Electromechanics of lipid-modulated gating of potassium channels

Experimental studies reveal that the anionic lipid phosphatidic acid (POPA), non-phospholipid cholesterol, and cationic lipid DOTAP inhibit the gating of voltage-sensitive potassium (Kv) channels. Here, we develop a continuum electromechanical model to investigate the interaction of these lipids with the ion channel. Our model suggests that: (i) POPA lipids may restrict the vertical motion of the voltage-sensor domain through direct electrostatic interactions; (ii) cholesterol may oppose the radial motion of the pore domain of the channel by increasing the mechanical rigidity of the membrane; and (iii) DOTAP can reduce the effect of electrostatic forces by regulating the dielectric constant at the channel–lipid interface. The electromechanical model predictions for the three lipid types match well with the experimental observations and provide mechanistic insights into lipid-dependent gating of Kv channels.

Sanguinarine Reverses Pulmonary Vascular Remolding of Hypoxia-Induced PH via Survivin/HIF1α-Attenuating Kv Channels

Background: Similarities in the biology of pulmonary hypertension and cancer suggest that anticancer therapies, such as sanguinarine, may also be effective in treating pulmonary hypertension. This, along with underlying biochemical pathways, is investigated in this study.Methods: Rats were subjected to 4-week hypoxia (or control) with or without sanguinarine treatment. In addition, pulmonary artery smooth muscle cells (PASMCs) were examined after 24–48 h hypoxia (with normoxic controls) and with or without sanguinirine. Pulmonary artery pressures and plasma survivin levels were measured in vivo. Ex vivo tissues were examined histologically with appropriate staining. mRNA and protein levels of survivin, HIF-1α, TGFb1, BMPR2, Smad3, P53, and Kv 1.2, 1.5, 2.1 were determined by real-time PCR and Western blot in PASMCs and distal PAs tissue. PASMC proliferation and changes of TGFb1 and pSmad3 induced by sanguinarine were studied using MTT and Western blot. Electrophysiology for Kv functions was measured by patch-clamp experiments.Results: Four-week hypoxia resulted in an increase in serum survivin and HIF-1α, pulmonary artery pressures, and pulmonary vascular remodeling with hypertrophy. These changes were all decreased by treatment with sanguinarine. Hypoxia induced a rise of proliferation in PASMCs which was prevented by sanguinarine treatment. Hypoxic PASMCs had elevated TGFb1, pSmad3, BMPR2, and HIF1α. These increases were all ameliorated by sanguinarine treatment. Hypoxia treatment resulted in reduced expression and function of Kv 1.2, 1.5, 2.1 channels, and these changes were also modulated by sanguinarine.Conclusion: Sanguinarine is effective in modulating hypoxic pulmonary vascular hypertrophy via the survivin pathway and Kv channels.

The role of K-channels in the formation of a nociceptive signal in the rat trigeminal nerve

The central problem of this work is to elucidate the mechanisms of pain in migraine and to establish the role of Kv channels in regulating the excitability of meningeal afferents of the trigeminal nerve that form a pain signal in migraine. The study was conducted on a preparation of an isolated rat skull. It was found that Kv-channel inhibitors 4-aminopyridine (100 microns and 1 mM) and tetraethylammonium (5mm) lead to an increase in the excitability of trigeminal nerve afferents, at the same time, this effect was partially removed by a nonsteroidal anti–inflammatory agent - naproxen, and was not sensitive to sumatriptan, a classic anti-migraine drug. Key words: migraine, K-channels, trigeminal nerve, 4-aminopyridine, tetraethylammonium, naproxen, sumatriptan.

Irisin relaxes rat thoracic aorta: MEK1/2 signaling pathway, KV channels, SKCa channels, and BKCa channels are involved in irisin-induced vasodilation

In this study, it was aimed to investigate the effects of irisin on vascular smooth muscle contractility in rat thoracic aorta, and the hypothesis that mitogen-activated protein kinase kinase (MEK1/2) signalling pathway, voltage-gated potassium (KV) channels, small-conductance calcium-activated potassium (SKCa) channels, and large-conductance calcium-activated potassium (BKCa) channels may have roles in these effects. Isometric contraction-relaxation responses of isolated thoracic aorta rings were measured with an organ bath model. The steady contraction was induced with 10-5 M phenylephrine (PHE), and then the concentration-dependent responses of irisin (10-9-10-6 M) were examined. Irisin exerted the vasorelaxant effects at concentrations of 10-8, 10-7, and 10-6 M compared to the control group (p<0.001). Besides, MEK1/2 inhibitor U0126, KV channel blocker XE-991, SKCa channel blocker apamin, and BKCa channel blocker tetraethylammonium (TEA) incubations significantly inhibited the irisin-induced relaxation responses. In conclusion, the first physiological findings were obtained regarding the functional relaxing effects of irisin in rat thoracic aorta. The findings demonstrated that irisin induces relaxation responses in endothelium-intact aortic rings in a concentration-dependent manner. Furthermore, this study is the first to report that irisin-induced relaxation responses are related to the activity of the MEK1/2 pathway, KV channels, and calcium-activated K+ (SKCa and BKCa) channels.

KCNQ and KCNE Isoform-Dependent Pharmacology Rationalizes Native American Dual Use of Specific Plants as Both Analgesics and Gastrointestinal Therapeutics

Indigenous peoples of the Americas are proficient in botanical medicine. KCNQ family voltage-gated potassium (Kv) channels are sensitive to a variety of ligands, including plant metabolites. Here, we screened methanolic extracts prepared from 40 Californian coastal redwood forest plants for effects on Kv current and membrane potential in Xenopus oocytes heterologously expressing KCNQ2/3, which regulates excitability of neurons, including those that sense pain. Extracts from 9 of the 40 plant species increased KCNQ2/3 current at –60 mV by ≥threefold (maximally, 15-fold by Urtica dioica) and/or hyperpolarized membrane potential by ≥-3 mV (maximally, –11 mV by Arctostaphylos glandulosa). All nine plants have traditionally been used as both analgesics and gastrointestinal therapeutics. Of two extracts tested, both acted as KCNQ-dependent analgesics in mice. KCNQ2/3 activation at physiologically relevant, subthreshold membrane potentials by tannic acid, gallic acid and quercetin provided molecular correlates for analgesic action of several of the plants. While tannic acid also activated KCNQ1 and KCNQ1-KCNE1 at hyperpolarized, negative membrane potentials, it inhibited KCNQ1-KCNE3 at both negative and positive membrane potentials, mechanistically rationalizing historical use of tannic acid-containing plants as gastrointestinal therapeutics. KCNE dependence of KCNQ channel modulation by plant metabolites therefore provides a molecular mechanistic basis for Native American use of specific plants as both analgesics and gastrointestinal aids.

Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristic of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily missing a representative structure of the T1 domain is the Kv2 family. We used X-ray crystallography to solve the structure of the human Kv2.1 T1 domain. The structure is similar to other T1 domains but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer as determined with in-line SEC-MALS-SAXS and negative stain EM. The Kv2.1 T1-T1 interface involves electrostatic interactions including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. We show that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains which may reflect the versatility of Kv2.1, the only Kv subfamily that can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Empagliflozin Relaxes Resistance Mesenteric Arteries by Stimulating Multiple Smooth Muscle Cell Voltage-Gated K+ (KV) Channels

The antidiabetic drug empagliflozin is reported to produce a range of cardiovascular effects, including a reduction in systemic blood pressure. However, whether empagliflozin directly modulates the contractility of resistance-size mesenteric arteries remains unclear. Here, we sought to investigate if empagliflozin could relax resistance-size rat mesenteric arteries and the associated underlying molecular mechanisms. We found that acute empagliflozin application produces a concentration-dependent vasodilation in myogenic, depolarized and phenylephrine (PE)-preconstricted mesenteric arteries. Selective inhibition of smooth muscle cell voltage-gated K+ channels KV1.5 and KV7 abolished empagliflozin-induced vasodilation. In contrast, pharmacological inhibition of large-conductance Ca2+-activated K+ (BKCa) channels and ATP-sensitive (KATP) channels did not abolish vasodilation. Inhibition of the vasodilatory signaling axis involving endothelial nitric oxide (NO), smooth muscle cell soluble guanylyl cyclase (sGC) and protein kinase G (PKG) did not abolish empagliflozin-evoked vasodilation. Inhibition of the endothelium-derived vasodilatory molecule prostacyclin (PGI2) had no effect on the vasodilation. Consistently, empagliflozin-evoked vasodilation remained unaltered by endothelium denudation. Overall, our data suggest that empagliflozin stimulates smooth muscle cell KV channels KV1.5 and KV7, resulting in vasodilation in resistance-size mesenteric arteries. This study demonstrates for the first time a novel mechanism whereby empagliflozin regulates arterial contractility, resulting in vasodilation. Due to known antihypertensive properties, treatment with empagliflozin may complement conventional antihypertensive therapy.

A different mechanism of C-type inactivation in the Kv-like KcsA mutant E71V

ABSTRACTA large class of K+ channels display a time-dependent phenomenon called C-type inactivation whereby prolonged activation by an external stimulus leads to a non-conductive conformation of the selectivity filter. C-type inactivation is of great physiological importance particularly in voltage-activated K+ channels (Kv), affecting the firing patterns of neurons and shaping cardiac action potentials. While understanding the molecular basis of inactivation has a direct impact on human health, its structural basis remains unresolved. Knowledge about C-type inactivation has been largely deduced from the pH-activated bacterial K+ channel KcsA, whose selectivity filter under inactivating conditions adopts a constricted conformation at the level of the central glycine (TTVGYGD) that is stabilized by tightly bound water molecules. However, C-type inactivation is highly sensitive to the molecular environment surrounding the selectivity filter in the pore domain, which is different in Kv channels than in the model KcsA. In particular, a glutamic acid residue at position 71 along the pore helix in KcsA is consistently substituted by a nonpolar valine in most Kv channels, suggesting that this side chain is an important molecular determinant of function. Here, a combination of X-ray crystallography, solid-state NMR and molecular dynamics simulations of the E71V mutant of KcsA is undertaken to explore the features associated with this Kv-like construct. In both X-ray and ssNMR data, it is observed that the filter of the Kv-like KcsA mutant does not adopt the familiar constricted conformation under inactivating conditions. Rather, the filter appears to adopt a conformation that is slightly narrowed and rigidified over its entire length. No structural inactivation water molecules are present. On the other hand, molecular dynamics simulations indicate that the familiar constricted conformation can nonetheless be stably established in the mutant channel. Together, these findings suggest that the Kv-like E71V mutation in the KcsA channel may be associated with different modes of C-type inactivation, showing that distinct selectivity filter environments entail distinct C-type inactivation mechanisms.

Structural basis of gating modulation of Kv4 channel complexes

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary β-subunits—intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)—to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1–5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2–DPP6S–KChIP1 dodecamer complex, the Kv4.2–KChIP1 and Kv4.2–DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2–KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1–S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2–KChIP1–DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.