Charged Residues between the Selectivity Filter and S6 Segments Contribute to the Permeation Phenotype of the Sodium Channel

The deep regions of the Na+ channel pore around the selectivity filter have been studied extensively; however, little is known about the adjacent linkers between the P loops and S6. The presence of conserved charged residues, including five in a row in domain III (D-III), hints that these linkers may play a role in permeation. To characterize the structural topology and function of these linkers, we neutralized the charged residues (from position 411 in D-I and its homologues in D-II, -III, and -IV to the putative start sites of S6) individually by cysteine substitution. Several cysteine mutants displayed enhanced sensitivities to Cd2+ block relative to wild-type and/or were modifiable by external sulfhydryl-specific methanethiosulfonate reagents when expressed in TSA-201 cells, indicating that these amino acids reside in the permeation pathway. While neutralization of positive charges did not alter single-channel conductance, negative charge neutralizations generally reduced conductance, suggesting that such charges facilitate ion permeation. The electrical distances for Cd2+ binding to these residues reveal a secondary “dip” into the membrane field of the linkers in domains II and IV. Our findings demonstrate significant functional roles and surprising structural features of these previously unexplored external charged residues

High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

Key pointsPatients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications.AbstractPatients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β2 -adrenergic receptor (β2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to adrenergic stimulation when compared with those of control mice fed normal chow. Genetic deletion of the β2 AR led to a normalized adrenergic response and preserved cardiac contractile reserve in HFD-fed mice. Together, these data indicate that HFD promotes phosphorylation of the β2 AR, contributing to impairment of cardiac contractile reserve before cardiac structural and functional remodelling, suggesting that early intervention in the insulin-adrenergic signalling network might be effective in prevention of cardiac complications in diabetes

The local translation of KNa in dendritic projections of auditory neurons and the roles of KNa in the transition from hidden to overt hearing loss

Local and privileged expression of dendritic proteins allows segregation of distinct functions in a single neuron but may represent one of the underlying mechanisms for early and insidious presentation of sensory neuropathy. Tangible characteristics of early hearing loss (HL) are defined in correlation with nascent hidden hearing loss (HHL) in humans and animal models. Despite the plethora of causes of HL, only two prevailing mechanisms for HHL have been identified, and in both cases, common structural deficits are implicated in inner hair cell synapses, and demyelination of the auditory nerve (AN). We uncovered that N

OPA1 mutation and late-onset cardiomyopathy: mitochondrial dysfunction and mtDNA instability.

BackgroundMitochondrial fusion protein mutations are a cause of inherited neuropathies such as Charcot-Marie-Tooth disease and dominant optic atrophy. Previously we reported that the fusion protein optic atrophy 1 (OPA1) is decreased in heart failure.Methods and resultsWe investigated cardiac function, mitochondrial function, and mtDNA stability in a mouse model of the disease with OPA1 mutation. The homozygous mutation is embryonic lethal. Heterozygous OPA(+/-) mice exhibit reduced mtDNA copy number and decreased expression of nuclear antioxidant genes at 3 to 4 months. Although initial cardiac function was normal, at 12 months the OPA1(+/-) mouse hearts had decreased fractional shortening, cardiac output, and myocyte contraction. This coincided with the onset of blindness. In addition to small fragmented mitochondria, aged OPA1(+/-) mice had impaired cardiac mitochondrial function compared with wild-type littermates.ConclusionsOPA1 mutation leads to deficiency in antioxidant transcripts, increased reactive oxygen species, mitochondrial dysfunction, and late-onset cardiomyopathy

Cardioprotection by Controlling Hyperamylinemia in a Humanized Diabetic Rat Model

BACKGROUND: Chronic hypersecretion of the pancreatic hormone amylin is common in humans with obesity or prediabetic insulin resistance and induces amylin aggregation and proteotoxicity in the pancreas. We recently showed that hyperamylinemia also affects the cardiovascular system. Here, we investigated whether amylin aggregates interact directly with cardiac myocytes and whether controlling hyperamylinemia protects the heart. METHODS AND RESULTS: By Western blot, we found abundant amylin aggregates in lysates of cardiac myocytes from obese patients, but not in controls. Aggregated amylin was elevated in failing hearts, suggesting a role in myocyte injury. Using rats overexpressing human amylin in the pancreas (HIP rats) and control myocytes incubated with human amylin, we show that amylin aggregation at the sarcolemma induces oxidative stress and Ca2+ dysregulation. In time, HIP rats developed cardiac hypertrophy and left-ventricular dilation. We then tested whether metabolites with antiaggregation properties, such as eicosanoid acids, limit myocardial amylin deposition. Rats were treated with an inhibitor of soluble epoxide hydrolase, the enzyme that degrades endogenous eicosanoids. Treatment doubled the blood concentration of eicosanoids, which drastically reduced incorporation of aggregated amylin in cardiac myocytes and blood cells, without affecting pancreatic amylin secretion. Animals in the treated group showed reduced cardiac hypertrophy and left-ventricular dilation. The cardioprotective mechanisms included the mitigation of amylin-induced cardiac oxidative stress and Ca2+ dysregulation. CONCLUSIONS: The results suggest blood amylin as a novel therapeutic target in diabetic heart disease and elevating blood levels of antiaggregation metabolites as a pharmacological strategy to reduce amylin aggregation and amylin-mediated cardiotoxicity

Cooperativity of Kv7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells.

The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered Kv7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-Kv7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered Kv7.4 channels mechanically sensitive, confirmed in consolidated Kv7.4 channels at the base of OHCs. Kv7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast Kv7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies

Substituted phenyl groups improve the pharmacokinetic profile and anti-inflammatory effect of urea-based soluble epoxide hydrolase inhibitors in murine models

a b s t r a c t Soluble epoxide hydrolase inhibitors (sEHIs) are anti-inflammatory, analgesic, anti-hypertensive, cardioand renal-protective in multiple animal models. However, the earlier adamantyl-containing urea-based inhibitors are rapidly metabolized. Therefore, new potent inhibitors with the adamantyl group replaced by a substituted phenyl group were synthesized to presumptively offer better pharmacokinetic (PK) properties. Here we describe the improved PK profile of these inhibitors and the anti-inflammatory effect of the most promising one in a murine model. The PK profiles of inhibitors were determined following p.o. administration and serial bleeding in mice. The anti-inflammatory effect of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), the most promising inhibitor among the five sEHIs tested, was investigated in a lipopolysaccharide (LPS)-challenged murine model. The earlier broadly-used adamantyl-containing sEHI, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), was used for comparison. Compared with the earlier adamantyl-containing urea-based inhibitors, substituted phenyl-containing urea-based inhibitors afford more favorable PK properties, such as higher C max s, larger AUCs and longer t 1/2 s, which, as expected, show more stable metabolic stability. Moreover, oral administration of TPPU dramatically reversed the shifts caused by LPS-challenge in plasma levels of inflammatory cytokines, epoxides and corresponding diols, which is more potent than t-AUCB. The substituted phenyl-containing sEHIs are more metabolically stable than those with adamantyl group, resulting in more potent efficacy in vivo. This indicates a new strategy for development of sEHIs for further study toward clinical trials