Kir Channel Family Research Articles

Cloning, functional expression, and pharmacological characterization of inwardly rectifying potassium channels (Kir) from Apis mellifera

Potassium channels belong to the super family of ion channels and play a fundamental role in cell excitability. Kir channels are potassium channels with an inwardly rectifying property. They play a role in setting the resting membrane potential of many excitable cells including neurons. Although putative Kir channel family genes can be found in the Apis mellifera genome, their functional expression, biophysical properties, and sensitivity to small molecules with insecticidal activity remain to be investigated. We cloned six Kir channel isoforms from Apis mellifera that derive from two Kir genes, AmKir1 and AmKir2, which are present in the Apis mellifera genome. We studied the tissue distribution, the electrophysiological and pharmacological characteristics of three isoforms that expressed functional currents (AmKir1.1, AmKir2.2, and AmKir2.3). AmKir1.1, AmKir2.2, and AmKir2.3 isoforms exhibited distinct characteristics when expressed in Xenopus oocytes. AmKir1.1 exhibited the largest potassium currents and was impermeable to cesium whereas AmKir2.2 and AmKir2.3 exhibited smaller currents but allowed cesium to permeate. AmKir1 exhibited faster opening kinetics than AmKir2. Pharmacological experiments revealed that both AmKir1.1 and AmKir2.2 are blocked by the divalent ion barium, with IC50 values of 10−5 and 10−6 M, respectively. The concentrations of VU041, a small molecule with insecticidal properties required to achieve a 50% current blockade for all three channels were higher than those needed to block Kir channels in other arthropods, such as the aphid Aphis gossypii and the mosquito Aedes aegypti. From this, we conclude that Apis mellifera AmKir channels exhibit lower sensitivity to VU041.

Insights into Activation Dynamics and Functional Sites of Inwardly Rectifying Potassium Channel Kir3.2 by an Elastic Network Model Combined with Perturbation Methods.

The inwardly rectifying potassium channel Kir3.2, a member of the inward rectifier potassium (Kir) channel family, exerts important biological functions through transporting potassium ions outside of the cell, during which a large-scale synergistic movement occurs among its different domains. Currently, it is not fully understood how the binding of the ligand to the Kir3.2 channel leads to the structural changes and which key residues are responsible for the channel gating and allosteric dynamics. Here, we construct the Gaussian network model (GNM) of the Kir3.2 channel with the secondary structure and covalent interaction information considered (sscGNM), which shows a better performance in reproducing the channel's flexibility compared with the traditional GNM. In addition, the sscANM-based perturbation method is used to simulate the channel's conformational transition caused by the activator PIP2's binding. By applying certain forces to the PIP2 binding pocket, the coarse-grained calculations generate the similar conformational changes to the experimental observation, suggesting that the topology structure as well as PIP2 binding are crucial to the allosteric activation of the Kir3.2 channel. We also utilize the sscGNM-based thermodynamic cycle method developed by us to identify the key residues whose mutations significantly alter the channel's binding free energy with PIP2. We identify not only the residues important for the specific binding but also the ones critical for the allosteric transition coupled with PIP2 binding. This study is helpful for understanding the working mechanism of Kir3.2 channels and can provide important information for related drug design.

Discovery and Characterization of VU0542270, the First Selective Inhibitor of Vascular Kir6.1/SUR2B KATP Channels.

Vascular smooth muscle KATP channels critically regulate blood flow and blood pressure by modulating vascular tone and therefore represent attractive drug targets for treating several cardiovascular disorders. However, the lack of potent inhibitors that can selectively inhibit Kir6.1/SUR2B (vascular KATP) over Kir6.2/SUR1 (pancreatic KATP) has eluded discovery despite decades of intensive research. We therefore screened 47,872 chemically diverse compounds for novel inhibitors of heterologously expressed Kir6.1/SUR2B channels. The most potent inhibitor identified in the screen was an N-aryl-N'-benzyl urea compound termed VU0542270. VU0542270 inhibits Kir6.1/SUR2B with an IC50 of approximately 100 nM but has no apparent activity toward Kir6.2/SUR1 or several other members of the Kir channel family at doses up to 30 µM (>300-fold selectivity). By expressing different combinations of Kir6.1 or Kir6.2 with SUR1, SUR2A, or SUR2B, the VU0542270 binding site was localized to SUR2. Initial structure-activity relationship exploration around VU0542270 revealed basic texture related to structural elements that are required for Kir6.1/SUR2B inhibition. Analysis of the pharmacokinetic properties of VU0542270 showed that it has a short in vivo half-life due to extensive metabolism. In pressure myography experiments on isolated mouse ductus arteriosus vessels, VU0542270 induced ductus arteriosus constriction in a dose-dependent manner similar to that of the nonspecific KATP channel inhibitor glibenclamide. The discovery of VU0542270 provides conceptual proof that SUR2-specific KATP channel inhibitors can be developed using a molecular target-based approach and offers hope for developing cardiovascular therapeutics targeting Kir6.1/SUR2B. SIGNIFICANCE STATEMENT: Small-molecule inhibitors of vascular smooth muscle KATP channels might represent novel therapeutics for patent ductus arteriosus, migraine headache, and sepsis; however, the lack of selective channel inhibitors has slowed progress in these therapeutic areas. Here, this study describes the discovery and characterization of the first vascular-specific KATP channel inhibitor, VU0542270.

The dynamics of the electrotactic reaction of mouse 3T3 fibroblasts

The molecular mechanisms behind electrotaxis remain largely unknown, with no identified primary direct current electric field (dcEF) sensor. Two leading hypotheses propose mechanisms involving the redistribution of charged components in the cell membrane (driven by electrophoresis or electroosmosis) and the asymmetric activation of ion channels. To investigate these mechanisms, we studied the dynamics of electrotactic behaviour of mouse 3T3 fibroblasts.We observed that 3T3 fibroblasts exhibit cathodal migration within just 1 min when exposed to physiological dcEF. This rapid response suggests the involvement of ion channels in the cell membrane. Our large-scale screening method identified several ion channel genes as potential key players, including the inwardly rectifying potassium channel Kir4.2. Blocking the Kir channel family with Ba2+ or silencing the Kcnj15 gene, encoding Kir4.2, significantly reduced the directional migration of 3T3 cells. Additionally, the levels of the intracellular regulators of Kir channels, spermine (SPM) and spermidine (SPD), had a significant impact on cell directionality. Interestingly, inhibiting Kir4.2 resulted in the temporary cessation of electrotaxis for approximately 1–2 h before its return. This observation suggests a two-phase mechanism for the electrotaxis of mouse 3T3 fibroblasts, where ion channel activation triggers the initial rapid response to dcEF, and the subsequent redistribution of membrane receptors sustains long-term directional movement.In summary, our study unveils the involvement of Kir channels and proposes a biphasic mechanism to explain the electrotactic behaviour of mouse 3T3 fibroblasts, shedding light on the molecular underpinnings of electrotaxis.

Kir5.1 channels: potential role in epilepsy and seizure disorders.

Inwardly rectifying potassium (Kir) channels are broadly expressed in many mammalian organ systems, where they contribute to critical physiological functions. However, the importance and function of the Kir5.1 channel (encoded by the KCNJ16 gene) have not been fully recognized. This review focuses on the recent advances in understanding the expression patterns and functional roles of Kir5.1 channels in fundamental physiological systems vital to potassium homeostasis and neurological disorders. Recent studies have described the role of Kir5.1-forming Kir channels in mouse and rat lines with mutations in the Kcnj16 gene. The animal research reveals distinct renal and neurological phenotypes, including pH and electrolyte imbalances, blunted ventilatory responses to hypercapnia/hypoxia, and seizure disorders. Furthermore, it was confirmed that these phenotypes are reminiscent of those in patient cohorts in which mutations in the KCNJ16 gene have also been identified, further suggesting a critical role for Kir5.1 channels in homeostatic/neural systems health and disease. Future studies that focus on the many functional roles of these channels, expanded genetic screening in human patients, and the development of selective small-molecule inhibitors for Kir5.1 channels, will continue to increase our understanding of this unique Kir channel family member.

SUMOylation of Kir7.1 participates in neuropathic pain through regulating its membrane expression in spinal cord neurons.

AimsPotassium (K+) channels have been demonstrated to play a prominent involvement in nociceptive processing. Kir7.1, the newest members of the Kir channel family, has not been extensively studied in the CNS, and its function remains largely unknown. The present study investigated the role of spinal Kir7.1 in the development of pathological pain.Methods and ResultsNeuropathic pain was induced by spared nerve injury (SNI). The mechanical sensitivity was assessed by von Frey test. Immunofluorescence staining assay revealed that Kir7.1 was predominantly expressed in spinal neurons but not astrocytes or microglia in normal rats. Western blot results showed that SNI markedly decreased the total and membrane expression of Kir7.1 in the spinal dorsal horn accompanied by mechanical hypersensitivity. Blocking Kir7.1 with the specific antagonist ML418 or knockdown kir7.1 by siRNA led to mechanical allodynia. Co‐IP results showed that the spinal kir7.1 channels were decorated by SUMO‐1 but not SUMO‐2/3, and Kir7.1 SUMOylation was upregulated following SNI. Moreover, inhibited SUMOylation by GA (E1 inhibitor) or 2‐D08 (UBC9 inhibitor) can increase the spinal surface Kir7.1 expression.ConclusionSUMOylation of the Kir7.1 in the spinal cord might contribute to the development of SNI‐induced mechanical allodynia by decreasing the Kir7.1 surface expression in rats.

Functional characterization of the inward-rectifier potassium (Kir) channel family in bone cells

Functional characterization of the inward-rectifier potassium (Kir) channel family in bone cells

Structural Determinants Mediating Tertiapin Block of Neuronal Kir3.2 Channels.

Tertiapin (TPN) is a 21 amino acid venom peptide from Apis mellifera that inhibits certain members of the inward rectifier potassium (Kir) channel family at a nanomolar affinity with limited specificity. Structure-based computational simulations predict that TPN behaves as a pore blocker; however, the molecular determinants mediating block of neuronal Kir3 channels have been inconclusive and unvalidated. Here, using molecular docking and molecular dynamics (MD) simulations with 'potential of mean force' (PMF) calculations, we investigated the energetically most favored interaction of TPN with several Kir3.x channel structures. The resulting binding model for Kir3.2-TPN complexes was then tested by targeted mutagenesis of the predicted contact sites, and their impact on the functional channel block was measured electrophysiologically. Together, our findings indicate that a high-affinity TPN block of Kir3.2 channels involves a pore-inserting lysine side chain requiring (1) hydrophobic interactions at a phenylalanine ring surrounding the channel pore and (2) electrostatic interactions with two adjacent Kir3.2 turret regions. Together, these interactions collectively stabilize high-affinity toxin binding to the Kir3.2 outer vestibule, which orients the ε-amino group of TPN-K21 to occupy the outermost K+ binding site of the selectivity filter. The structural determinants for the TPN block described here also revealed a favored subunit arrangement for assembled Kir3.x heteromeric channels, in addition to a multimodal binding capacity of TPN variants consistent with the functional dyad model for polybasic peptide pore blockers. These novel findings will aid efforts in re-engineering the TPN pharmacophore to develop peptide variants having unique and distinct Kir channel blocking properties.

G protein-coupled and ATP-sensitive inwardly rectifying potassium ion channels are essential for HIV entry

The high genetic diversity of Human Immunodeficiency virus (HIV), has hindered the development of effective vaccines or antiviral drugs against it. Hence, there is a continuous need for identification of new antiviral targets. HIV exploits specific host proteins also known as HIV-dependency factors during its replication inside the cell. Potassium channels play a crucial role in the life cycle of several viruses by modulating ion homeostasis, cell signaling, cell cycle, and cell death. In this study, using pharmacological tools, we have identified that HIV utilizes distinct cellular potassium channels at various steps in its life cycle. Members of inwardly rectifying potassium (Kir) channel family, G protein-coupled (GIRK), and ATP-sensitive (KATP) are involved in HIV entry. Blocking these channels using specific inhibitors reduces HIV entry. Another member, Kir 1.1 plays a role post entry as inhibiting this channel inhibits virus production and release. These inhibitors are not toxic to the cells at the concentration used in the study. We have further identified the possible mechanism through which these potassium channels regulate HIV entry by using a slow-response potential-sensitive probe DIBAC4(3) and have observed that blocking these potassium channels inhibits membrane depolarization which then inhibits HIV entry and virus release as well. These results demonstrate for the first time, the important role of Kir channel members in HIV-1 infection and suggest that these K+ channels could serve as a safe therapeutic target for treatment of HIV/AIDS.

Kir Channel Blockages by Proflavine Derivatives via Multiple Modes of Interaction.

Many compounds inhibit tetrameric and pseudo-tetrameric cation channels by associating with the central cavity located in the middle of the membrane plane. They traverse the ion conduction pathway from the intracellular side and through access to the cavity. Previously, we reported that the bacteriostatic agent, proflavine, preferentially blocked a subset of inward rectifier K+ (Kir) channels. However, the development of the inhibition of Kir1.1 by the compound was obviously different from that operating in Kir3.2 as a pore blocker. To gain mechanistic insights into the compound-channel interaction, we analyzed its chemical specificity, subunit selectivity, and voltage dependency using 13 different combinations of Kir-channel family members and 11 proflavine derivatives. The Kir-channel family members were classified into three groups: 1) Kir2.2, Kir3.x, Kir4.2, and Kir6.2Δ36, which exhibited Kir3.2-type inhibition (slow onset and recovery, irreversible, and voltage-dependent blockage); 2) Kir1.1 and Kir4.1/Kir5.1 (prompt onset and recovery, reversible, and voltage-independent blockage); and 3) Kir2.1, Kir2.3, Kir4.1, and Kir7.1 (no response). The degree of current inhibition depended on the combination of compounds and channels. Chimera between proflavine-sensitive Kir1.1 and -insensitive Kir4.1 revealed that the extracellular portion of Kir1.1 is crucial for the recognition of the proflavine derivative acrinol. In conclusion, preferential blockage of Kir-channel family members by proflavine derivatives is based on multiple modes of action. This raises the possibility of designing subunit-specific inhibitors.

Knockout of Kcnj16 (Kir5.1) in Dahl Salt‐Sensitive Rats Produces Seizure Phenotype

Kir5.1 is a member of an inwardly rectifying potassium (Kir) channel family notably present in the kidney and brain. We previously established that a knockout rat model of Kcnj16 (gene encoding Kir5.1) on a Dahl salt‐sensitive background (SSKcnj16−/−) exhibits a severe cardiovascular phenotype (Palygin et al., JCI Insight, 2017). Mutations in ion channel genes can alter neuronal excitability and mutations in Kir genes have been linked to seizure disorders in humans. Since Kir5.1 is known to be expressed in the brain and may contribute to neuronal membrane potential and spatial potassium buffering, we hypothesized that in addition to cardiovascular and renal dysfunction, neurological phenotypes, including seizures, would be prevalent in the SSKcnj16−/−rats. We found that SSKcnj16−/− rats (but not control SS rats) experience tonic‐clonic seizures when exposed to a 10 kHz tone (86 dB for 2 min), whereas other frequencies (0.1 or 1 kHz) did not elicit seizures. When exposed to the seizure‐inducing acoustic stimulus once/day for 10 days, we noted that SSKcnj16−/− rats experienced a seizure in response to 92% of stimuli. SSKcnj16−/− rats also showed spontaneous mortality within hours after a stimulus with a survival rate of 67% (N=21) for the duration of the 10 day protocol. Control rats (N=10) had no incidence of seizure or death during the 10 days of acoustic stimulation. We found no difference among male and female SSKcnj16−/− rats, where both sexes had a similar audiogenic seizure response and reduced survival. Behavioral tests including a modified Irwin screen and open field test revealed that before and after seizure stimulation SSKcnj16−/− rats tended to have altered activity levels, gait, piloerection, and respiration rates. Analysis of blood electrolytes revealed critically low serum potassium levels in SSKcnj16−/− compared to SS controls, a difference that appeared to be exacerbated by repeated seizures. We hypothesize that seizures may be intensifying potassium imbalance and disrupting the homeostasis that is particularly essential for excitable cells such as neurons. We conclude that knockout of Kir5.1 leads to distinct neurological phenotypes including a seizure disorder and subsequent spontaneous death, which may have relevance to channelopathies associated with seizure disorders in humans.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute and Chronic Respiratory Effects from Repeated Audiogenic Seizures in SSKcnj16−/− Rats

Kir5.1 is a member of an inwardly rectifying potassium (Kir) channel family highly expressed in the kidney and brain. Mutations in ion channel genes can alter neuronal excitability, and mutations in related Kir genes have been linked to seizure disorders in humans. We previously established that a knockout rat model of Kcnj16 (gene encoding Kir5.1) on a Dahl salt‐sensitive background (SSKcnj16−/−) exhibits a severe cardiovascular phenotype (Palygin et al., JCI Insight, 2017) as well as an audiogenic reflex epilepsy phenotype. SSKcnj16−/− rats, but not control SS rats, experience tonic‐clonic seizures when exposed to a 10 kHz tone (86 dB for 2 min). Although seizures are typically considered benign events, patients with uncontrolled epilepsy are 27 times more likely die of Sudden Unexplained Death in Epilepsy (SUDEP) compared to those without epilepsy, presumably due to cardiorespiratory failure associated with repeated seizures. Here, we tested the hypothesis that repeated audiogenic seizures would acutely and chronically alter breathing in SSKcnj16−/− but not control SS rats. SSKcnj16−/− and control rats were exposed to the acoustic stimulus once/day for 10 days while using plethysmography to measure breathing before, during, and after exposure. SSKcnj16−/− rats show a marked increase in tidal volume (1.4 ± 0.1 to 3.2 ± 1.2 mL/100g) after acoustic stimulation which becomes more pronounced after exposure to 10 acoustic stimuli (1.7 ± 0.1 to 4.2 ± 1.1 mL/100g). Minute ventilation and drive to breathe show similar increases with successive acoustic exposures. SS control rats do not show changes in tidal volume or minute ventilation over the 10 days. In addition, we see a substantial increase in resting VO2 between day 1 (7.4 ± 0.4 mL/min/100g) and day 10 (11.8 ± 0.6 mL/min/100g) of acoustic stimulation in SSKcnj16−/−/− rats but not controls. During the 10 days of stimulation, we observed spontaneous mortality in 33% of SSKcnj16−/− rats (N = 7 of 21 total) within hours of a seizure, while 100% (N=12) of control SS rats survived. We found no difference among male and female SSKcnj16−/− rats, where both sexes had a similar audiogenic seizure response and reduced survival. Further studies into the mechanisms behind seizure‐induced death in SSKcnj16−/− rats could have central relevance to the pathophysiology of SUDEP, which remains poorly understood.Support or Funding InformationNIH HL122358This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay.

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gαs-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gαs and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl+ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl+ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.

N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K+ channels

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC50 = 3.5nM [control] vs.1.0nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.

N‐(2‐Methoxyphenyl) Benzenesulfonamide, a Novel Agonist of Neuronal G Protein‐Gated Inward Rectifier K+ Channels

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin (SST), dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs such as tolbutamide and diazoxide. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the SST type-2 and 5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by SST. In order to determine the mechanism for the augmentation in the fluorescent signal, GIRK currents were recorded in the AtT20 cells using the whole-cell patch clamp technique (Figure 1). In control solution, application of a low concentration (0.5 nM) of SST produced little if any activation of the GIRK channel (Figure 1a). As expected, the subsequent addition of a high concentration (100 nM) of SST caused a robust activation of the current. In contrast, application of 0.5 nM SST caused a strong activation of the current in cells pretreated with MPBS (Figure 1b). In conclusion, MPBS represents a novel prototypic SST-dependent agonist of neuronal GIRK channels. Structural analogs of MPBS are currently being tested for GIRK channel potency, selectivity and mechanism of action. Support or Funding InformationSupported by NSF grant CBET-1606882 to KW. Figure 1Open in figure viewerPowerPoint GIRK currents measured in AtT20 cells during stimulation by somatostatin (SST). Currents were recorded during voltage steps applied from a holding potential of −40 mV to −100 mV during stimulation with either 0.5 or 100 nM SST.

Hydrogen Sulfide (H2S) Regulation of Kir (Inwardly Rectifying K+) Channels

Inwardly rectifying potassium channels establish and regulate the resting membrane potential of excitable cells in tissues such as brain and heart. A wealth of electrophysiological, structural, and biochemical studies has revealed that a biological phospholipid, Phosphatidylinositol 4,5-bisphosphate(PIP2), is a key direct activator of Kir(Inwardly-rectifying K+) channels. Here we show that hydrogen sulfide(H2S), an emerging gasotransmitter, produced in cells, regulates the activity of several members of the Kir channel family differentially via s-sulfhydration of its cytoplasmic cysteine residues and the extent of regulation depends on the strength of channel-PIP2 interaction. These findings have used two independent methods to demonstrate H2S effects: direct administration of an exogenous donor, sodium hydrogen sulfide(NaHS), and expression of cystathionine gamma lyase(CSE), the principle producer of endogenous H2S in peripheral tissues, such as in the cardiovascular system and specific regions of the brain. We show here that H2S regulation depends on the strength of channel-PIP2 interactions by employing techniques that alter the extent of channel-PIP2 interactions. H2S regulation is attenuated when strengthening channel-PIP2 interactions either through point mutations in Kir3.2(altering channel structure intrinsically), interacting proteins(e.g. G-beta-gamma) or by increasing intracellular PIP2 levels by co-expression of PIP5Kinase(PIP5K), the enzyme that converts endogenous PIP(4) into PIP2(4,5). Conversely, our results show increased H2S regulation when channel-PIP2 interactions are weakened through the administration of Wortmannin(Wort), a known blocker of PIP4K, which leads to abatement of intracellular PIP2. In addition, activation of co-expressed Ci-VSP, a voltage activated PIP5 phosphatase, also reduces intracellular PIP2, and H2S is able to exert greater effects on the channel. These H2S effects depend on the presence of specific cytoplasmic cysteine residues in Kir3.2(GIRK2), as their replacement(by other non-polar amino acids) abolishes the H2S effect(without affecting channel-PIP2 interactions). Reintroduction of specific cysteine residues back in to the background of this GIRK2 cysteine less mutant rescues the H2S effect. In order to monitor H2S modification, we employ the biochemical technique, Tag Switch Assay(TSA), showing that Kir3.2 is directly s-sulfhydrated at specific cysteine residues upon addition of exogenous H2S via administration of NaHS to wild-type Kir3.2 and Kir3.2 mutants lacking specific cysteine residues. Lastly, molecular dynamics simulation experiments are providing mechanistic insight in how s-sulfhydration of these specific cysteine residues would lead to changes in affecting channel-PIP2 interactions and channel gating. These studies reveal for the first time the intricate interplay between Kir channels(proteins), PIP2(biological lipids) and H2S(gaseous molecules).

Emerging complexities of lipid regulation of potassium channels.

Inwardly rectifying potassium (Kir) channels are important regulators of cellular excitability, enabling cells to alter electrical activity in response to diverse autonomic and metabolic signals ([Hibino et al., 2010][1]). Fairly or not, the Kir channel family is sometimes derided as the least

A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway.

Non-basic amino acids in the ROMK1 channels via an appropriate distance modulate PIP2 regulated pHi-gating

The ROMK1 (Kir1.1) channel activity is predominantly regulated by intracellular pH (pHi) and phosphatidylinositol 4,5-bisphosphate (PIP2). Although several residues were reported to be involved in the regulation of pHi associated with PIP2 interaction, the detailed molecular mechanism remains unclear. We perform experiments in ROMK1 pHi-gating with electrophysiology combined with mutational and structural analysis. In the present study, non basic residues of C-terminal region (S219, N215, I192, L216 and L220) in ROMK1 channels have been found to mediate channel-PIP2 interaction and pHi gating. Further, our structural results show these residues with an appropriate distance to interact with membrane PIP2. Meanwhile, a cluster of basic residues (R188, R217 and K218), which was previously discovered regarding the interaction with PIP2, exists in this appropriate distance to discriminate the regulation of channel-PIP2 interaction and pHi-gating. This appropriate distance can be observed with high conservation in the Kir channel family. Our results provide insight that an appropriate distance cooperates with the electrostatics interaction of channel-PIP2 to regulate pHi-gating.

Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val168 or Asn171 in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.