The activation of GABAergic neurons in the hypothalamic tuberomammillary nucleus attenuates sevoflurane and propofol-induced anesthesia in mice.

Background: The histaminergic neurons in the hypothalamic tuberomammillary nucleus (TMN) have been suggested to play a vital role in maintaining a rising state. But the neuronal types of the TMN are in debate and the role of GABAergic neurons remains unclear. Methods: In the present study, we examined the role of TMN GABAergic neurons in general anesthesia using chemogenetics and optogenetics strategies to regulate the activity of TMN GABAergic neurons. Results: The results indicated that either chemogenetic or optogenetic activation of TMN GABAergic neurons in mice decreased the effect of sevoflurane and propofol anesthesia. In contrast, inhibition of the TMN GABAergic neurons facilitates the sevoflurane anesthesia effect. Conclusion: Our results suggest that the activity of TMN GABAergic neurons produces an anti-anesthesia effect in loss of consciousness and analgesia.

Identification of Sodium- and Chloride-Sensitive Sites in the Slack Channel.

The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.

Directed Asymmetric Nickel-Catalyzed Reductive 1,2-Diarylation of Electronically Unactivated Alkenes.

Transition-metal catalyzed intermolecular 1,2-diarylation of electronically unactivated alkenes has emerged as an extensive research topic in organic synthesis. However, most examples are mainly limited to terminal alkenes. Furthermore, transition-metal catalyzed asymmetric 1,2-diarylation of unactivated alkenes still remains unsolved and is a formidable challenge. Herein, we describe a highly efficient directed nickel-catalyzed reductive 1,2-diarylation of unactivated internal alkenes with high diastereoselectivities. More importantly, our further effort towards enantioselective 1,2-diarylation of the unactivated terminal and challenging internal alkenes is achieved, furnishing various polyarylalkanes featuring benzylic stereocenters in high yields and with good to high enantioselectivities and high diastereoselectivities. Interestingly, the generation of cationic Ni-catalyst by adding alkali metal fluoride is the key to increased efficiency of this enantioselective reaction.

Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel.

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate.

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

Effectiveness of five interventions used for prevention of gestational diabetes: A network meta-analysis.

BACKGROUND: Gestational diabetes mellitus (GDM) is associated with short- and long-term health issues for mother and child; preventing these complications is crucially important. This study aimed to perform a systematic review and network meta-analysis of the relationships among 5 interventions used to prevent GDM. MATERIALS AND METHODS: A comprehensive literature search was performed to pool evidence from inception to June 30, 2020. The type of studies was confined to randomized control trials and quasi-randomized control trials published in English investigating the interventions for preventing GDM, including physical activity, dietary intervention, probiotic intervention, mixed intervention, and inositol supplementation. The data were pooled together to report the odds ratio (OR) of GDM with a corresponding 95% credible interval (CrI) and generate a network plot, the surface under the cumulative ranking curve plot, and contribution plot. In addition, loop inconsistency was examined, and a funnel plot combined with Egger test was used to measure heterogeneity. RESULTS: The network meta-analysis included 46 randomized control trials involving 16,545 patients. Compared with placebo, physical activity (OR: 0.64, 95% CrI: 0.46-0.88) and probiotic intervention (OR: 0.57, 95% CrI: 0.34-0.96) reduced the incidence of GDM significantly. However, dietary intervention, a combination of physical activity and diet intervention, and inositol supplementation did not significantly alter GDM risk. CONCLUSIONS: Physical activity and probiotic intervention are more effective than placebo in reducing the risk of developing GDM. Future work should focus on the type, duration, frequency, and timing of physical activity and probiotic intervention.

The Slack Channel Deletion Causes Mechanical Pain Hypersensitivity in Mice.

The role of the Slack (also known as Slo2.2, KNa1.1, or KCNT1) channel in pain-sensing is still in debate on which kind of pain it regulates. In the present study, we found that the Slack-/- mice exhibited decreased mechanical pain threshold but normal heat and cold pain sensitivity. Subsequently, X-gal staining, in situ hybridization, and immunofluorescence staining revealed high expression of the Slack channel in Isolectin B4 positive (IB4+) neurons in the dorsal root ganglion (DRG) and somatostatin-positive (SOM+) neurons in the spinal cord. Patch-clamp recordings indicated the firing frequency was increased in both small neurons in DRG and spinal SOM+ neurons in the Slack-/- mice whereas no obvious slow afterhyperpolarization was observed in both WT mice and Slack-/- mice. Furthermore, we found Kcnt1 gene expression in spinal SOM+ neurons in Slack-/- mice partially relieved the mechanical pain hypersensitivity of Slack-/- mice and decreased AP firing rates of the spinal SOM+ neurons. Finally, deletion of the Slack channel in spinal SOM+ neurons is sufficient to result in mechanical pain hypersensitivity in mice. In summary, our results suggest the important role of the Slack channel in the regulation of mechanical pain-sensing both in small neurons in DRG and SOM+ neurons in the spinal dorsal horn.

The Slack Channel Regulates Anxiety-Like Behaviors via Basolateral Amygdala Glutamatergic Projections to Ventral Hippocampus.

Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. In the present study, we find that the global Slack KO male mice exhibit anxious behaviors, whereas the Slack Y777H male mice manifest anxiolytic behaviors. The expression of Slack channels is rich in basolateral amygdala (BLA) glutamatergic neurons and downregulated in chronic corticosterone-treated mice. In addition, electrophysiological data show enhanced excitability of BLA glutamatergic neurons in the Slack KO mice and decreased excitability of these neurons in the Slack Y777H mice. Furthermore, the Slack channel deletion in BLA glutamatergic neurons is sufficient to result in enhanced avoidance behaviors, whereas Kcnt1 gene expression in the BLA or BLA-ventral hippocampus (vHPC) glutamatergic projections reverses anxious behaviors of the Slack KO mice. Our study identifies the role of the Slack channel in controlling anxious behaviors by decreasing the excitability of BLA-vHPC glutamatergic projections, providing a potential target for anxiolytic therapies.SIGNIFICANCE STATEMENT Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. Here, we examined the behaviors of loss- and gain-of-function of Slack channel mice in elevated plus maze and open field tests and found the anxiolytic role of the Slack channel. By altering the Slack channel expression in the specific neuronal circuit, we demonstrated that the Slack channel played its anxiolytic role by decreasing the excitability of BLA-vHPC glutamatergic projections. Our data reveal the role of the Slack channel in the regulation of anxiety, which may provide a potential molecular target for anxiolytic therapies.

Efficacy of continuous positive airway pressure in neonates with respiratory distress syndrome: A protocol for systematic review and meta-analysis.

BACKGROUND: Respiratory distress syndrome (RDS) is a condition caused by a deficiency in pulmonary surfactant. Many interventions, including pulmonary surfactant, non-invasive respiratory support, and other supportive treatments have been used to prevent RDS. However, recent studies have focused on the continuous positive airway pressure as a significant potential agent for preventing RDS. However, its safety and effectiveness are yet to be assessed. To this end, the current study aims to perform to explore the safety and effectiveness of continuous positive airways in treating neonates with RDS. METHODS: We will conduct comprehensive literature searches on MEDLINE, Cochrane Library, EMBASE, Chinese National Knowledge Infrastructure, and Chinese BioMedical Literature from their inception to April 2021. The search aims to identify all the randomized controlled studies on continuous positive airway pressure in treating neonates with RDS. In addition, we aim to search the gray literature to establish any available potential studies. We will use 2 independent authors to determine study eligibility, extract data using the structured pro-forma table, analyze data, and utilize suitable tools in assessing the risk of bias in the selected studies. Accordingly, we will conduct all statistical analyses using RevMan 5.3 software. RESULTS: The current study aims to provide high-quality synthesis of existing evidence concerning the continuous positive airway pressure to treat neonates suffering from RDS. CONCLUSION: Our findings seek to provide evidence to establish whether continuous positive airway pressure can ascertain safety and effectiveness for neonates with RDS. ETHICS AND DISSEMINATION: The study will require ethical approval. OSF REGISTRATION NUMBER: May 20, 2021.osf.io/7nj8s. (https://osf.io/7nj8s/).

Evaluation of efficiency and safety of combined montelukast sodium and budesonide in children with cough variant asthma: A protocol for systematic review and meta-analysis.

BACKGROUND: Cough variant asthma (CVA) is classified as a distinct form of asthma. As the primary or only symptom, cough is the leading cause for the most prevalent chronic cough among kids. The American College of Clinical Pharmacy, British Thoracic Society, and Chinese guidelines established for diagnosing and treating chronic cough in kids recommend inhaled corticosteroids, combined with leukotriene receptor antagonists when necessary. METHODS: We will conduct a comprehensive search in major databases using keywords to find studies related to the analysis of montelukast sodium and budesonide for treating CVA in kids. Two reviewers will independently assess the quality of the selected research articles and perform data extraction. Next, we will use the RevMan software (version: 5.3) to conduct the statistical analysis of the present study. RESULTS: This study will assess the efficacy and safeness of using montelukast sodium and budesonide to treat kids with CVA by pooling the results of individual studies. CONCLUSION: Our findings will provide vigorous evidence to judge whether montelukast sodium and budesonide therapy is an efficient form of therapy for CVA patients. ETHICS AND DISSEMINATION: Ethics approval is not needed for the present meta-analysis. OSF REGISTRATION NUMBER: May 17, 2021.osf.io/cuvjz (https://osf.io/cuvjz/).

Cloning and characterization of the rat Slo3 (KCa 5.1) channel: From biophysics to pharmacology.

BACKGROUND AND PURPOSE: The Slo3 potassium (KCa 5.1) channel, which is specifically expressed in the testis and sperm, is essential for mammalian male fertilization. The sequence divergence of the bovine, mouse and human Slo3 α-subunit revealed a rapid evolution rate across different species. The rat Slo3 (rSlo3) channel has not been cloned and characterized previously. EXPERIMENTAL APPROACH: We used molecular cloning, electrophysiology (inside-out patches and outside-out patches) and mutagenesis to investigate the biophysical properties and pharmacological characteristics of the rSlo3 channel. KEY RESULTS: The rat Slo3 channel (rSlo3) is gated by voltage and cytosolic pH rather than intracellular calcium. The characteristics of voltage-dependent, pH-sensitivity and activation kinetics of the rSlo3 channel differ from the characteristics of other Slo3 orthologues. In terms of pharmacology, the 4-AP blockade of the rSlo3 channel also shows properties distinct from its blockade of the mSlo3 channel. Iberiotoxin and progesterone weakly inhibit the rSlo3 channel. Finally, we found that propofol, one of the widely used general anaesthetics, blocks the rSlo3 channel from both intracellular and extracellular sides, whereas ketamine only blocks the rSlo3 channel at the extracellular side. CONCLUSION AND IMPLICATIONS: Our findings suggest that the rSlo3 channel possesses unique biophysical and pharmacological properties. Our results provide new insights into the diversities of the Slo3 family of channels, which are valuable for estimating the effects of the use of these drugs to improve sperm quality.

The neuropeptide GsMTx4 inhibits a mechanosensitive BK channel through the voltage-dependent modification specific to mechano-gating.

The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.

Up-regulation of antioxidative proteins TRX1, TXNL1 and TXNRD1 in the cortex of PTZ kindling seizure model mice.

Oxidative stress has been considered as one of pathogenesis of brain damage led by epilepsy. Reducing oxidative stress can ameliorate brain damage during seizures. However, expression levels of important antioxidative enzymes such as thioredoxin-1 (TRX1), thioredoxin-like 1 protein (TXNL1) and thioredoxin reductase 1 (TXNRD1) during seizures have not been investigated. In this study, we examined protein and mRNA expression levels of TRX1, TXNL1 and TXNRD1 in different brain regions in PTZ induced seizure model mice. We found that protein expression levels of TRX1, TXNL1 and TXNRD1 are simultaneously up-regulated by 2- or 3-fold in the cortex of both acute and chronic seizure model mice. But there is no unified expression pattern change of these enzymes in the hippocampus, cerebellum and diencephalon in the seizure model mice. Less extent up-regulation of mRNA expression of these enzymes were also observed in the cortex of seizure mice. These data suggest that antioxidative enzymes may provide a protective effect against oxidative stress in the cortex during seizures.

Preparation of micro-electrolysis material from flotation waste of copper slag and its application for degradation of organic contaminants in water.

Flotation waste of copper slag (FWCS) was used as a raw material for the preparation of a micro-electrolysis material (MEM) through a carbothermal reduction process. The performance of MEM was evaluated for the degradation of organic contaminants in water. The effects of preparation conditions on the performance of MEM were investigated. Results showed that the MEM prepared under the conditions of calcination temperature of 1100 °C, calcination time of 60 min, and coal dosage of 25% presented the best performance for degrading methyl orange (MO). The decolorization process was enhanced by increasing the MEM dosage, decreasing the initial pH of the solution, and raising the solution temperature. Moreover, the MEM presented good capability for the degradation of methylene blue, eosin Y, and acid fuchsin. X-ray diffraction (XRD) analysis showed that increasing the roasting temperature was beneficial to the formation of zero-valent iron (ZVI). Scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) showed that micro-sized ZVI particles were formed in the MEM, and they contained a small amount of copper element. Meanwhile, the mechanism analysis showed that a redox reaction of the MEM and MO occurred, the azo bond of MO was destroyed, and sulfanilic acid was generated.

Regulatory Effect of General Anesthetics on Activity of Potassium Channels.

General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian nervous system have been investigated during past decades. In recent years, K+ channels have been identified as important targets of both volatile and intravenous anesthetics. This review covers achievements that have been made both on the regulatory effect of general anesthetics on the activity of K+ channels and their underlying mechanisms. Advances in research on the modulation of K+ channels by general anesthetics are summarized and categorized according to four large K+ channel families based on their amino-acid sequence homology. In addition, research achievements on the roles of K+ channels in general anesthesia in vivo, especially with regard to studies using mice with K+ channel knockout, are particularly emphasized.

Correction: +mRNA expression of LRRC55 protein (leucine-rich repeat-containing protein 55) in the adult mouse brain.

[This corrects the article DOI: 10.1371/journal.pone.0191749.].

+mRNA expression of LRRC55 protein (leucine-rich repeat-containing protein 55) in the adult mouse brain.

LRRC55 (leucine-rich repeat-containing protein 55) protein is an auxiliary γ subunit of BK (Big conductance potassium channel) channels, which leftward shifts GVs of BK channels around 50 mV in the absence of cytosolic Ca2+. LRRC55 protein is also the only γ subunit of BK channels that is expressed in mammalian nervous system. However, the expression pattern of LRRC55 gene in adult mammalian brain remains elusive. In this study, we investigated the distribution of LRRC55 mRNA in the adult mouse brain by using in situ hybridization. We found that LRRC55 mRNA is richly expressed in the adult mouse medial habenula nucleus (MHb), cerebellum and pons. However, the potential role of LRRC55 in MHb and cerebellum could be different based on the function of BK channels in these brain regions.

Allosteric-activation mechanism of BK channel gating ring triggered by calcium ions.

Calcium ions bind at the gating ring which triggers the gating of BK channels. However, the allosteric mechanism by which Ca2+ regulates the gating of BK channels remains obscure. Here, we applied Molecular Dynamics (MD) and Targeted MD to the integrated gating ring of BK channels, and achieved the transition from the closed state to a half-open state. Our date show that the distances of the diagonal subunits increase from 41.0 Å at closed state to 45.7Å or 46.4 Å at a half-open state. It is the rotatory motion and flower-opening like motion of the gating rings which are thought to pull the bundle crossing gate to open ultimately. Compared with the 'Ca2+ bowl' at RCK2, the RCK1 Ca2+ sites make more contribution to opening the channel. The allosteric motions of the gating ring are regulated by three group of interactions. The first weakened group is thought to stabilize the close state; the second strengthened group is thought to stabilize the open state; the third group thought to lead AC region forming the CTD pore to coordinated motion, which exquisitely regulates the conformational changes during the opening of BK channels by Ca2+.

Epilepsy-Related Slack Channel Mutants Lead to Channel Over-Activity by Two Different Mechanisms.

Twelve sodium-activated potassium channel (KCNT1, Slack) genetic mutants have been identified from severe early-onset epilepsy patients. The changes in biophysical properties of these mutants and the underlying mechanisms causing disease remain elusive. Here, we report that seven of the 12 mutations increase, whereas one mutation decreases, the channel's sodium sensitivity. Two of the mutants exhibit channel over-activity only when the intracellular Na(+) ([Na(+)]i) concentration is ∼80 mM. In contrast, single-channel data reveal that all 12 mutants increase the maximal open probability (Po). We conclude that these mutant channels lead to channel over-activity predominantly by increasing the ability of sodium binding to activate the channel, which is indicated by its maximal Po. The sodium sensitivity of these epilepsy causing mutants probably determines the [Na(+)]i concentration at which these mutants exert their pathological effects.

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels.

All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification.