Inhibitor Of Kv1 Research Articles

Characterization of relaxant mechanism of H2S in mouse corpus cavernosum

The aim of this study was to investigate the mechanism of H2 S-induced relaxation in mouse corpus cavernosal tissue. l-cysteine (10(-6) × 10(-3) mol/L) and exogenous H2 S (NaHS; 10(-6) to 10(-3) mol/L) induced concentration-dependent relaxation. l-cysteine-induced relaxations was reduced by d,l-propargylglycine, a cystathionine gamma lyase (CSE) inhibitor but not influenced by aminooxyacetic acid, a cystathionine beta synthase (CBS) inhibitor. l-cysteine induced relaxations, but not of those of H2 S diminished in endothelium-denuded tissues. N(ω) -nitro-l-arginine (l-NA; 10(-4) mol/L), a nitric oxide synthase inhibitor, and ODQ (10(-4) mol/L), a guanylyl cyclase inhibitor, increased the H2 S-induced relaxation. Zaprinast (5 × 10(-6) mol/L) and sildenafil (10(-6) mol/L), phosphodiesterase inhibitors, inhibited H2 S-induced relaxation. Adenylyl cyclase inhibitors N-ethylmaleimide (2.5 × 10(-5) mol/L) and SQ22536 (10(-4) mol/L) reduced relaxation to H2 S. Also, H2 S-induced relaxation was reduced by KCl (50 mmol/L), 4-aminopyridine (10(-3) mol/L), a Kv inhibitor, glibenclamide (10(-5) mol/L), a KATP inhibitor or barium chloride (10(-5) mol/L), a KIR inhibitor. However, H2 S-induced relaxation was not influenced by apamin (10(-6) mol/L), a SKC a (2+) inhibitor, charybdotoxin (10(-7) mol/L), an IKC a (2+) and BKC a (2+) inhibitor or combination of apamin and charybdotoxin. Nifedipine (10(-6) mol/L), an L-type calcium channel blocker and atropine (10(-6) mol/L), a muscarinic receptor blocker, inhibited H2 S-induced relaxation. However, H2 S-induced relaxation was not influenced by ouabain (10(-4) mol/L), a Na(+) /K(+) -ATPase inhibitor. This study suggests that H2 S endogenously synthesizes from l-cysteine by CSE endothelium-dependent in mouse corpus cavernosum tissue, and exogenous H2 S may cause endothelium-independent relaxations via activation of K channels (KATP channel, KV channels, KIR channels), L-type voltage-gated Ca(2+) channels, adenylyl cyclase/cAMP pathway and muscarinic receptor, and there is the interaction between H2 S and NO/cGMP.

K V 7 Channels are Potential Regulators of the Exercise Pressor Reflex

The exercise pressor reflex (EPR) is a peripheral neural reflex that originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure, ventilation, and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease and heart disease to increase the incidence of myocardial infarction. The goal of this research was to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Certain chemicals such as bradykinin are released within contracting muscle and enhance the excitability of group III/IV afferents to evoke the EPR. However, the mechanism by which bradykinin elicits the EPR is unknown. KV7 channels strongly regulate neuronal excitability and have been shown to be modulated by bradykinin. We have identified KV currents in identified muscle afferents by their characteristic activation/deactivation kinetics and block by KV7 specific inhibitor XE991. The KV7 current was also inhibited by 300 nM bradykinin. A KV7 channel antibody labeled group IV muscle afferent fibers within muscle, which demonstrated axonal expression of these channels. KV7 likely modulates the EPR. The inhibition of KV7 by bradykinin could be a mechanism by which this metabolic mediator enhances EPR excitability. Furthermore, our results suggest that the recently FDA approved KV7 activator, retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease. This study is funded by NIH grant AR059397 and the ATSU-KCOM Graduate Program Committee.

Inhibition of Kv1.3 Channels in Human Jurkat T Cells by Xanthohumol and Isoxanthohumol.

Using whole-cell patch-clamp technique, we investigated influence of selected compounds from groups of prenylated chalcones and flavonoids: xanthohumol and isoxanthohumol on the activity of Kv1.3 channels in human leukemic Jurkat T cells. Obtained results provide evidence that both examined compounds were inhibitors of Kv1.3 channels in these cells. The inhibitory effects occurred in a concentration-dependent manner. The estimated value of the half-blocking concentration (EC50) was about 3 μM for xanthohumol and about 7.8 μM for isoxanthohumol. The inhibition of Kv1.3 channels by examined compounds was not complete. Upon an application of the compounds at the maximal concentrations equal to 30 μM, the activity of Kv1.3 channels was inhibited to about 0.13 of the control value. The inhibitory effect was reversible. The application of xanthohumol and isoxanthohumol did not change the currents’ activation and inactivation rate. These results may confirm our earlier hypothesis that the presence of a prenyl group in a molecule is a factor that facilitates the inhibition of Kv1.3 channels by compounds from the groups of flavonoids and chalcones. The inhibition of Kv1.3 channels might be involved in antiproliferative and proapoptotic effects of the compounds observed in cancer cell lines expressing these channels.

Ca2+ dependent but PKC independent signalling mediates UTP induced contraction of rat mesenteric arteries.

Uridine triphosphate (UTP) can be released from damaged cells to cause vasoconstriction. Although UTP is known to act through P2Y receptors and PLC activation in vascular smooth muscle, the role of PKC in generating the response is somewhat unclear. Here we have used Tat-linked membrane permeable peptide inhibitors of PKC to assess the general role of PKC and also of specific isoforms of PKC in the UTP induced contraction of rat mesenteric artery. We examined the effect of PKC inhibition on UTP induced contraction, increased cytoplasmic Ca2+ and reduction of K+ currents and found that PKC inhibition caused a relatively small attenuation of contraction but had little effect on changes in cytoplasmic Ca2+. UTP attenuation of both voltage-gated (Kv) and ATP-dependent (KATP) K+ currents was abolished when intracellular Ca2+ was decreased from 100 to 20 nM. PKC inhibition reduced slightly the ability of UTP to attenuate Kv currents but had no effect on KATP current inhibition. In conclusion, both UTP induced contraction of mesenteric artery and the inhibition of Kv and KATP currents of mesenteric artery smooth muscle cells by UTP are relatively independent of PKC activation; furthermore, the inhibition of both Kv and KATP currents requires intracellular Ca2+.

Mechanisms of the dilator action of the Erigerontis Herba on rat aorta

Ethnopharmacological relevanceErigerontis Herba is widely used as a traditional Chinese medicine and is commonly used for neuroprotection and vascular protection. Aim of studyIn this study, the vasodilator effects of Erigerontis Herba (DZXX) were investigated using rat isolated aorta rings. Material and methodThe involvement of endothelium in the vasorelaxation was studied by comparing response of endothelium-intact and endothelium-denuded aorta rings which precontracted with U46619. The involvement of K+ channels was studied by pretreatment of the aorta rings with various K+ channel inhibitors. The involvement of Ca2+ channel was studied by incubating aorta rings with Ca2+-free solution, primed with U46619 prior to elicit contraction by addition of Ca2+ solution. ResultsDZXX (0.2–2mg/ml) induced a concentration-dependent relaxation on U44619-precontracted aorta rings with EC50 of 0.354±0.036mg/ml. Removal of endothelium or pretreatment with a BKCa inhibitor iberiotoxin, KIR inhibitor barium chloride or Kv inhibitor 4-aminopyridine produced no effect on the DZXX-induced vasorelaxation. However, pretreatment with a KATP inhibitor glibenclamide or a non-selective K+ channel inhibitor tetraethylammonium produced significant inhibition on the DZXX-induced vasorelaxation by 29.9% and 21.3%, respectively. Pretreatment with DZXX (0.4, 1.2 and 2mg/ml) produced a concentration-dependent inhibition on Ca2+-induced vasoconstriction. ConclusionsThese results suggest that the vasodilator effect of DZXX was endothelium-independent, mediated by decreasing the influx of Ca2+ by calcium channel inhibition and increasing the influx of K+ by opening of a KATP channel.

From pan-reactive KV7 channel opener to subtype selective opener/inhibitor by addition of a methyl group.

The voltage-gated potassium channels of the KV7 family (KV7.1–5) play important roles in controlling neuronal excitability and are therefore attractive targets for treatment of CNS disorders linked to hyperexcitability. One of the main challenges in developing KV7 channel active drugs has been to identify compounds capable of discriminating between the neuronally expressed subtypes (KV7.2–5), aiding the identification of the subunit composition of KV7 currents in various tissues, and possessing better therapeutic potential for particular indications. By taking advantage of the structure-activity relationship of acrylamide KV7 channel openers and the effects of these compounds on mutant KV7 channels, we have designed and synthesized a novel KV7 channel modulator with a unique profile. The compound, named SMB-1, is an inhibitor of KV7.2 and an activator of KV7.4. SMB-1 inhibits KV7.2 by reducing the current amplitude and increasing the time constant for the slow component of the activation kinetics. The activation of KV7.4 is seen as an increase in the current amplitude and a slowing of the deactivation kinetics. Experiments studying mutant channels with a compromised binding site for the KV7.2–5 opener retigabine indicate that SMB-1 binds within the same pocket as retigabine for both inhibition of KV7.2 and activation of KV7.4. SMB-1 may serve as a valuable tool for KV7 channel research and may be used as a template for further design of better subtype selective KV7 channel modulators. A compound with this profile could hold novel therapeutic potential such as the treatment of both positive and cognitive symptoms in schizophrenia.

Hesperetin inhibits rat coronary constriction by inhibiting Ca2+ influx and enhancing voltage-gated K+ channel currents of the myocytes

Hesperetin (HSP, one of the most common flavonoids in Citrus) has been reported to possess many benificial effects and is indicated for many diseases both as a therapeutic drug and as a supplement. Although its vascular effects have been extensively studied, little is known about its effects and the underlying mechanisms on coronary artery. In the present study, the myogenic effects of HSP were studied with a wire myograph in isolated rat coronary artery (RCA). Molecular probe and the patch clamp technique were used to study effects of HSP on intracellular free Ca2+ concentration, inward Ca2+ currents through L-type voltage-gated Ca2+ channels (LVGC) and outward K+ currents through voltage-gated K+ channels (KV). HSP (0.01–0.1mM) concentration-dependently depressed concentration–contraction curves of both KCl and thromboxane receptor agonist 9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U46619), and relaxed RCA precontracted by the both vasoconstrictors. The vasospasmolytic effect was more potent in KCl- than in U46619-induced contraction. The vasorelaxation was attenuated by 4-aminopyridine, a specific KV inhibitor, but not affected by NG-nitro-L-arginine methylester ester, indomethacin, glibenclamide, iberiotoxin, BaCl2 or endothelium denudation. At the same concentrations, HSP inhibited extracellular Ca2+ influx-induced contraction, reduced intracellular free Ca2+ concentration, inhibited inward Ca2+ currents through LVGC and increased outward K+ currents through KV in the vascular smooth muscle cells (VSMCs) freshly isolated from RCA. Collectively, our results show that HSP is vasospasmolytic in RCA and suggest that the vasospasmolysis is mediated by inhibition of LVGC and enhancement of KV currents in RCA VSMCs.

Tamoxifen Inhibition of Kv7.2/Kv7.3 Channels

Tamoxifen Inhibition of Kv7.2/Kv7.3 Channels

Amplified Cold Transduction in Native Nociceptors by M-Channel Inhibition

Topically applied camphor elicits a sensation of cool, but nothing is known about how it affects cold temperature sensing. We found that camphor sensitizes a subpopulation of menthol-sensitive native cutaneous nociceptors in the mouse to cold, but desensitizes and partially blocks heterologously expressed TRPM8 (transient receptor potential cation channel subfamily M member 8). In contrast, camphor reduces potassium outward currents in cultured sensory neurons and, in cold nociceptors, the cold-sensitizing effects of camphor and menthol are additive. Using a membrane potential dye-based screening assay and heterologously expressed potassium channels, we found that the effects of camphor are mediated by inhibition of Kv7.2/3 channels subtypes that generate the M-current in neurons. In line with this finding, the specific M-current blocker XE991 reproduced the cold-sensitizing effect of camphor in nociceptors. However, the M-channel blocking effects of XE991 and camphor are not sufficient to initiate cold transduction but require a cold-activated inward current generated by TRPM8. The cold-sensitizing effects of XE991 and camphor are largest in high-threshold cold nociceptors. Low-threshold corneal cold thermoreceptors that express high levels of TRPM8 and lack potassium channels are not affected by camphor. We also found that menthol--like camphor--potently inhibits Kv7.2/3 channels. The apparent functional synergism arising from TRPM8 activation and M-current block can improve the effectiveness of topical coolants and cooling lotions, and may also enhance TRPM8-mediated analgesia.

Structure–Activity Relationship of a Highly Selective Peptidyl Inhibitor of Kv1.3 Voltage-Gated K+-Channel from Scorpion (B. sindicus) Venom

Venomous fauna of the world is a unique source of protein and peptide toxins with a wide range of pharmacological and physiological activities targeting in particular the bio-signaling system. Thus the most widely known source of peptidyl neurotoxins used for callipering different ion channels (e.g. Na+, K+, Ca++ or Cl− etc.) as well as many medicinally important peptides which have been accepted as drugs or are in clinical trials originate from the venom of scorpions of Buthidae family. In the present study, structure–activity relationship of highly selective short-chain neurotoxin from scorpion Buthus sindicus (a common yellow scorpion of Sindh, Pakistan) has been established. The toxin named as Bs-KTx6 (4,115.4 Da) has been isolated, synthesized and found to be a potent as well as selective inhibitor of voltage gated potassium channel Kv1.3 (IC50 = 7.7 pM). The structural studies on channel selective modulators like Bs-KTx6 may serve as a possible template in establishing libraries of peptidyl toxins for treating diseases such as multiple sclerosis, type-1 diabetes mellitus, rheumatoid arthritis, allergic contact dermatitis, bone resorption due to periodontitis and delayed type hypersensitivity. It is also worth mentioning that the peptide based drugs have excellent safety profiles as well as selectivity compared to other non-protein molecules. In conclusion, identified peptide Bs-KTx6 is a specific as well as very selective blocker of Kv1.3 and provides a starting template for the synthesis of peptidyl drugs to treat autoimmune diseases.

Disappearance of Hypoxic Pulmonary Vasoconstriction and O2-Sensitive Nonselective Cationic Current in Arterial Myocytes of Rats Under Ambient Hypoxia

Acute hypoxia induces contraction of pulmonary artery (PA) to protect ventilation/perfusion mismatch in lungs. As for the cellular mechanism of hypoxic pulmonary vasoconstriction (HPV), hypoxic inhibition of voltage-gated K+ channel (Kv) in PA smooth muscle cell (PASMC) has been suggested. In addition, our recent study showed that thromboxane A2 (TXA2) and hypoxia-activated nonselective cation channel (INSC) is also essential for HPV. However, it is not well understood whether HPV is maintained in the animals exposed to ambient hypoxia for two days (2d-H). Specifically, the associated electrophysiological changes in PASMCs have not been studied. Here we investigate the effects of 2d-H on HPV in isolated ventilated/perfused lungs (V/P lungs) from rats. HPV was almost abolished without structural remodeling of PA in 2d-H rats, and the lost HPV was not recovered by Kv inhibitor, 4-aminopyridine. Patch clamp study showed that the hypoxic inhibition of Kv current in PASMC was similar between 2d-H and control. In contrast, hypoxia and TXA2-activated INSC was not observed in PASMCs of 2d-H. From above results, it is suggested that the decreased INSC might be the primary functional cause of HPV disappearance in the relatively early period (2 d) of hypoxia.

Abstracts from the TOXINS 2012

Abstracts from the TOXINS 2012

Synthesis and evaluation of diphenylphosphinic amides and diphenylphosphine oxides as inhibitors of Kv1.5

Diphenylphosphinic amides and diphenylphosphine oxides have been synthesized and tested as inhibitors of the Kv1.5 potassium ion channel as a possible treatment for atrial fibrillation. In vitro structure–activity relationships are discussed and several compounds with Kv1.5 IC50 values of <0.5μM were discovered. Selectivity over the ventricular IKs current was monitored and selective compounds were found. Results from a rabbit PD-model are included.

Crotamine Toxicity Revisited: Novel Insights Based on KV Channel Inhibition

Crotamine, a 5KDa peptide possesses a unique biological versatility. Not only its cell-penetrating activity has become of clinical interest but moreover, its potential selective anti-tumor activity is of great pharmacological importance. Before, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltage-gated potassium (KV) channels in an effort to explain its in vivo effects.Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 KV channels and 4 NaV channels), expressed in Xenopus laevis oocytes.Crotamine selectively inhibits KV1.1, KV1.2 and KV1.3 channels with an IC50 of ∼300 nM and the key amino acids responsible for this molecular interaction are highlighted. Our results demonstrate for the first time that the symptoms which are observed in the typical crotamine syndrome may result from the inhibition of KV channels.The ability of crotamine to inhibit the potassium current through KV channels unravels it as the first snake peptide with the unique multifunctionality such as cell penetrating, antitumoral activity and KV channel inhibiting properties. The potent and selective KV channel inhibiting properties, as demonstrated in this work, can be an advantage for the use of crotamine or its derivatives as anti-tumor drug. This new property of crotamine might explain some experimental observations and opens new perspectives of pharmacological uses.

Different K+ Channels Are Involved in Relaxation of Arterial and Venous Graft Induced by Nicorandil

The drug nicorandil is a vasodilator approved for the treatment of angina. In addition to its well-known effect on the opening of ATP-sensitive K (KATP) channels, nicorandil-induced vasorelaxation also involves the opening of Ca-activated K channels. The aim of this study was to investigate the effects of nicorandil on the isolated human internal mammary artery (HIMA) and the human saphenous vein (HSV) and to define the contribution of different K channel subtypes in the nicorandil action on these arterial and venous grafts. Our results show that nicorandil induced a concentration-dependent relaxation of HSV and HIMA rings precontracted by phenylephrine. Glibenclamide, a selective KATP channels inhibitor, partially inhibited the response to nicorandil in both HSV and HIMA. Iberiotoxin, a most selective blocker of large-conductance Ca-activated K (BKCa) channels, partly antagonized relaxation of HIMA. A nonselective blocker of voltage-gated K channels, 4-aminopyridine caused partial inhibition of the nicorandil-induced relaxation of HSV but did not antagonize relaxation of HIMA induced by nicorandil. Margatoxin, a potent inhibitor of KV1.3 channels, did not abolish the effect of nicorandil on HSV and HIMA. Our results showed that nicorandil induced strong endothelium-independent relaxation of HSV and HIMA contracted by phenylephrine. It seems that KATP and 4-aminopyridine-sensitive K channels located in the smooth muscle of HSV mediated relaxation induced by nicorandil. In addition, KATP and BKCa channels are probably involved in the nicorandil action on HIMA.

Abstract 16945: Role of Thromboxane A 2 -Activated Nonselective Cation Channels in Hypoxic Pulmonary Vasoconstriction of Rat

Hypoxia-induced pulmonary vasoconstriction (HPV) is a physiological mechanism responsible for controlling the ventilation-perfusion ratio in the lungs. Maintenance of the ventilation-perfusion ratio at 0.8-1 is critical for the optimization and maximization of gas exchange in the lung. Inhibition of K + channels has been shown to be a mechanism for hypoxia-induced membrane depolarization in pulmonary arterial smooth muscle cells (PASMCs). Pharmacological inhibition of K + channels, however, does not induce significant contraction in isolated rat pulmonary arteries, but significantly enhances HPV. These results suggest that, in addition to K + channel inhibition, other mechanisms are involved in hypoxia-induced membrane depolarization. We recently found that a partial contraction by thromboxane A 2 (TXA 2 ) is required to induce HPV. Therefore we hypothesize that TXA 2 receptor (TP) stimulation is involved in eliciting membrane polarization by activating nonselective cation (NSC) channels. Consistently, we found that 5-10 nM U46619, a stable agonist of TP, was essential for the contraction of isolated rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K + channel (Kv). Whole-cell voltage clamp experiments with rat PASMCs revealed that U46619 induced a weakly outward-rectifying NSC current (I NSC,TXA2 ), which was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid (TRPV) subfamily. In addition, 2-aminoethoxydiphenyl borate, an agonist for TRPV1-3, activated NSC channels. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (4α-PDD) rarely increased the membrane conductance. RT-PCR analysis showed the expression of transcripts for TRPV2 and TRPV4 in rat PASMCs. Finally, it was confirmed that the pretreatment with RR and tranilast (a TRPV2 inhibitor) both significantly inhibited HPV in the presence of U46619. Taken together, these data suggest that the concerted effects of I NSC,TXA2 increase and Kv inhibition during under hypoxia are required to induce membrane depolarization sufficient for eliciting HPV. Our results also imply that TXA 2 -activated NSC might be formed by TRPV2 in rat PASMCs and contribute to the hypoxia-induced membrane depolarization in PASMCs.

Role of thromboxane A2-activated nonselective cation channels in hypoxic pulmonary vasoconstriction of rat

Hypoxia-induced pulmonary vasoconstriction (HPV) is critical for matching of ventilation/perfusion in lungs. Although hypoxic inhibition of K(+) channels has been a leading hypothesis for depolarization of pulmonary arterial smooth muscle cells (PASMCs) under hypoxia, pharmacological inhibition of K(+) channels does not induce significant contraction in rat pulmonary arteries. Because a partial contraction by thromboxane A(2) (TXA(2)) is required for induction of HPV, we hypothesize that TXA(2) receptor (TP) stimulation might activate depolarizing nonselective cation channels (NSCs). Consistently, we found that 5-10 nM U46619, a stable agonist for TP, was indispensible for contraction of rat pulmonary arteries by 4-aminopyridine, a blocker of voltage-gated K(+) channel (K(v)). Whole cell voltage clamp with rat PASMC revealed that U46619 induced a NSC current (I(NSC,TXA2)) with weakly outward rectifying current-voltage relation. I(NSC,TXA2) was blocked by ruthenium red (RR), an antagonist of the transient receptor potential vanilloid-related channel (TRPV) subfamily. 2-Aminoethoxydiphenyl borate, an agonist for TRPV1-3, consistently activated NSC channels in PASMCs. In contrast, agonists for TRPV1 (capsaicin), TRPV3 (camphor), or TRPV4 (α-PDD) rarely induced an increase in the membrane conductance of PASMCs. RT-PCR analysis showed the expression of transcripts for TRPV2 and -4 in rat PASMCs. Finally, it was confirmed that pretreatment with RR largely inhibited HPV in the presence of U46619. The pretreatment with agonists for TRPV1 (capsaicin) and TRPV4 (α-PDD) was ineffective as pretone agents for HPV. Taken together, it is suggested that the concerted effects of I(NSC,TXA2) activation and K(v) inhibition under hypoxia induce membrane depolarization sufficient for HPV. TRPV2 is carefully suggested as the TXA(2)-activated NSC in rat PASMC.

Effects of the potassium ion channel modulators BMS-204352 Maxipost and its R-enantiomer on salicylate-induced tinnitus in rats

Currently, there are no effective pharmacological therapies for chronic tinnitus despite a number of efforts from clinical studies and more recently, studies in animals using compounds to enhance endogenous inhibition or reduce central hyperactivity. The purpose of the current study was to evaluate the therapeutic efficacy of a novel anxiolytic with potassium channel activity in suppressing salicylate induced tinnitus in animals. Kv7 potassium channels are present in the peripheral and central auditory system where they are believed to modulate neural activity. Maxipost, a compound which attenuates hyperexcitability via positive modulation of Kv7.2–Kv7.5 channels, was administered to rats with behavioral evidence of salicylate induced tinnitus. Tinnitus was measured using our previously established animal model, Schedule Induced Polydipsia Avoidance Conditioning, a paradigm where rats were conditioned to drink only during quiet and suppress drinking in the presence of sound. Salicylate alone significantly suppressed licks in quiet but had no effect on licks in sound; results consistent with the presence of tinnitus. Maxipost at 10mg/kg suppressed behavioral evidence of tinnitus as it completely reversed salicylate's suppression of licks in quiet. Unexpectedly, the R-enantiomer of Maxipost, R-Maxipost, which has no anxiolytic effects and negatively modulates Kv7.2–Kv7.5, also suppressed behavioral evidence of tinnitus. Our original hypothesis was that Kv7.2–Kv7.5 channels might play a key role in tinnitus generation and that Maxipost but not R-Maxipost would suppress tinnitus; however, it appears that a shared mechanism between Maxipost and R-xMaxipost, such as inhibition of Kv7.1 channels or activation of BK channels or some novel mechanism common to both compounds, underlies salicylate induced tinnitus as both compounds completely abolished behavioral evidence of tinnitus in a dose-dependent manner. Further studies with specific BK channel agonists/antagonists are necessary to determine the contribution of these channels to other forms of tinnitus or determine novel targets that could be related to tinnitus.

Elucidating new mechanisms in vasoconstrictor signal transduction: inhibition of Kv7.5 channel activity by PKCα

Physiological concentrations of arginine8-vasopressin (AVP) induce vasoconstriction by increasing the influx of calcium (Ca2+) through voltage-sensitive Ca2+ channels (VSCC) in vascular smooth muscle cells (VSMCs). We recently reported that AVP activates VSCCs through a protein kinase C (PKC)-dependent suppression of Kv7.5 potassium channel activity. However, the PKC isoform that mediates these responses has yet to be identified. Here, we tested the hypothesis that PKCα mediates the suppression of Kv7.5 currents using cultured rat aortic (A7r5) or freshly isolated mesenteric artery VSMCs. VSMCs were transduced with a rapamycin-inducible, GFP-expressing PKCα adenoviral vector. Kv7.5 currents were measured by patch clamp electrophysiology, and PKCα translocation was measured as an increase in the ratio of peripheral to whole cell fluorescence intensity. Under voltage clamp conditions (−20mV holding potential), Kv7.5 currents were significantly reduced upon rapamycin treatment, as compared to the same cells prior to treatment. The time course of Kv7.5 current inhibition ran parallel to the rapamycin-induced PKCα translocation. We found that Kv7.5 protein co-immunoprecipitates with a scaffolding protein, A-kinase anchoring protein 150 (AKAP150). We propose that PKCα and Kv7.5 interact within an AKAP150-containing signaling complex, enabling PKCα-mediated suppression of Kv7.5 currents.

P2Y1receptors mediate an activation of neuronal calcium-dependent K+channels

Molecularly defined P2Y receptor subtypes are known to regulate the functions of neurons through an inhibition of K(V)7 K(+) and Ca(V)2 Ca(2+) channels and via an activation or inhibition of Kir3 channels. Here, we searched for additional neuronal ion channels as targets for P2Y receptors. Rat P2Y(1) receptors were expressed in PC12 cells via an inducible expression system, and the effects of nucleotides on membrane currents and intracellular Ca(2+) were investigated. At a membrane potential of 30 mV, ADP induced transient outward currents in a concentration-dependent manner with half-maximal effects at 4 μm. These currents had reversal potentials close to the K(+) equilibrium potential and changed direction when extracellular Na(+) was largely replaced by K(+), but remained unaltered when extracellular Cl() was changed. Currents were abolished by P2Y(1) antagonists and by blockade of phospholipase C. ADP also caused rises in intracellular Ca(2+), and ADP-evoked currents were abolished when inositol trisphosphate-sensitive Ca(2+) stores were depleted. Blockers of K(Ca)2, but not those of K(Ca)1.1 or K(Ca)3.1, channels largely reduced ADP-evoked currents. In hippocampal neurons, ADP also triggered outward currents at 30 mV which were attenuated by P2Y(1) antagonists, depletion of Ca(2+) stores, or a blocker of K(Ca)2 channels. These results demonstrate that activation of neuronal P2Y(1) receptors may gate Ca(2+)-dependent K(+) (K(Ca)2) channels via phospholipase C-dependent increases in intracellular Ca(2+) and thereby define an additional class of neuronal ion channels as novel effectors for P2Y receptors. This mechanism may form the basis for the control of synaptic plasticity via P2Y(1) receptors.