering basal synaptic transmission or other neuronal properties. Altogether, our results reveal a novel and selective anticonvulsant pathway that promptly terminates seizures, suggesting that SGK1.1 activation can be a potent factor to secure the brain against permanent neuronal damage associated to epilepsy.

Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor.

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

Physiological Roles and Therapeutic Potential of Ca2+ Activated Potassium Channels in the Nervous System.

Within the potassium ion channel family, calcium activated potassium (KCa) channels are unique in their ability to couple intracellular Ca2+ signals to membrane potential variations. KCa channels are diversely distributed throughout the central nervous system and play fundamental roles ranging from regulating neuronal excitability to controlling neurotransmitter release. The physiological versatility of KCa channels is enhanced by alternative splicing and co-assembly with auxiliary subunits, leading to fundamental differences in distribution, subunit composition and pharmacological profiles. Thus, understanding specific KCa channels' mechanisms in neuronal function is challenging. Based on their single channel conductance, KCa channels are divided into three subtypes: small (SK, 4-14 pS), intermediate (IK, 32-39 pS) and big potassium (BK, 200-300 pS) channels. This review describes the biophysical characteristics of these KCa channels, as well as their physiological roles and pathological implications. In addition, we also discuss the current pharmacological strategies and challenges to target KCa channels for the treatment of various neurological and psychiatric disorders.

Phenotypic Modulation of Cultured Primary Human Aortic Vascular Smooth Muscle Cells by Uremic Serum.

Patients with chronic kidney disease (CKD) have a markedly increased incidence of cardiovascular disease (CVD). The high concentration of circulating uremic toxins and alterations in mineral metabolism and hormone levels produce vascular wall remodeling and significant vascular damage. Medial calcification is an early vascular event in CKD patients and is associated to apoptosis or necrosis and trans-differentiation of vascular smooth muscle cells (VSMC) to an osteogenic phenotype. VSMC obtained from bovine or rat aorta and cultured in the presence of increased inorganic phosphate (Pi) have been extensively used to study these processes. In this study we used human aortic VSMC primary cultures to compare the effects of increased Pi to treatment with serum obtained from uremic patients. Uremic serum induced calcification, trans-differentiation and phenotypic remodeling even with normal Pi levels. In spite of similar calcification kinetics, there were fundamental differences in osteochondrogenic marker expression and alkaline phosphatase induction between Pi and uremic serum-treated cells. Moreover, high Pi induced a dramatic decrease in cell viability, while uremic serum preserved it. In summary, our data suggests that primary cultures of human VSMC treated with serum from uremic patients provides a more informative model for the study of vascular calcification secondary to CKD.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Functional effects of proinflammatory factors present in Sjögren's syndrome salivary microenvironment in an in vitro model of human salivary gland.

Primary Sjögren's syndrome (pSS) is an autoimmune exocrinopathy in which the role that the immune response plays in reducing exocrine gland function, including the glandular microenvironment of cytokines, has not been fully understood. Epithelial cells from biopsies of human parotid gland (HPG) were used to establish a model of human salivary gland in vitro. In this model, the functional consequences of several proinflammatory soluble factors present in the pSS glandular microenvironment were assessed. Stimulation with isoproterenol and calcium produced a significant increase in the basal activity of amylase in the HPG cell supernatants. Under these conditions, the presence of TNF-α and CXCL12 increased amylase mRNA cellular abundance, but reduced the amylase activity in the cell-free supernatant in a dose-dependent manner. IL-1ß and IFN-γ, but not TGF-ß, also diminished amylase secretion by HPG cells. These results suggest that the glandular microenvironment of cytokine, by acting post-transcriptionally, may be responsible, at least in part, for the reduced exocrine function observed in pSS patients. These data may help to a better understanding of the pathogenesis of SS, which in turn would facilitate the identification of new therapeutic targets for this disorder.

Understanding the conformational motions of RCK gating rings.

Regulator of conduction of K+ (RCK) domains are ubiquitous regulators of channel and transporter activity in prokaryotes and eukaryotes. In humans, RCK domains form an integral component of large-conductance calcium-activated K channels (BK channels), key modulators of nerve, muscle, and endocrine cell function. In this review, we explore how the study of RCK domains in bacterial and human channels has contributed to our understanding of the structural basis of channel function. This knowledge will be critical in identifying mechanisms that underlie BK channelopathies that lead to epilepsy and other diseases, as well as regions of the channel that might be successfully targeted to treat such diseases.

Interactions of divalent cations with calcium binding sites of BK channels reveal independent motions within the gating ring.

Large-conductance voltage- and calcium-activated K+ (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca2+ and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. In contrast to Ca2+ that binds to both RCK domains, Mg2+, Cd2+, or Ba2+ interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg2+ and Ba2+ selectively induce structural movement of the RCK2 domain, whereas Cd2+ causes motions of RCK1, in all cases substantially smaller than those elicited by Ca2+ By combining divalent species interacting with unique sites, we demonstrate that RCK1 and RCK2 domains move independently when their specific binding sites are occupied. Moreover, binding of chemically distinct cations to both RCK domains is additive, emulating the effect of fully occupied Ca2+ binding sites.

Histone Deacetylase 6-Controlled Hsp90 Acetylation Significantly Alters Mineralocorticoid Receptor Subcellular Dynamics But Not its Transcriptional Activity.

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily that transduces the biological effects of corticosteroids. Its best-characterized role is to enhance transepithelial sodium reabsorption in response to increased aldosterone levels. In addition, MR participates in other aldosterone- or glucocorticoid-controlled processes such as cardiovascular homeostasis, adipocyte differentiation or neurogenesis, and regulation of neuronal activity in the hippocampus. Like other steroid receptors, MR forms cytosolic heterocomplexes with heat shock protein (Hsp) 90), Hsp70, and other proteins such as immunophilins. Interaction with Hsp90 is thought to maintain MR in a ligand-binding competent conformation and to regulate ligand-dependent and -independent nucleocytoplasmatic shuttling. It has previously been shown that acetylation of residue K295 in Hsp90 regulates its interaction with the androgen receptor and glucocorticoid receptor (GR). In this work we hypothesized that Hsp90 acetylation provides a regulatory step to modulate MR cellular dynamics and activity. We used Hsp90 acetylation mimic mutant K295Q or nonacetylatable mutant K295R to examine whether MR nucleocytoplasmatic shuttling and gene transactivation are affected. Furthermore, we manipulated endogenous Hsp90 acetylation levels by controlling expression or activity of histone deacetylase 6 (HDAC6), the enzyme responsible for deacetylation of Hsp90-K295. Our data demonstrates that HDAC6-mediated Hsp90 acetylation regulates MR cellular dynamics but it does not alter its function. This stands in contrast with the down-regulation of GR by HDAC6, suggesting that Hsp90 acetylation may play a role in balancing relative MR and GR activity when both factors are co-expressed in the same cell.

Plasma membrane insertion of epithelial sodium channels occurs with dual kinetics.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for Na(+) transport across electrically tight epithelia. Regulation of ENaC activity is critical for electrolyte and extracellular volume homeostasis, as well as for lung liquid clearance and colon Na(+) handling. ENaC activity is tightly controlled by a combination of mechanisms involving changes in open probability and plasma membrane abundance. The latter reflects a combination in channel biosynthesis and trafficking to and from the membrane. Studying ENaC trafficking with different techniques in a variety of expression systems has yielded inconsistent results, indicating either fast or slow rates of insertion and retrieval, which range from the order of minutes to several hours. Here, we use Xenopus oocytes as ENaC expression system to study channel insertion rate in the membrane using two different techniques under comparable conditions: (1) confocal microscopy coupled to fluorescence recovery after photobleaching (FRAP) measurements; and (2) fluorescent bungarotoxin (BTX) binding to ENaC subunits modified to include BTX binding sites (BBSs) in their extracellular domain, a technique that has not been previously used to study ENaC trafficking. Our confocal-FRAP data indicate a fast rate of ENaC incorporation to the membrane in a process conditioned by channel subunit composition. On the other hand, BTX binding experiments indicate much slower channel insertion rates, with matching slow ENaC retrieval rates. The data support a model that includes fast recycling of endocytosed ENaC with parallel incorporation of newly synthesized channels at a slower rate.