Phosphatidylinositol 3-phosphate indirectly activates KCa3.1 via 14 amino acids in the carboxy terminus of KCa3.1

KCa3.1 is an intermediate conductance Ca2+-activated K+ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1.     

Novel Phenolic Inhibitors of Small/Intermediate-Conductance Ca2+-Activated K+ Channels,KCa3.1 and KCa2.3

Background

KCa3.1 channels are calcium/calmodulin-regulated voltage-independent K⁺ channels that produce membrane hyperpolarization and shape Ca²⁺-signaling and thereby physiological functions in epithelia, blood vessels, and white and red blood cells. Up-regulation of KCa3.1 is evident in fibrotic and inflamed tissues and some tumors rendering the channel a potential drug target. In the present study, we searched for novel potent small molecule inhibitors of KCa3.1 by testing a series of 20 selected natural and synthetic (poly)phenols, synthetic benzoic acids, and non-steroidal anti-inflammatory drugs (NSAIDs), with known cytoprotective, anti-inflammatory, and/or cytostatic activities.

Methodology/Principal Findings

In electrophysiological experiments, we identified the natural phenols, caffeic acid (EC50 1.3 µM) and resveratrol (EC50 10 µM) as KCa3.1 inhibitors with moderate potency. The phenols, vanillic acid, gallic acid, and hydroxytyrosol had weak or no blocking effects. Out of the NSAIDs, flufenamic acid was moderately potent (EC50 1.6 µM), followed by mesalamine (EC50≥10 µM). The synthetic fluoro-trivanillic ester, 13b ([3,5-bis[(3-fluoro-4-hydroxy-benzoyl)oxymethyl]phenyl]methyl 3-fluoro-4-hydroxy-benzoate), was identified as a potent mixed KCa2/3 channel inhibitor with an EC50 of 19 nM for KCa3.1 and 360 pM for KCa2.3, which affected KCa1.1 and Kv channels only at micromolar concentrations. The KCa3.1/KCa2-activator SKA-31 antagonized the 13b-blockade. In proliferation assays, 13b was not cytotoxic and reduced proliferation of 3T3 fibroblasts as well as caffeic acid. In isometric vessel myography, 13b increased contractions of porcine coronary arteries to serotonin and antagonized endothelium-derived hyperpolarization-mediated vasorelaxation to pharmacological KCa3.1/KCa2.3 activation.

Conclusions/Significance

We identified the natural phenols, caffeic acid and resveratrol, the NSAID, flufenamic acid, and the polyphenol 13b as novel KCa3.1 inhibitors. The high potency of 13b with pan-activity on KCa3.1/KCa2 channels makes 13b a new pharmacological tool to manipulate inflammation and cancer growth through KCa3.1/KCa2 blockade and a promising template for new drug design.     

Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation.     

Pulmonary Hypertension in Wild Type Mice and Animals with Genetic Deficit in KCa2.3 and KCa3.1 Channels

Objective

In vascular biology, endothelial K_Ca2.3 and K_Ca3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of K_Ca2.3 and K_Ca3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of K_Ca2.3 and K_Ca3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension.

Approach and Result

Male wild type and K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The K_Ca3.1^−/−/K_Ca2.3^T/T(+DOX) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially- and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the K_Ca2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the K_Ca2.3 and K_Ca3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of K_Ca2.3 and K_Ca3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype.

Conclusion

Despite the deficits of the K_Ca2.3 and K_Ca3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of K_Ca2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of K_Ca2.3/K_Ca3.1 activators for the treatment of pulmonary hypertension.     

Hydrophobic interactions as key determinants to the KCa3.1 channel closed configuration. An analysis of KCa3.1 mutants constitutively active in zero Ca2+

In this study we present evidence that residue Val282 in the S6 transmembrane segment of the calcium-activated KCa3.1 channel constitutes a key determinant of channel gating. A Gly scan of the S6 transmembrane segment first revealed that the substitutions A279G and V282G cause the channel to become constitutively active in zero Ca2+. Constitutive activity was not observed when residues extending from Cys276 to Ala286, other than Ala279 and Val282, were substituted to Gly. The accessibility of Cys engineered at Val275 deep in the channel cavity was next investigated for the ion-conducting V275C/V282G mutant and closed V275C channel in zero Ca2+ using Ag+ as probe. These experiments demonstrated that internal Ag+ ions have free access to the channel cavity independently of the channel conducting state, arguing against an activation gate located at the S6 segment C-terminal end. Experiments were also conducted where Val282 was substituted by residues differing in size and/or hydrophobicity. We found a strong correlation between constitutive activity in zero Ca2+ and the hydrophobic energy for side chain burial. Single channel recordings showed finally that constitutive activation in zero Ca2+ is better explained by a model where the channel is locked in a low conducting state with a high open probability rather than resulting from a change in the open/closed energy balance that would favor channel openings to a full conducting state in the absence of Ca2+. We conclude that hydrophobic interactions involving Val282 constitute key determinants to KCa3.1 gating by modulating the ion conducting state of the selectivity filter through an effect on the S6 transmembrane segment.     

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia

Endothelial dysfunction is associated with K_Ca3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial K_Ca3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial K_Ca3.1 dysfunction occurs in vessels from women with preeclampsia. We compared K_Ca3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on K_Ca3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial K_Ca3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in K_Ca3.1 current. Plasma from PE induced K_Ca3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced K_Ca3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced K_Ca3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1–NOX2-mediated pathway and downregulates endothelial K_Ca3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests K_Ca3.1as a novel target for patients with preeclampsia.     

KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells

The K_Ca3.1 K⁺ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the K_Ca3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed K_Ca3.1 mRNA and protein, and robust K_Ca3.1 ion currents. K_Ca3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and K_Ca3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective K_Ca3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by K_Ca3.1 blockade. Treatment with K_Ca3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT.     

Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1

The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K(+) channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca(2+)-activated K(+) channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine, or charge reversal mutations results in a rapid degradation (<30 min) of total protein, confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch clamp experiments revealed that this Arg/His mutation had a dramatically reduced open probability (P(o)), relative to wild type channels. Additionally, we demonstrate, using a combination of short hairpin RNA, dominant negative, and co-immunoprecipitation studies, that both KCa3.1 and KCa2.3 are translocated out of the endoplasmic reticulum associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97, and targeted for proteasomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Furthermore, these improperly folded K(+) channels are translocated out of the endoplasmic reticulum in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated, and targeted for proteasomal degradation.     

Pharmacological gating modulation of small- and intermediate-conductance Ca2+-activated K+ channels (KCa2.x and KCa3.1)

This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca²⁺-activated K⁺ channels (K_Ca2 and K_Ca3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite K_Ca channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative K_Ca2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective K_Ca modulators entering into drug development for these indications.     

KCa3.1 upregulation preserves endothelium‐dependent vasorelaxation during aging and oxidative stress

Endothelial oxidative stress develops with aging and reactive oxygen species impair endothelium‐dependent relaxation (EDR) by decreasing nitric oxide (NO) availability. Endothelial K_Ca3.1, which contributes to EDR, is upregulated by H₂O₂. We investigated whether K_Ca3.1 upregulation compensates for diminished EDR to NO during aging‐related oxidative stress. Previous studies identified that the levels of ceramide synthase 5 (CerS5), sphingosine, and sphingosine 1‐phosphate were increased in aged wild‐type and CerS2 mice. In primary mouse aortic endothelial cells (MAECs) from aged wild‐type and CerS2 null mice, superoxide dismutase (SOD) was upregulated, and catalase and glutathione peroxidase 1 (GPX1) were downregulated, when compared to MAECs from young and age‐matched wild‐type mice. Increased H₂O₂ levels induced Fyn and extracellular signal‐regulated kinases (ERKs) phosphorylation and K_Ca3.1 upregulation. Catalase/GPX1 double knockout (catalase^?/?/GPX1^?/?) upregulated K_Ca3.1 in MAECs. NO production was decreased in aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? MAECs. However, K_Ca3.1 activation‐induced, N^G‐nitro‐l ‐arginine‐, and indomethacin‐resistant EDR was increased without a change in acetylcholine‐induced EDR in aortic rings from aged wild‐type, CerS2 null, and catalase^?/?/GPX1^?/? mice. CerS5 transfection or exogenous application of sphingosine or sphingosine 1‐phosphate induced similar changes in levels of the antioxidant enzymes and upregulated K_Ca3.1. Our findings suggest that, during aging‐related oxidative stress, SOD upregulation and downregulation of catalase and GPX1, which occur upon altering the sphingolipid composition or acyl chain length, generate H₂O₂ and thereby upregulate K_Ca3.1 expression and function via a H₂O₂/Fyn‐mediated pathway. Altogether, enhanced K_Ca3.1 activity may compensate for decreased NO signaling during vascular aging.     

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca²⁺-dependent K⁺ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.     

KCa3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma

Background

The Ca²⁺-activated K⁺ channel K_Ca3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K_Ca3.1 modifies experimental asthma in sheep.

Methodology and Principal Findings

Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K_Ca3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling.

Conclusions

These findings suggest that K_Ca3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K_Ca3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.     

The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K_Ca3.1 K⁺ channel-dependent cell processes contribute to IPF pathophysiology.

Methods

K_Ca3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K_Ca3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K_Ca3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).

Results

Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K_Ca3.1 channel mRNA and protein. K_Ca3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K_Ca3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K_Ca3.1 was expressed strongly in IPF tissue. K_Ca3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca²⁺.

Conclusions

K_Ca3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K_Ca3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.     

KCa3.1 and TRPM7 Channels at the Uropod Regulate Migration of Activated Human T Cells

The migration of T lymphocytes is an essential part of the adaptive immune response as T cells circulate around the body to carry out immune surveillance. During the migration process T cells polarize, forming a leading edge at the cell front and a uropod at the cell rear. Our interest was in studying the involvement of ion channels in the migration of activated human T lymphocytes as they modulate intracellular Ca(2+) levels. Ca(2+) is a key regulator of cellular motility. To this purpose, we created protein surfaces made of the bio-polymer PNMP and coated with ICAM-1, ligand of LFA-1. The LFA-1 and ICAM-1 interaction facilitates T cell movement from blood into tissues and it is critical in immune surveillance and inflammation. Activated human T lymphocytes polarized and migrated on ICAM-1 surfaces by random walk with a mean velocity of ～6 μm/min. Confocal microscopy indicated that Kv1.3, CRAC, and TRPM4 channels positioned in the leading-edge, whereas KCa3.1 and TRPM7 channels accumulated in the uropod. The localization of KCa3.1 and TRPM7 at the uropod was associated with oscillations in intracellular Ca(2+) levels that we measured in this cell compartment. Further studies with blockers against Kv1.3 (ShK), KCa3.1 (TRAM-34), CRAC (SKF-96365), TRPM7 (2-APB), and TRPM4 (glibenclamide) indicated that blockade of KCa3.1 and TRPM7, and not Kv1.3, CRAC or TRPM4, inhibits the T cell migration. The involvement of TRPM7 in cell migration was confirmed with siRNAs against TRPM7. Downregulation of TRPM7 significantly reduced the number of migrating T cells and the mean velocity of the migrating T cells. These results indicate that KCa3.1 and TRPM7 selectively localize at the uropod of migrating T lymphocytes and are key components of the T cell migration machinery.     

Modulation of KCa3.1 Channels by Eicosanoids,Omega-3 Fatty Acids,and Molecular Determinants

Background

Cytochrome P450- and ω-hydrolase products (epoxyeicosatrienoic acids (EETs), hydroxyeicosatetraeonic acid (20-HETE)), natural omega-3 fatty acids (ω3), and pentacyclic triterpenes have been proposed to contribute to a wide range of vaso-protective and anti-fibrotic/anti-cancer signaling pathways including the modula-tion of membrane ion channels. Here we studied the modulation of intermediate-conductance Ca²⁺/calmodulin-regulated K⁺ channels (K_Ca3.1) by EETs, 20-HETE, ω3, and pentacyclic triterpenes and the structural requirements of these fatty acids to exert channel blockade.

Methodology/Principal Findings

We studied modulation of cloned human hK_Ca3.1 and the mutant hK_Ca3.1^V275A in HEK-293 cells, of rK_Ca3.1 in aortic endothelial cells, and of mK_Ca3.1 in 3T3-fibroblasts by inside-out and whole-cell patch-clamp experiments, respectively. In inside-out patches, Ca²⁺-activated hK_Ca3.1 were inhibited by the ω3, DHA and α-LA, and the ω6, AA, in the lower µmolar range and with similar potencies. 5,6-EET, 8,9-EET, 5,6-DiHETE, and saturated arachidic acid, had no appreciable effects. In contrast, 14,15-EET, its stable derivative, 14,15-EEZE, and 20-HETE produced channel inhibition. 11,12-EET displayed less inhibitory activity. The K_Ca3.1^V275A mutant channel was insensitive to any of the blocking EETs. Non-blocking 5,6-EET antagonized the inhibition caused by AA and augmented cloned hK_Ca3.1 and rK_Ca3.1 whole-cell currents. Pentacyclic triterpenes did not modulate K_Ca3.1 currents.

Conclusions/Significance

Inhibition of K_Ca3.1 by EETs (14,15-EET), 20-HETE, and ω3 critically depended on the presence of electron double bonds and hydrophobicity within the 10 carbons preceding the carboxyl-head of the molecules. From the physiological perspective, metabolism of AA to non-blocking 5,6,- and 8,9-EET may cause AA-de-blockade and contribute to cellular signal transduction processes influenced by these fatty acids.     

New insights on the voltage dependence of the KCa3.1 channel block by internal TBA

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.     

Protein/Protein Interactions (PDZ) in Proximal Tubules

The Journal of Membrane Biology -