Elevated Potassium Ion Concentration Research Articles

Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus

Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the dynamic endosomal environment, which triggers conformational changes in the spikes that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition, and endosome-resident proteins, and these cues dictate when and where viral fusion occurs. We recently reported that infection with an enveloped bunyavirus requires elevated potassium ion concentrations [K+], controlled by cellular K+ channels, that are encountered during viral transit through maturing endosomes. Here we reveal the molecular basis for the K+ requirement of bunyaviruses through the first direct visualization of a member of the Nairoviridae family, namely Hazara virus (HAZV), using cryo-EM. Using cryo-electron tomography, we observed HAZV spike glycoproteins within infectious HAZV particles exposed to both high and low [K+], which showed that exposure to K+ alone results in dramatic changes to the ultrastructural architecture of the virion surface. In low [K+], the spikes adopted a compact conformation arranged in locally ordered arrays, whereas, following exposure to high [K+], the spikes became extended, and spike–membrane interactions were observed. Viruses exposed to high [K+] also displayed enhanced infectivity, thus identifying K+ as a newly defined trigger that helps promote viral infection. Finally, we confirmed that K+ channel blockers are inhibitory to HAZV infection, highlighting the potential of K+ channels as anti-bunyavirus targets.

Leaching from the stratum corneum does not explain the previously reported elevated potassium ion concentration in sweat.

The purpose of this study was to determine if K+ is leached from the stratum corneum when sweat is present on the skin's surface. The results will help address whether sweat [K+] previously reported in the literature are artifactually elevated as a result of K+ leaching. Twelve (six female, six male) healthy volunteers participated in this study. After thorough skin cleansing and preparation with isopropyl alcohol and high-performance liquid chromatography-grade distilled water, three sites were chosen and a 50 μL drop of artificial sweat was pipetted directly onto the skin. The artificial sweat had a [K+] of 4 mEq·L-1, an osmolality of 120 mosm·L-1, and a pH of 6.0. Immediately following, a clear plastic cover slip (~6 cm2) with a shallow 0.8 cm2 convex impression in the center was applied over each drop, preventing evaporation. Each sample was allowed to sit on the forearm, under the plastic cover slip, for 10 min. The mean (±SD) [K+] in 'artificial' sweat not exposed to the skin was measured to be 4.2±0.4 mEq·L-1. After 10 min of exposure to the stratum corneum of the forearm, the artificial sweat had a mean (±SD) [K+] of 3.9±0.3 mEq·L-1. There was no significant difference (p>0.05) in the [K+] between the control artificial sweat and the samples collected after 10 min of exposure to forearm skin. These results do not support the hypothesis that significant K+ leaching from the stratum corneum into standing sweat is the cause for the previously reported elevated sweat [K+].

Optical mapping of propagation changes induced by elevated extracellular potassium ion concentration in genetically altered mouse hearts

Diabetes is associated with high rates of cardiovascular disease and sudden death. Therefore, dissecting specific mechanisms, such as the effects of impaired insulin signaling on cardiac electrophysiology may lead to better diagnosis and treatment. Lack of insulin receptors in mouse myocytes has been shown to reduce repolarizing potassium currents and prolong action potential duration. We hypothesized that these changes would manifest as rate-related effects on electrical propagation in the intact heart. This study employed optical mapping to characterize propagation changes in intact mouse hearts with cardiomyocyte-restricted knock out of insulin receptors (CIRKO). Methods: Fluorescent signals emitted from excited Di-4-ANEPPS in isolated Langendorff perfused mouse hearts were recorded from the left ventricular epicardium using an 8 by 8 photo diode array. The study included hearts from 8 CIRKO mice and 8 wild type (WT) littermate controls. Hearts were stimulated from the right atrium or the left ventricle at basic cycle lengths ranging from 160 to 280 ms under normal conditions and then after 5 minutes of perfusion with elevated potassium ion concentration (9.4 mM). Results: None of the 8 CIRKO hearts maintained regular responses to atrial stimulation at the 160 ms cycle length under normal conditions; however, all of the WT hearts were captured at this rate. Total activation time for a 4 mm by 4 mm area was longer for CIRKO hearts when compared with WT. Average epicardial conduction velocity was slower for the CIRKO when compared to WT. Propagation delay due to the presence of high [K+] e was significant in both CIRKO and WT mice, but significantly longer for the CIRKO hearts. Conclusions: These results show that in addition to reducing repolarization currents, impaired myocardial insulin signaling leads to impaired electrical impulse propagation particularly at increased heart rates. These data suggest a link between impaired myocardial insulin signaling and the increased risk of arrhythmia and sudden death in patients with diabetes.

The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration

The effects of K+-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing ‘low’ K+ (5 or 10 mM), as well as in the presence of ‘high’ K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in ‘high’ as compared with ‘low’ K+-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-d-glucose accumulation and increased N-methyl-d-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in ‘high’ K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K+-induced membrane depolarization.

Metorphamide, a novel endogenous adrenal opioid peptide, inhibits nicotine-induced secretion from bovine adrenal chromaffin cells

Opioid peptides are found in high concentrations in the adrenal medulla. Recently, a novel opioid octapeptide, metorphamide, possessing an amidated C-terminal, was characterized and also found to be present in adrenal tissue. We have studied the ability of this novel peptide to modify nicotine-induced secretion from isolated bovine adrenal chromaffin cells. Exocytosis was monitored by measuring adenosine triphosphate (ATP) release on-line by the luciferin-luciferase bioluminescence method, or by measuring endogenous catecholamine release by high-performance liquid chromatography (HPLC) with electrochemical detection. Metorphamide inhibited 5 μM nicotine-induced ATP release from fresh chromaffin cells by almost 50% at 5 μM. Metorphamide at concentrations less than 1 μM had no effect on 5 μM nicotine-induced adrenaline and noradrenaline release from cultured cells, but at higher concentrations inhibited their release equally, with an IC 50 of approximately 10 μM. By contrast, Met 5-enkephalin inhibited the release of both catecholamines equally with an IC 50 of 1mM, making metorphamide greater than 100-fold more potent than Met 5-enkephalin in this system. Naloxone (10 μM) and diprenorphine (1 μM) failed to antagonise the inhibitory action of metorphamide on nicotine-induced catecholamine release. Metorphamide inhibited the nicotinic response in a non-competitive manner, and failed to affect either adrenaline or noradrenaline release induced by elevated potassium ion concentrations. The results suggest metorphamide acts on naloxone- and diprenorphine-resistant receptors to inhibit chromaffin cell nicotinic secretion and that the novel amidated C-terminal of the peptide is important for this action.

Release of gonadotropin-releasing hormone from the Japanese quail hypothalamus in vitro

The regulation of GnRH (gonadotropin-releasing hormone) secretion in Japanese quail was studied by maintaining excised hypothalamic slices containing the median eminence region in a continuous flow superfusion system. GnRH released into the superfusate was measured by radioimmunoassay using an antibody generated against synthetic GnRH. GnRH secretion increased in a dose-response manner when hypothalamic slices from female quail were exposed to superfusion medium containing elevated potassium ion concentrations. The potassium-induced GnRH release was found to be calcium dependent. GnRH was also increased when either 1 × 10 −6 M norepinephrine, epinephrine, or 1 × 10 −5 M of the β-adrenergic agonist, isoproterenol was added to the superfusion medium. GnRH secretion from hypothalami of castrate and intact male quail was measured on Days 1, 10, and 21 of photostimulation and was elevated on Day 21. No differences were observed between the castrate and intact groups. It is concluded that GnRH is released from the quail hypothalamus in a manner analogous to mammals and that its release is subject to adrenergic stimulation.

Is GABA release modulated by presynaptic receptors?

THERE is considerable evidence, in both the peripheral and central nervous systems, that presynaptic α-adrenoreceptors modulate noradrenaline release induced by both electrical and potassium ion stimulation; α-agonists inhibit and α-antagonists facilitate this release1–5. There is also evidence that similar control mechanisms occur in dopaminergic4,6–9 and cholinergic10 neurones. Here we report that the release of γ-aminobutyric acid (GABA) from in vitro rat frontal cortex, caused by elevated potassium ion concentrations, is reduced by the GABA agonists muscimol and 3-aminopropane sulphonic acid, and by GABA itself, and also that the effect of muscimol is antagonised by the GABA antagonists bicuculline and picrotoxin. This in vitro study demonstrates that the release of GABA is subject to a negative feedback control which may be important in vivo, and further, that this mechanism can be modified by drugs.

Effect of elevated potassium ion concentrations on electrically evoked release of [ 3H]acetylcholine in slices of rat hippocampus

To establish the effect of raising the concentration of extracellular potassium ions on axonal conduction and transmitter release in a mammalian central pathway, the septohippocampal cholinergic tract, the rate of [ 3H]acetylcholine release evoked by electrical stimulation was measured in rat hippocampal slices superfused with Krebs' solution containing 3–15 m m K +. The evoked release of [ 3H]acetylcholine was abolished by the presence of tetrodotoxin or by the omission of Ca 2+ in the superfusion medium, indicating that it originated from terminals depolarized by conducted action potentials. Potassium concentrations between 3 and 8m m had no effect but 10–15 m m K + reduced the rate of evoked release and decreased the size of the releasable pool of [ 3H]acetylcholine. Decreasing the sodium content of the Krebs' solution to 97 m m or less had effects similar to those of elevated [K +]. Elevated K + (10–15 m m) reversibly reduced the size of compound action potentials in the fimbria and the alveus. The results suggest that extracellular potassium concentrations occurring under physiological conditions do not affect axonal conduction and transmitter release but that both are reduced in pathological states when extracellular [K +] above 8 m m occur.

Differential effects of elevated potassium ion concentration and of theophylline on growth hormone release by rat adenohypophyses in vitro.

The effects of 27 mM K+ and of 6.7 mM theophylline on the release of growth hormone (GH) by rat hemipituitaries in vitro were investigated using bioassay (rat tibia test) and radioimmunoassay (RIA). Both agents markedly increased the release of immunoreactive GH. High K+ also promoted the release of bioactive GH but to a much lesser degree than RIA-GH. Theophylline did not consistently affect the release of bioassay-detectable GH. The results suggest that these agents promote massive release of a form of immunoreactive GH (possibly "immature") that has little or no activity in the bioassay. Theophylline is relatively more effective in this regard than is elevated K+.

Tracer and nontracer potassium fluxes in squid giant axons and the effects of changes in external potassium concentration and membrane potential.

The efflux of labeled and unlabeled potassium ions from the squid giant axon has been measured under a variety of experimental conditions. Axons soaked in sea water containing 42K ions lost radioactivity when placed in inactive sea water according to kinetics which indicate the presence of at least two cellular compartments. A rapidly equilibrating superficial compartment, probably the Schwann cell, was observed to elevate the specific activity of 42K lost from such axons to K-free sea water for a period of hours. The extra radioactive potassium loss from such axons during stimulation, however, was shown to have a specific activity identical within error to that measured in the axoplasm at the end of the experiment. The same was shown for the extra potassium loss occurring during passage of a steady depolarizing current. Axons placed in sea water with an elevated potassium ion concentration (50 mM) showed an increased potassium efflux that was in general agreement with the accompanying increase in membrane conductance. The efflux of potassium ions observed in 50 mM K sea water at different membrane potentials did not support the theory that the potassium fluxes obey the independence principle.