IDOR-1104-0086, a novel Kv7 channel activator with antiseizure effects in rodents.

Kv7 (KCNQ) channel openers induce hypothermia in the mouse

Diabetic peripheral neuropathy (DPN) is the most incapacitating complication of diabetes mellitus. Up to 50% of patients with DPN develop peripheral neuropathic pain (PNP). The underlying ionic and molecular mechanisms of diabetic PNP (DPNP) are poorly understood. However, voltage gated potassium (Kv7) channels which have been implicated in the pathogenesis of other types of PNP are likely to be involved. Here we examined, in the streptozotocin (STZ) rat model of DPNP, whether activating the Kv7 channels with a potent activator retigabine (ezogabine) would reverse/attenuate behavioural signs of DPNP. STZ rats exhibited behavioural indices of mechanical and heat hypersensitivity, but not cold hypersensitivity or spontaneous pain, 35 days after STZ injection. Retigabine given at a dose of 15 mg/kg (but not at 7.5 mg/kg, i.p.) significantly attenuated mechanical, but not heat hypersensitivity in DPNP rats, and was as effective as the positive control gabapentin. This analgesic effect of retigabine was completely reversed by the Kv7/M channel blocker XE991 (3 mg/kg, i.p.) indicating that the anti-allodynic effects of retigabine were mediated by Kv7 channels. In conclusion, the findings suggest that Kv7 channels are involved in DPNP pathogenesis, and that strategies that target their activation may prove to be effective in treating DPNP.

Voltage gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. Comprised of four identical subunits, Kv channels contain four voltage sensing domains arranged on the periphery of a central pore domain. Each voltage sensor is comprised of four transmembrane helices, numbered S1 through S4. The S4 helix, containing four to six highly-conserved, positively-charged arginine or lysine residues, is responsible for voltage sensitivity in Kv channels. The pore domain consists of two transmembrane helices, S5 and S6. The S5 helix constitutes the periphery of the pore domain and is believed to be relatively immobile. The S6 helices, lining the interior of the channel, gate the protein and regulate K+ permeation. Because each subunit of Kv channels contains six transmembrane helices, they are often referred to as 6TM Kv channels. The depolarization of an action potential is initiated as sodium ions enter the cell. At the cellular resting potential of -70 mV, potassium ion channels are closed, and the S4 helix is in its “down” state. As the electrochemical gradient changes, the S4 helices of Kv channels begin to reorient within the membrane. At the peak of the action potential (roughly +20 mV), the S4 helices exist in their “up” state. This conformational transition of the S4 helix is coupled to the pore domain via the S4-S5 linker, a short, amphipathic helix along the intracellular membrane-water interface. By bridging the C-terminus of the voltage sensor to the N-terminus of the pore domain, the S4-S5 linker couples the voltage sensitivity of the voltage sensor to K+ conduction in the pore domain. Because they begin opening at voltages less than 0 mV, all crystal structures of Kv channels contain an open pore domain. With no structure in the closed conformation, the mechanism of gating in Kv channels remains unclear. Nevertheless, significant biophysical studies have revealed insights into both the closed conformation and the gating transition itself. In this dissertation, I will explore questions relevant to the gating mechanism in voltage gated potassium ion channels through fully-atomistic molecular dynamics (MD) simulations. First, in Chapter 2, I will address the potential role of the 310 helical conformation found in the C-terminal end of S4 in the crystal structures of Kv channels. Spanning eight or more residues, these 310 helices are both uncharacteristically long and conserved in K+ channel crystal structures. By simulating the Kv1.2/2.1 chimera channel’s voltage sensor embedded in a lipid bilayer, I find that an alpha to 310 helical interconversion of the S4 helix reproduces many experimental measurements of the open and closed states of Kv channels. In Chapter 3, I perform molecular dynamics simulations of the entire Kv1.2/2.1 chimera channel. First, I examine the impact of an alpha to 310 helical interconversion of the S4 helix on the pore domain of the channel. Though the results are consistent with the results in Chapter 2 (and the corresponding experimental measurements), I find that this secondary structural modification is insufficient to influence the pore domain of the channel on the timescale of my simulations. In the second half of Chapter 3, I use molecular dynamics simulations to generate a closed state model of the Kv1.2/2.1 chimera from luminescence resonance energy transfer (LRET) measurements of the closed conformation of KvAP. The resulting structure is indeed closed, and also recapitulates a number of experimentally determined measurements of the closed channel. In Chapter 4, I focus on the pore domain. First, using targeted molecular dynamics simulations, I generate a transition between a closed model of the KvAP linker and pore domain to the open conformation. Then, using an umbrella sampling method, I quantify the energetics of the gating transition in KvAP and assess the physiological implications. In agreement with experimental studies of Kv channel energetics, I find that the open pore is roughly 2.7 kcal/mol lower in free energy than the closed conformation. The targeted molecular dynamics and umbrella sampling simulations reveal additional insights into the gating mechanism of KvAP. Lastly, in Chapter 5, I use MD simulations to gain insights into the binding mechanism of VSTx1, a Kv channel inhibitor. By using the experimentally determined neutron scattering density profile of the VSTx1 toxin bound to a lipid bilayer as a restraint for molecular dynamics simulations, I recreate the experimental scattering density profile, and also offer insight into the binding of VSTx1 to a lipid membrane.

Iloperidone, a second-generation atypical antipsychotic drug, is widely used in the treatment of schizophrenia. However, the side-effects of iloperidone on vascular K+ channels remain to be determined. Therefore, we explored the effect of iloperidone on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Iloperidone inhibited vascular Kv channels in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 2.11 ± 0.5 μM and a Hill coefficient of 0.68 ± 0.03. Iloperidone had no effect on the steady-state inactivation kinetics. However, it shifted the steady-state activation curve to the right, indicating that iloperidone inhibited Kv channels by influencing the voltage sensors. Application of 20 repetitive depolarizing pulses (1 and 2 Hz) progressively increased the inhibition of the Kv current in the presence of iloperidone. Furthermore, iloperidone increased the recovery time constant from Kv channel inactivation, suggesting that iloperidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with a Kv1.5 inhibitor (diphenyl phosphine oxide 1 [DPO-1]) inhibited the Kv current to a level similar to that with iloperidone alone. However, pretreatment with a Kv2.1 or Kv7.X inhibitor (guangxitoxin or linopirdine) did not affect the inhibitory effect of iloperidone on Kv channels. Therefore, iloperidone directly inhibits Kv channels in a concentration- and use (state)-dependent manner independently of its antagonism of serotonin and dopamine receptors. Furthermore, the primary target of iloperidone is the Kv1.5 subtype.

Paliperidone, an atypical antipsychotic, is widely used to treat schizophrenia. In this study, we explored whether paliperidone inhibited the voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells. Paliperidone reduced Kv channel activity in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 16.58 ± 3.03 μM and a Hill coefficient of 0.60 ±0.04. It did not significantly shift the steady-state activation or inactivation curves, suggesting that the drug did not affect the gating properties of Kv channels. In the presence of paliperidone, the application of 20 repetitive depolarizing pulses at 1 and 2Hz gradually increased the inhibition of the Kv current. Further, the recovery time constant after Kv channel inactivation was increased by paliperidone, indicating that it inhibited the Kv channel in a use (state)-dependent manner. Its inhibitory effects were reduced by pretreatment with a Kv1.5 subtype inhibitor. However, pretreatment with a Kv2.1 or Kv7 inhibitor did not reduce its inhibitory effect. We conclude that paliperidone inhibits Kv channels (mainly Kv1.5 subtype channels) in a concentration- and use (state)-dependent manner without changing channel gating.

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.

The voltage-gated proton channel, Hv1, is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels, but uniquely lacks a central pore domain (Ramsey et al., Nature 440, 1213, 2006; Sasaki et al., Science 312, 589, 2006). To begin to understand voltage-dependent activation in the Hv1 channel, we perform molecular dynamics simulations of a homology model for the transmembrane region of the human Hv1 in a hydrated lipid bilayer under a membrane potential. Under a depolarizing potential (positive on the intracellular side), the channel pathway exhibits a robust transmembrane hydrogen bonded water wire connecting the intracellular and extracellular sides. Under a hyperpolarizing potential, a cluster of hydrophobic side chains occludes the central region of the channel and prevents water wire formation. Assuming that a water wire plays a role in proton conduction by Hv1, these observations are consistent with experiments showing that the channel opens at depolarizing potentials. The basic side chains in the VSD S4 segment carry most of the gating charge during the activation of Kv channels. We observe translocation of the third arginine in the Hv1 S4 segment (Arg211) relative to a highly conserved phenylalanine side chain (Phe150) located at the center of the putative channel permeation pathway. Phe150 is homologous to a highly conserved Phe side chain in Kv channels, which is thought to modulate channel activation (Tao et al., Science 328, 67, 2010; Lacroix and Bezanilla, PNAS 108, 12313, 2011). Thus, our simulations suggest that these residues may be involved in voltage activation of Hv1. This work is supported by grants from the NIH (GM74637, GM86685 and GM72507) and NSF (CHE-0750175), and Teragrid resources provided by the NSF-supported NICS.

Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons, where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. Here, we show that Kv7 channel activity is upregulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons from rats of both sex, thus resulting in a synergistic long-term depression of intrinsic excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for induction. However, although LTD is dependent on cannabinoid type 1 receptors, LTD-IE is not. Molecular modeling shows a strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.SIGNIFICANCE STATEMENT In principal cells, Kv7 channels are essentially located at the axon initial segment. In contrast, in O-LM interneurons, Kv7 channels are highly expressed in the dendrites, suggesting a singular role of these channels in O-LM cell function. Here, we show that LTD of excitatory inputs in O-LM interneurons is associated with an upregulation of Kv7 channels, thus resulting in a synergistic LTD of LTD-IE. Both forms of plasticity are mediated by the biosynthesis of eCBs. Stimulation of CB1 receptors induces LTD, whereas the direct interaction of eCBs with Kv7 channels induces LTD-IE. Our results thus provide a previously unexpected involvement of eCBs in long-lasting plasticity of intrinsic excitability in GABAergic interneurons.

Aims: Voltage-gated K + (K v ) channels in vascular smooth muscle cells (VSMCs) play an important role in the regulation of coronary microcirculation. Atorvastatin (ATV) has shown some beneficial effects on vascular function, but the effect of ATV on K v channels-mediated coronary vasodilation remains unknown. The present study was designed to investigate the role of ATV in improving K v channels-mediated coronary dilator function in diabetes and the underlying mechanisms. Methods: Isolated VSMCs were incubated in normal or high glucose medium plus a different dose of ATV for 24 h at 37 o C. Patch-clamp recording and molecular biological techniques were used to assess the function and expression of K v channels. Control or type 2 diabetic rats were treated with ATV (50 mg/kg daily) by oral gavage for 10 weeks. Vasodilation of isolated rat coronary arteries was measured using a pressurized myograph. GW9662, the peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, was used to determine whether the mechanism of ATV-improved K v channel function can be explained by upregulation of PPARγ pathway. Results: Patch-clamp analysis revealed that high glucose reduced K v current density by 58.7 ± 4.6%, which was accompanied by a downregulation of K v 1.2 and K v 1.5 expression. Treatment with ATV reversed the inhibitory effect of high glucose on K v current density in a dose-dependent manner (1 μmol/L, 15.0 ± 2.6%; 10 μmol/L, 49.1 ± 3.8%; 100 μmol/L, 69.9 ± 4.8%; P < 0.05). In addition, ATV restored high glucose-induced downregulation of K v channel protein expression, and the difference was significant in both 10 μmol/L and 100 μmol/L groups. For in vivo study, K v channels-mediated coronary vasodilation was decreased in diabetic rats, compared with controls (9.1 ± 1.3 vs. 36.7 ± 1.4%, P < 0.05), whereas this decrease was partly corrected by ATV (25.0 ± 2.8 vs. 9.1 ± 1.3%, P < 0.05). Treatment with ATV prominently increased protein expression of PPARγ both in vitro and in vivo . The effect of ATV in regulating K v channels and K v channels-mediated vasodilation was markedly blunted by GW9662. Conclusions: In conclusion, treatment with ATV activates PPARγ pathway and preserves K v channel activity in VSMCs, thus providing improvement of coronary dilator function in diabetic rats.

Voltage-gated potassium (Kv) channels are widely expressed in the central and peripheral nervous system and are crucial mediators of neuronal excitability. Importantly, these channels also actively participate in cellular and molecular signaling pathways that regulate the life and death of neurons. Injury-mediated increased K(+) efflux through Kv2.1 channels promotes neuronal apoptosis, contributing to widespread neuronal loss in neurodegenerative disorders such as Alzheimer's disease and stroke. In contrast, some forms of neuronal activity can dramatically alter Kv2.1 channel phosphorylation levels and influence their localization. These changes are normally accompanied by modifications in channel voltage dependence, which may be neuroprotective within the context of ischemic injury. Kv1 and Kv7 channel dysfunction leads to neuronal hyperexcitability that critically contributes to the pathophysiology of human clinical disorders such as episodic ataxia and epilepsy. This review summarizes the neurotoxic, neuroprotective, and neuroregulatory roles of Kv channels and highlights the consequences of Kv channel dysfunction on neuronal physiology. The studies described in this review thus underscore the importance of normal Kv channel function in neurons and emphasize the therapeutic potential of targeting Kv channels in the treatment of a wide range of neurological diseases.

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K(+) (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [I(K(V))] is selective to PASMC; hypoxia has little effect on I(K(V)) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming alpha-subunits and regulatory beta-subunits. KCNA5 is a Kv channel alpha-subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits I(K(V)) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased I(K(V)) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect I(K(V)) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of Po(2) from 145 to 35 mmHg reduced I(K(V)) by approximately 40% in rat PASMC transfected with human KCNA5 but had no effect on I(K(V)) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on I(K(V)) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels.

Neural activity is important for establishing proper connectivity in the developing visual system. Tetrodotoxin blockade of sodium (Na(+))-dependent action potentials impairs the refining of synaptic connections made by developing retinal ganglion cells (RGCs), but does not affect their ability to get out to their target. Although this may suggest neural activity is not required for the directed extension of RGC axons, in many species developing RGCs express additional, Na(+)-independent ionic mechanisms. To test whether the ability of RGC axons to extend in a directed fashion is influenced by membrane excitability, we blocked the principal modulators of the neural activity of a neuron, voltage-dependent potassium (Kv) channels. First, we showed that RGCs and their growth cones express Kv channels when they are growing through the brain on the way to their main midbrain target, the optic tectum. Second, a Kv channel blocker, 4-aminopyridine (4-AP), was applied to the developing Xenopus optic projection. Blocking Kv channels inhibited RGC axon extension and caused aberrant routing of many RGC fibers. With the higher doses, <25% of embryos had a normal optic projection. These data suggest that Kv channel activity regulates the guidance of growing axons in the vertebrate brain.

Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPepsilon and PTPalpha and the mechanisms by which they regulate K+ channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting an effect on channel number or organization. PTPalpha inhibits Kv channels more strongly than PTPepsilon; this correlates with constitutive association of PTPalpha with Kv2.1, driven by membranal localization of PTPalpha. PTPalpha, but not PTPepsilon, activates Src in sciatic nerve extracts, suggesting Src deregulation is not responsible exclusively for the observed phenotypes and highlighting an unexpected difference between both PTPs. Developmentally, sciatic nerve myelination is reduced transiently in mice lacking either PTP and more so in mice lacking both PTPs, suggesting both PTPs support myelination but are not fully redundant. We conclude that PTPepsilon and PTPalpha differ significantly in their regulation of Kv channels and Src in the system examined and that similarity between PTPs does not necessarily result in full functional redundancy in vivo.

Voltage-gated potassium (Kv) channels are known regulators of cellular electrical properties in excitable cells such as neurons and muscle cells. The Kv channels also play roles in cell proliferation, differentiation and cell survival in non-excitable cells. In the present study, we focused on the role of Kv channels in osteogenic differentiation using human mesenchymal stem cells (hMSCs) and the osteoblast-like MG-63 and SaOS-2 cell lines. Use of a non-specific Kv channel blocker, tetraethylammonium (TEA), confirmed that blockade of Kv channels in hMSCs increased their mineral contents density, an indicator of osteogenic differentiation. We also used two osteoblast-like cell lines, MG-63 and SaOS-2 cells, to focus on the regulation of osteogenic properties by Kv channel blockers. We screened the mRNA and protein expression of Kv channels in MG-63 and SaOS-2 cells and compared the relative expression of Kv channels during osteogenic differentiation. We confirmed that the expression level of Kv7.3 was significantly changed, while blockade of Kv7.3 using the selective Kv7.3 blocker, linopirdine and XE991, increased osteogenic differentiation in MG-63 and SaOS-2 cells. In contrast, the use of Kv7 opener, flupirtine, caused no significant change in osteogenic differentiation in either cell line. In conclusion, Kv channels, especially the Kv7 families, are involved in osteogenic differentiation and have potential functions in hMSCs, MG-63 and SaOS-2 cells during osteogenic differentiation. Blockade of Kv7.3 may be important in regulation of osteogenic differentiation.