Loss-of-function KCa2.2 mutations abolish channel activity.

Small-conductance Ca2+-activated potassium channels subtype 2 (KCa2.2, also called SK2) are operated exclusively by a Ca2+-calmodulin gating mechanism. Heterozygous genetic mutations of KCa2.2 channels have been associated with autosomal dominant neurodevelopmental disorders including cerebellar ataxia and tremor in humans and rodents. Taking advantage of these pathogenic mutations, we performed structure-function studies of the rat KCa2.2 channel. No measurable current was detected from HEK293 cells heterologously expressing these pathogenic KCa2.2 mutants. When coexpressed with the KCa2.2_WT channel, mutations of the pore-lining amino acid residues (I360M, Y362C, G363S, and I389V) and two proline substitutions (L174P and L433P) dominant negatively suppressed and completely abolished the activity of the coexpressed KCa2.2_WT channel. Coexpression of the KCa2.2_I289N and the KCa2.2_WT channels reduced the apparent Ca2+ sensitivity compared with the KCa2.2_WT channel, which was rescued by a KCa2.2 positive modulator.

KCa 2.2 (KCNN2): A physiologically and therapeutically important potassium channel.

One group of the K+ ion channels, the small-conductance Ca2+ -activated potassium channels (KCa 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+ -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+ . According to the phylogenic research done on the KCa 2.x channels family, there are three channels' subtypes: KCa 2.1, KCa 2.2, and KCa 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa 2.2 modulators are promising potential therapeutics for SCAs.

Development and validation of an ultrahigh-performance liquid chromatography-tandem mass spectrometry method to investigate the plasma pharmacokinetics of a KCa 2.2/KCa 2.3 positive allosteric modulator in mice.

RATIONALE: There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of KCa 2.2/KCa 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently developed positive allosteric modulator of KCa 2.2/KCa 2.3 channels (compound 2q) in mouse plasma. METHODS: Mouse plasma samples (10 µL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C18 ultrahigh-performance liquid chromatography column and detected by a tandem mass spectrometer. The method was validated using US Food and Drug Administration (FDA) guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined. RESULTS: The calibration standards were linear (r2 ≥ 0.99) in the range of 1.56-200 nM of 2q with intra- and inter-run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were >94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at -80°C or the processed samples stored in the autosampler at 10°C were stable for at least 3 weeks and 36 h, respectively. After intravenous injection, 2q showed a bi-exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half-life of 45 min. CONCLUSIONS: The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias.

Channelopathy of small- and intermediate-conductance Ca2+-activated K+ channels.

Small- and intermediate-conductance Ca2+-activated K+ (KCa2.x/KCa3.1 also called SK/IK) channels are gated exclusively by intracellular Ca2+. The Ca2+ binding protein calmodulin confers sub-micromolar Ca2+ sensitivity to the channel-calmodulin complex. The calmodulin C-lobe is constitutively associated with the proximal C-terminus of the channel. Interactions between calmodulin N-lobe and the channel S4-S5 linker are Ca2+-dependent, which subsequently trigger conformational changes in the channel pore and open the gate. KCNN genes encode four subtypes, including KCNN1 for KCa2.1 (SK1), KCNN2 for KCa2.2 (SK2), KCNN3 for KCa2.3 (SK3), and KCNN4 for KCa3.1 (IK). The three KCa2.x channel subtypes are expressed in the central nervous system and the heart. The KCa3.1 subtype is expressed in the erythrocytes and the lymphocytes, among other peripheral tissues. The impact of dysfunctional KCa2.x/KCa3.1 channels on human health has not been well documented. Human loss-of-function KCa2.2 mutations have been linked with neurodevelopmental disorders. Human gain-of-function mutations that increase the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels have been associated with Zimmermann-Laband syndrome and hereditary xerocytosis, respectively. This review article discusses the physiological significance of KCa2.x/KCa3.1 channels, the pathophysiology of the diseases linked with KCa2.x/KCa3.1 mutations, the structure-function relationship of the mutant KCa2.x/KCa3.1 channels, and potential pharmacological therapeutics for the KCa2.x/KCa3.1 channelopathy.

Spatial ensemble modeling for predicting the potential distribution of Lymantria dispar asiatica (Lepidoptera: Erebidae: Lymantriinae) in South Korea.

The spongy moth, Lymantria dispar, is a pest that damages various tree species throughout North America and Eurasia, has recently emerged in South Korea, threatening local forests and landscapes. The establishment of effective countermeasures against this species' outbreak requires predicting its potential distribution with climate change. In this study, we used species distribution models (CLIMEX and MaxEnt) to predict the potential distribution of the spongy moth and identify areas at risk of exposure to a sustained occurrence of the pest by constructing an ensemble map that simultaneously projected the outcomes of the two models. The results showed that the spongy moth could be distributed over the entire country under the current climate, but the number of suitable areas would decrease under a climate change scenario. This study is expected to provide basic data that can predict areas requiring intensive control and monitoring in advance with methodologically improved modeling technique.

Crystal Structure and Substrate Recognition of Cellobionic Acid Phosphorylase, Which Plays a Key Role in Oxidative Cellulose Degradation by Microbes.

The microbial oxidative cellulose degradation system is attracting significant research attention after the recent discovery of lytic polysaccharide mono-oxygenases. A primary product of the oxidative and hydrolytic cellulose degradation system is cellobionic acid (CbA), the aldonic acid form of cellobiose. We previously demonstrated that the intracellular enzyme belonging to glycoside hydrolase family 94 from cellulolytic fungus and bacterium is cellobionic acid phosphorylase (CBAP), which catalyzes reversible phosphorolysis of CbA into glucose 1-phosphate and gluconic acid (GlcA). In this report, we describe the biochemical characterization and the three-dimensional structure of CBAP from the marine cellulolytic bacterium Saccharophagus degradans. Structures of ligand-free and complex forms with CbA, GlcA, and a synthetic disaccharide product from glucuronic acid were determined at resolutions of up to 1.6 Å. The active site is located near the dimer interface. At subsite +1, the carboxylate group of GlcA and CbA is recognized by Arg-609 and Lys-613. Additionally, one residue from the neighboring protomer (Gln-190) is involved in the carboxylate recognition of GlcA. A mutational analysis indicated that these residues are critical for the binding and catalysis of the aldonic and uronic acid acceptors GlcA and glucuronic acid. Structural and sequence comparisons with other glycoside hydrolase family 94 phosphorylases revealed that CBAPs have a unique subsite +1 with a distinct amino acid residue conservation pattern at this site. This study provides molecular insight into the energetically efficient metabolic pathway of oxidized sugars that links the oxidative cellulolytic pathway to the glycolytic and pentose phosphate pathways in cellulolytic microbes.

Open-close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase.

Infant gut-associated bifidobacteria possess a metabolic pathway to utilize lacto-N-biose (Gal-ß1,3-GlcNAc) and galacto-N-biose (Gal-ß1,3-GalNAc) from human milk and glycoconjugates specifically. In this pathway, N-acetylhexosamine 1-kinase (NahK) catalyzes the phosphorylation of GlcNAc or GalNAc at the anomeric C1 position with ATP. Crystal structures of NahK have only been determined in the closed state. In this study, we determined open state structures of NahK in three different forms (apo, ADP complex, and ATP complex). A comparison of the open and closed state structures revealed an induced fit structural change defined by two rigid domains. ATP binds to the small N-terminal domain, and binding of the N-acetylhexosamine substrate to the large C-terminal domain induces a closing conformational change with a rotation angle of 16°. In the nucleotide binding site, two magnesium ions bridging the α-γ and ß-γ phosphates were identified. A mutational analysis indicated that a residue coordinating both of the two magnesium ions (Asp228) is essential for catalysis. The involvement of two magnesium ions in the catalytic machinery is structurally similar to the catalytic structures of protein kinases and aminoglycoside phosphotransferases, but distinct from the structures of other anomeric kinases or sugar 6-kinases. These findings help to elucidate the possible evolutionary adaptation of substrate specificities and induced fit mechanism.

A Model for Predicting Spring Emergence of Monochamus saltuarius (Coleoptera: Cerambycidae) from Korean white pine, Pinus koraiensis.

Monochamus saltuarius Gebler is a vector that transmits the pine wood nematode, Bursaphelenchus xylophilus, to Korean white pine, Pinus koraiensis, in Korea. To reduce the damage caused by this nematode in pine forests, timely control measures are needed to suppress the cerambycid beetle population. This study sought to construct a forecasting model to predict beetle emergence based on spring temperature. Logs of Korean white pine were infested with M. saltuarius in 2009, and the infested logs were overwintered. In February 2010, infested logs were then moved into incubators held at constant temperature conditions of 16, 20, 23, 25, 27, 30 or 34°C until all adults had emerged. The developmental rate of the beetles was estimated by linear and nonlinear equations and a forecasting model for emergence of the beetle was constructed by pooling data based on normalized developmental rate. The lower threshold temperature for development was 8.3°C. The forecasting model relatively well predicted the emergence pattern of M. saltuarius collected from four areas in northern Republic of Korea. The median emergence dates predicted by the model were 2.2-5.9 d earlier than the observed median dates.

Sulfolobus tokodaii ST2133 is characterized as a thioredoxin reductase-like ferredoxin:NADP+ oxidoreductase.

The putative gene (st2133) for ferredoxin:NADP(+) oxidoreductase (FNR) from Sulfolobus tokodaii, a thermoacidophilic crenarchaeon, was heterologously expressed. About 90% of the purified product was a homodimer containing 0.46 mol FAD/mol subunit, and showing NADPH:DCPIP oxidoreductase activity, V max being 1.38 and 21.8 U/mg (70 °C) in the absence and presence of 1 mM FMN. NADPH was a much better electron donor than NADH with various electron acceptors, such as oxygen, hydrogen peroxide, DCPIP, cytochrome c, and dithiobisnitrobenzoate. Most of the reactions were activated by 15- to 140-fold on addition of FMN, while FAD was 5-10 times less effective. Ferredoxin (Fd) from S. tokodaii served as an electron carrier in both Fd-dependent NADPH formation and NADPH-dependent Fd reduction. ST2133 belongs to the thioredoxin reductase-like protein family, which is slightly distantly related to FNR family proteins from bacteria, plants and man. This is the first report on FNR from a crenarchaeon, providing a clue to the recycling of Fd during archaeal metabolism.

Crystal structures of glycoside hydrolase family 3 ß-glucosidase 1 from Aspergillus aculeatus.

GH3 (glycoside hydrolase family 3) BGLs (ß-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

An empirical predictive model for the spring emergence of Thecodiplosis japonensis (Diptera: Cecidomyiidae): model construction and validation on the basis of 25 years of field observation data.

The reliability of the empirical phenology model proposed by Nam et al. (2013) was tested using spring emergence data of Thecodiplosis japonensis Uchida et Inouye (Diptera: Cecidomyiidae) from 1986 to 2011 in Yeongcheon, Korea. First, the lower developmental threshold temperature (LDT) was estimated using spring emergence of T. japonensis and field temperatures. Then a degree-day model to predict spring emergence of T. japonensis was constructed, based on LDTs estimated from field observations and laboratory temperature-dependent development data. Based on field observations, the estimated LDT value for postdiapause development of T. japonensis was 6.1 degrees C, only 0.2 degrees C higher than that estimated from laboratory data. The explanatory power of the empirical degree-day model constructed on the basis of LDT from field observations was 87.2%, similar to the 87.0% of a degree-day model based on laboratory data, suggesting that both models had similar explanatory power. Furthermore, the predictions of median date of emergence in both models were only five days earlier than the observed median date in 2011. These results show that the empirical phenology model based on field observations is a reliable method for predicting the phenology of insects.

Temperature-Dependent Oviposition Models for Monochamus saltuarius (Coleoptera: Cerambycidae).

Monochamus saltuarius Gebler is a serious insect pest in Europe and East Asia regions, including Portugal, Spain, China, Japan, and Korea. It transfers the pine wood nematode Bursaphelenchus xylophilus to conifer trees, resulting in pine wilt disease (PWD). As temperature is a key factor influencing insect population dynamics, temperature-dependent models describing M. saltuarius oviposition could estimate population growth potential and evaluate outbreak risks. In this study, the longevity and fecundity of M. saltuarius females were measured under constant temperature conditions ranging from 20 to 32 °C, and temperature-dependent models were constructed. The longevity of M. saltuarius females ranged from 83.36 days to 22.92 days, with a total fecundity of 141 eggs and 52.77 eggs at 20 °C and 32 °C, respectively. To describe oviposition, we used a single-phase simulation describing oviposition as a single model and a two-phase simulation describing sexual maturation and oviposition as two separate models. These models effectively described M. saltuarius oviposition (r2 > 0.96) under constant temperature conditions, with the two-phase simulation demonstrating greater accuracy overall. Such models could facilitate assessments of PWD risks. The modeling framework of this study shows potential for predicting threats from various forestry and agricultural pests.

Oxidative stress and ion channels in neurodegenerative diseases.

Numerous neurodegenerative diseases result from altered ion channel function and mutations. The intracellular redox status can significantly alter the gating characteristics of ion channels. Abundant neurodegenerative diseases associated with oxidative stress have been documented, including Parkinson's, Alzheimer's, spinocerebellar ataxia, amyotrophic lateral sclerosis, and Huntington's disease. Reactive oxygen and nitrogen species compounds trigger posttranslational alterations that target specific sites within the subunits responsible for channel assembly. These alterations include the adjustment of cysteine residues through redox reactions induced by reactive oxygen species (ROS), nitration, and S-nitrosylation assisted by nitric oxide of tyrosine residues through peroxynitrite. Several ion channels have been directly investigated for their functional responses to oxidizing agents and oxidative stress. This review primarily explores the relationship and potential links between oxidative stress and ion channels in neurodegenerative conditions, such as cerebellar ataxias and Parkinson's disease. The potential correlation between oxidative stress and ion channels could hold promise for developing innovative therapies for common neurodegenerative diseases.

Deep learning-based optic disc classification is affected by optic-disc tilt.

We aimed to determine the effect of optic disc tilt on deep learning-based optic disc classification. A total of 2507 fundus photographs were acquired from 2236 eyes of 1809 subjects (mean age of 46 years; 53% men). Among all photographs, 1010 (40.3%) had tilted optic discs. Image annotation was performed to label pathologic changes of the optic disc (normal, glaucomatous optic disc changes, disc swelling, and disc pallor). Deep learning-based classification modeling was implemented to develop optic-disc appearance classification models with the photographs of all subjects and those with and without tilted optic discs. Regardless of deep learning algorithms, the classification models showed better overall performance when developed based on data from subjects with non-tilted discs (AUC, 0.988 ± 0.002, 0.991 ± 0.003, and 0.986 ± 0.003 for VGG16, VGG19, and DenseNet121, respectively) than when developed based on data with tilted discs (AUC, 0.924 ± 0.046, 0.928 ± 0.017, and 0.935 ± 0.008). In classification of each pathologic change, non-tilted disc models had better sensitivity and specificity than the tilted disc models. The optic disc appearance classification models developed based all-subject data demonstrated lower accuracy in patients with the appearance of tilted discs than in those with non-tilted discs. Our findings suggested the need to identify and adjust for the effect of optic disc tilt on the optic disc classification algorithm in future development.

Extended genetic effects of ADH cluster genes on the risk of alcohol dependence: from GWAS to replication.

Alcohol dependence (AD) is a multifactorial and polygenic disorder involving complex gene-to-gene and gene-to-environment interactions. Several genome-wide association studies have reported numerous risk factors for AD, but replication results following these studies have been controversial. To identify new candidate genes, the present study used GWAS and replication studies in a Korean cohort with AD. Genome-wide association analysis revealed that two chromosome regions on Chr. 4q22-q23 (ADH gene cluster, including ADH5, ADH4, ADH6, ADH1A, ADH1B, and ADH7) and Chr. 12q24 (ALDH2) showed multiple association signals for the risk of AD. To investigate detailed genetic effects of these ADH genes on AD, a follow-up study of the ADH gene cluster on 4q22-q23 was performed. A total of 90 SNPs, including ADH1B rs1229984 (H47R), were genotyped in an additional 975 Korean subjects. In case-control analysis, ADH1B rs1229984 (H47R) showed the most significant association with the risk of AD (p = 2.63 × 10(-21), OR = 2.35). Moreover, subsequent conditional analyses revealed that all positive associations of other ADH genes in the cluster disappeared, which suggested that ADH1B rs1229984 (H47R) might be the sole functional genetic marker across the ADH gene cluster. Our findings could provide additional information on the ADH gene cluster regarding the risk of AD, as well as a new and important insight into the genetic factors associated with AD.

Spatial analysis of the occurrence of the western conifer seed bug Leptoglossus occidentalis (Heteroptera: Coreidae) in Europe based on multiple environmental variables.

The western conifer seed bug (WCSB) Leptoglossus occidentalis (Heidemann) (Heteroptera: Coreidae) is a pest insect that causes significant losses of coniferous trees worldwide. In this study, we sought to project the potential distribution of the WCSB based on dual CLIMEX modeling and random forest (RF) analysis to obtain basic data for WCSB monitoring strategies. The CLIMEX model, a semimechanistic niche model that responds to climate-based environmental parameters, is a species distribution model that focuses on regional climatic suitability. Given that this model can be used to select areas that are likely to reflect the climatically favorable spread of species, which we initially used CLIMEX to evaluate the potential distribution of the WCSB. The RF algorithm was used to predict the potential occurrence of WCSB and to evaluate the relative importance of environmental variables for WCSB occurrence. Using the RF model, land cover was found to be the most important variable for classifying the presence/pseudo-absence of the WCSB, with an accuracy of 77.1%. Climatic suitability for the WCSB was predicted to be 2.4-fold higher in Southern Europe than in Western Europe, and the WCSB was predicted to occur primarily near coniferous forests. Given that CLIMEX and RF analyses yielded different prediction results, using the findings of both models may compensate for the shortcomings of these models when used independently. Consequently, to ensure greater prediction reliability, we believe that it would be beneficial to base predictions on the combined potential distribution data obtained using both modeling approaches.

KCa2 and KCa3.1 Channels in the Airways: A New Therapeutic Target.

K+ channels are involved in many critical functions in lung physiology. Recently, the family of Ca2+-activated K+ channels (KCa) has received more attention, and a massive amount of effort has been devoted to developing selective medications targeting these channels. Within the family of KCa channels, three small-conductance Ca2+-activated K+ (KCa2) channel subtypes, together with the intermediate-conductance KCa3.1 channel, are voltage-independent K+ channels, and they mediate Ca2+-induced membrane hyperpolarization. Many KCa2 channel members are involved in crucial roles in physiological and pathological systems throughout the body. In this article, different subtypes of KCa2 and KCa3.1 channels and their functions in respiratory diseases are discussed. Additionally, the pharmacology of the KCa2 and KCa3.1 channels and the link between these channels and respiratory ciliary regulations will be explained in more detail. In the future, specific modulators for small or intermediate Ca2+-activated K+ channels may offer a unique therapeutic opportunity to treat muco-obstructive lung diseases.

Channelopathy-causing mutations in the S45A/S45B and HA/HB helices of KCa2.3 and KCa3.1 channels alter their apparent Ca2+ sensitivity.

Small- and intermediate-conductance Ca2+-activated potassium (KCa2.x and KCa3.1, also called SK and IK) channels are activated exclusively by a Ca2+-calmodulin gating mechanism. Wild-type KCa2.3 channels have a Ca2+ EC50 value of ∼0.3 µM, while the apparent Ca2+ sensitivity of wild-type KCa3.1 channels is ∼0.27 µM. Heterozygous genetic mutations of KCa2.3 channels have been associated with Zimmermann-Laband syndrome and idiopathic noncirrhotic portal hypertension, while KCa3.1 channel mutations were reported in hereditary xerocytosis patients. KCa2.3_S436C and KCa2.3_V450L channels with mutations in the S45A/S45B helices exhibited hypersensitivity to Ca2+. The corresponding mutations in KCa3.1 channels also elevated the apparent Ca2+ sensitivity. KCa3.1_S314P, KCa3.1_A322V and KCa3.1_R352H channels with mutations in the HA/HB helices are hypersensitive to Ca2+, whereas KCa2.3 channels with the equivalent mutations are not. The different effects of the equivalent mutations in the HA/HB helices on the apparent Ca2+ sensitivity of KCa2.3 and KCa3.1 channels may imply distinct modulation of the two channel subtypes by the HA/HB helices. AP14145 reduced the apparent Ca2+ sensitivity of the hypersensitive mutant KCa2.3 channels, suggesting the potential therapeutic usefulness of negative gating modulators.

Subtype-selective positive modulation of KCa 2 channels depends on the HA/HB helices.

BACKGROUND AND PURPOSE: In the activated state of small-conductance Ca2+ -activated potassium (KCa 2) channels, calmodulin interacts with the HA/HB helices and the S4-S5 linker. CyPPA potentiates KCa 2.2a and KCa 2.3 channel activity but not the KCa 2.1 and KCa 3.1 subtypes. EXPERIMENTAL APPROACH: Site-directed mutagenesis, patch-clamp recordings and in silico modelling were utilised to explore the structural determinants for the subtype-selective modulation of KCa 2 channels by CyPPA. KEY RESULTS: Mutating residues in the HA (V420) and HB (K467) helices of KCa 2.2a channels to their equivalent residues in KCa 3.1 channels diminished the potency of CyPPA. CyPPA elicited prominent responses on mutant KCa 3.1 channels with an arginine residue in the HB helix substituted for its equivalent lysine residue in the KCa 2.2a channels (R355K). KCa 2.1 channels harbouring a three-amino-acid insertion upstream of the cognate R438 residues in the HB helix showed no response to CyPPA, whereas the deletion mutant (KCa 2.1_ΔA434/Q435/K436) became sensitive to CyPPA. In molecular dynamics simulations, CyPPA docked between calmodulin C-lobe and the HA/HB helices widens the cytoplasmic gate of KCa 2.2a channels. CONCLUSION AND IMPLICATIONS: Selectivity of CyPPA among KCa 2 and KCa 3.1 channel subtypes relies on the HA/HB helices.

Subtype-Selective Positive Modulation of KCa2.3 Channels Increases Cilia Length.

Small-conductance Ca2+-activated potassium (KCa2.x) channels are gated exclusively by intracellular Ca2+. The activation of KCa2.3 channels induces hyperpolarization, which augments Ca2+ signaling in endothelial cells. Cilia are specialized Ca2+ signaling compartments. Here, we identified compound 4 that potentiates human KCa2.3 channels selectively. The subtype selectivity of compound 4 for human KCa2.3 over rat KCa2.2a channels relies on an isoleucine residue in the HA/HB helices. Positive modulation of KCa2.3 channels by compound 4 increased flow-induced Ca2+ signaling and cilia length, while negative modulation by AP14145 reduced flow-induced Ca2+ signaling and cilia length. These findings were corroborated by the increased cilia length due to the expression of Ca2+-hypersensitive KCa2.3_G351D mutant channels and the reduced cilia length resulting from the expression of Ca2+-hyposensitive KCa2.3_I438N channels. Collectively, we were able to associate functions of KCa2.3 channels and cilia, two crucial components in the flow-induced Ca2+ signaling of endothelial cells, with potential implications in vasodilation and ciliopathic hypertension.