Potassium Channels In Membrane Research Articles

Die okuläre Neuromyotonie – klinisches Bild und Gedanken zur Pathogenese

Ocular neuromyotonia (ONM) is a rare neurogenic disorder of ocular motility. The affected patients complain of recurrent transient diplopia secondary to a sudden, painless deviation of one eye. This deviation occurs in the direction of action of an extraocular muscle, which is being stimulated at high frequency. ONM is usually seen after radiation therapy, but may also be caused by compression of the affected cranial nerve. The pathophysiology of ONM is incompletely understood, potential mechanisms include 1) ephaptic transmission along the affected nerve, 2) disturbances of potassium channels in the neuronal cell membrane, and 3) central neural re-organisation. The diagnosis of ONM is made based on clinical findings and can be supported by electrophysiological characteristics. Neuroimaging with attention to the affected cranial nerve should be obtained in order to exclude a compressive cause. Therapy of ONM with carbamazepine is usually effective.

The role of cGMP and PKG in cardioprotection

Interrupting blood flow to a heart (ischemia) for several minutes followed by several minutes of restored flow renders it very resistant to cell death from a subsequent much more prolonged ischemic episode. This phenomena, termed ischemic preconditioning, is the result of a complex signal transduction pathway which ultimately prevents formation of lethal mitochondrial permeability transition pores when blood flow is restored. In mapping this pathway we found that NOS was in the pathway [1] and proposed that it was involved in opening ATP-sensitive potassium channels (KATP) in the mitochondrial inner membrane. Those channels play a key role in preconditioning's mechanism. Incubating isolated heart mitochondria in cGMP + PKG + ATP opened the channel as indicated by mitochondrial swelling [2]. To test this hypothesis further the cGMP analog 8-CPT-cGMP (10 μM) was infused for 30 min starting 5 min prior to the end of 30 min of regional ischemia in an isolated rabbit heart [3]. It greatly attenuated the resulting infarction by an amount similar to that seen with preconditioning (infarct size approximately 50% of that in untreated hearts). 8-CPT's protection could be blocked by the KATP blocker 5 hydroxydecanoate confirming its position in the pathway. Because there is a medical need for an intervention that prevents myocardial infarction when given at reperfusion we looked for a method of raising cGMP that would be clinically feasible. We gave the direct guanylyl cyclase activator BAY 58-2667 (cinaciguat) to open-chest rabbits at 53 μg/kg bolus just prior to reperfusion followed by a 60 min 1.25 μg/min infusion. It reduced infarct size from 40.6% of the ischemic zone to only 16.0%. In another experiment we tested Vardenafil infusion of 140 μg/kg/h starting 5 min before coronary occlusion in the open-chest rabbit. It also greatly protected the hearts against infarction. Because it is controversial as to whether heart muscle expresses PDE 5, we transfected isolated rabbit ventricular myocytes with CNG channels selective for cGMP. In the resting cells cGMP as monitored with patch clamping was near zero and did not increase when they were exposed to 1 μM vardenafil. However, when cGMP was first increased with 1 μM BAY 58-2667 addition of vardenafil greatly increased the cGMP-dependent membrane current indicating that a vardenafil-sensitive PDE was present which is presumed to be PDE5. We conclude that raising cGMP in the reperfused heart is an effective anti-infarct strategy.

Large‐conductance calcium‐activated potassium channels in purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains

Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

The spectrum of ABCC8 mutations in Norwegian patients with congenital hyperinsulinism of infancy

Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11, respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty-six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11. Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6-3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8-CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.

The AP‐2 adaptin α/σ2 hemicomplex binds to a di‐isoleucine motif to mediate Kir2.3 endocytosis

We have previously shown that a prototype basolateral membrane potassium channel, Kir2.3, contains a C‐terminal trafficking cassette comprised of a biosynthetic sorting signal, a PDZ binding site, and a novel di‐isoleucine endocytic signal. Present evidence indicates these signals sequentially interact with intracellular sorting machinery and Lin‐7/CASK to drive basolateral‐directed traffic in the biosynthetic and endocytotic pathways. Although channel endocytosis is dependent on the AP‐2 adaptor, it is unknown if and how the di‐isoleucine signal is recognized by the adaptin. To explore these questions, the subunits of AP‐1 and AP‐2 were expressed either individually or as hemicomplex forms and tested for interaction with Kir2.3 in a GST pull down assay. We found Kir2.3C binds strongly and preferentially to the AP‐2 α/σ 2 hemicomplex and modestly binds to the AP‐1γ/σ 1 hemicomplex. Kir2.3 did not recognize the β2/μ2 (AP‐2) hemicomplex. Replacement of the di‐isoleucine motif with di‐alanine significantly reduced the interaction. By contrast, the canonical dileucine motif binds preferentially to the γ/σ 1 (AP‐1) hemicomplex, with a weaker binding to α/σ 2 (AP‐2). In conclusion, the di‐isoleucine signal in Kir2.3 differs from canonical di‐leucine signals and may act purely as an endocytic signal, providing new insights into the trafficking mechanism of di‐hydrophobic sorting signals. Research supported by NIH.

Plant ion channels: gene families, physiology, and functional genomics analyses.

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

Barium, Tea and Sodium Sensitive Potassium Channels are Present in the Human Placental Syncytiotrophoblast Apical Membrane

The human placental syncytiotrophoblast (hSTB) is a polarized epithelial structure, without paracellular routes, forming the main barrier for materno–fetal exchange. There is ample evidence suggesting the presence of potassium (K +) channels in the placental apical membrane; which could contribute to membrane potential and volume regulation. We have therefore examined the K + currents of isolated apical membranes from human term placenta using electrophysiological methods: reconstitution of ion channels from apical membranes into giant liposomes (single channel recordings, patch clamp method) or their functional transplantation into Xenopus laevis oocytes (total currents recording, voltage clamp method). Single channel recording experiments show the presence of K + channels in the hSTB microvillous membrane sensitive to Tetraethylammonium (TEA) and Barium (Ba +2). Patch current activity was diminished 50% and 70% by 20 mmol/L TEA and 5 mmol/L Ba +2 respectively. The more frequent conductance was approximately 73 pS, however several levels of current were detected suggesting the presence of more than one type of K + channel. In addition, sodium (Na +) sensitivity was detected in the patch current thus, over 10 mmol/L Na + reduced the seal current to 38%. These results were corroborated by the total current experiments where the K + current elicited in injected oocytes with apical purified membrane was blocked by Ba +2 and TEA. The total current was also affected by Na +, becoming larger when a Na +-free solution was used. Our results show the existence of at least two types of Ba +2-sensitive K + channels including a TEA sensitive sub-population, and some of them Na + sensitive K + channels. These channels could be the conductive pathways proposed previously for this cation in placental hSTB. Our novel contribution has been to successfully obtain K + channel recordings in systems suitable for electrophysiological studies of isolated apical membranes.

제노푸스 Cx38 세포막채널의 단일채널분석

간극결합(intercellular channel)은 인접하는 두 세포사이에 형성된 이온채널이며 이를 통해서 각종 이온, 이차 신호전달물질, 그리고 1 kDa 미만의 대사물질들이 통과한다. 아울러, sodium 혹은 potassium 이온채널처럼 반쪽의 간극결합(connexon 혹은 hemichannel)도 세포막채널로서 작용을 한다. 현재까지 간극결합을 구성하는 connexin (Cx) 단위체는 26종류 이상이 확인되었다. 이 가운데, Cx32, Cx38, Cx46 그리고 Cx50 만이 간극결합채널뿐만 아니라 세포막채널로서도 기능을 수행한다. Xenopus oocytes에서 connexin 38 (Cx38)이 발현하는 것으로 알려져 있지만 Cx38의 생물리학적 특성이 단일채널수준에서 연구가 진행된 경우는 없다. 이번 연구에서는 Cx38 채널의 생물리학적 특성, 즉 전압-의존적 개폐와 투과성(전기전도도와 이온선택성)을 알아보고자 단일채널기록을 수행하였다. Cx38 hemichannel은 전압-의존적인 빠른 개폐와 느린 개폐의 특성을 보였다. 양성전압 환경에서는 Cx38 채널이 낮은 열릴 확률(open probability)로 빠른 개폐가 유도된 반면, 음성전압에서는 느린 개폐가 높은 열릴 확률로 유도되었다. bi-ionic 실험을 통하여, Cx38 채널은 양이온보다 음이온을 더 선택 적으로 통과시킨다는 점을 알게 되었다. Cx38의 아미노산서열을 살펴보면, 아미노말단부위에 전하를 띠는 5개의 아미노산 잔기가 존재한다. 앞으로 이들 잔기를 치환시킨 돌연변이 Cx38 채널을 이용하여 과연 이들 아미노산 부위가 전압-의존적 개폐와 투과성에 관여하는 지 여부를 조사하는 연구는 매우 흥미로운 결과를 도출할 것으로 기대한다. Gap junction channels formed by two adjacent cells allow the passage of small molecules up to <TEX>${\sim}\;1\;kDa$</TEX> between them. Hemichannel (connexon or half of gap junction) also behaves as a membrane channel like sodium or potassium channels in a single cell membrane. Among 26 types of connexin (Cx), <TEX>$Cx32^*43E1$</TEX> (a chimera in which the first extracellular loop of Cx32 has been replaced with that of Cx43), Cx38, Cx46, and Cx50 form functional hemichannels as well as gap junction channels. Although it is known that Xenopus oocytes express endogenous connexin 38 (Cx38), its biophysical characteristics at single channel level are poorly understood. In this study, we performed single channel recordings from single Xenopus oocytes to acquire the biophysical properties of Cx38 including voltage-dependent gating and permeation (conductance and selectivity). The voltage-dependent fast and slow gatings of Cx38 hemichannel are distinct. Fast gating events occur at positive potentials and their open probabilities are low. In contrast, slow gatings dominate at negative potentials with high open probabilites. Based on hi-ionic experiments, Cx38 hemichannel is anion-selective. It will be interesting to test whether charged amino acid residues in the amino terminus of Cx38 are responsible for voltage gatings and permeation.

Regulating Glucose

Increased extracellular glucose stimulates firing of a subset of proopiomelanocortin (POMC)-containing neurons in the hypothalamus through a mechanism thought to depend, like insulin secretion in pancreatic β cells, on the adenosine 5′-triphosphate (ATP)-dependent closure of ATP-sensitive potassium (K ATP ) channels in the plasma membrane. Parton et al . generated transgenic mice (POMC-mut-Kir6.2 mice) that expressed a mutant K ATP Kir6.2 subunit under the transcriptional control of the POMC promoter, so that relatively ATP-insensitive K ATP channels were selectively expressed in hypothalamic POMC neurons (and in the pituitary). Although the body weight of POMC-mut-Kir6.2 mice was normal, glucose homeostasis was impaired so that they showed an exaggerated increase in blood glucose after intraperitoneal glucose injection. Whereas the firing rate of about half the POMC neurons in wild-type mice was stimulated by physiologically relevant increases in glucose concentration, only 1 of 27 POMC neurons in POMC-mut-Kir6.2 mice was glucose-sensitive. Glucose stimulated release of α-melanocyte–stimulating hormone (α-MSH) from hypothalamic slices from wild-type, but not POMC-mut-Kir6.2, mice. Moreover, it failed to stimulate the release of α-MSH from hypothalamic slices of wild-type mice fed a high-fat diet, and its effect on the firing rate of their POMC neurons was severely attenuated. Expression of the mRNA encoding uncoupling protein 2 (UCP2, a mitochondrial protein that decreases ATP production from glucose) was increased in the hypothalami of obese mice, and pharmacological inhibition of UCP with genipin stimulated firing in glucose-sensitive POMC neurons from wild-type mice. Genipin had little effect on POMC neuronal firing in POMC-mut-Kir6.2 mice, but it restored glucose-dependent release of hypothalamic α-MSH in mice fed a high-fat diet. Furthermore, although mice lacking UCP2 became obese when fed a high-fat diet, the ability of glucose to elicit release of their hypothalamic α-MSH was intact. Thus, the authors conclude that glucose sensing by hypothalamic POMC neurons is implicated in systemic glucose homeostasis, a sensitivity that is lost through a mechanism involving UCP2 in obese mice fed a high-fat diet. They further note that these findings may be pertinent to the pathogenesis of type 2 diabetes. L. E. Parton, C. P. Ye, R. Coppari, P. J. Enriori, B. Choi, C.-Y. Zhang, C. Xu, C. R. Vianna, N. Balthasar, C. E. Lee, J. K. Elmquist, M. A. Cowley, B. B. Lowell, Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity. Nature 449 , 228-232 (2007). [PubMed]

Caspase-1 inflammasomes: choosing between death and taxis

Among the arsenal of the innate immune system are the Nodlike receptors (NLRs), which detect intracellular pathogens or other ‘alarms’ and participate in the formation of the inflammasome, an oligomeric platform that dimerizes and thereby activates caspase-1. The caspase, in turn, processes pro-IL1b and pro-IL18, key cytokines for the recruitment and engagement of inflammatory cells (hence, ‘taxis’). But the activation of caspase-1 can also trigger a form of cell death with characteristics of necrosis and apoptosis that has been called ‘pyroptosis’. Two papers in this issue (FernandesAlnemri et al. and Petrilli et al.) address the role of Kþ efflux in the control of caspase-1 activation through the regulation of oligomerization of the ASC adapter molecule. In mammals, there are approximately 20 NLRs, including NALP1b, NALP3/cryopyrin, and IPAF. Upon activation, these can oligomerize and recruit ASC, which in turn binds to caspase-1 to trigger its protease activity. Such complexes were proposed as the inflammasomes, the platforms on which caspase-1 is activated. It has been known for over a decade that the activation of caspase-1 is inhibited by Kþ ions, which efflux from the cell when the cell surface receptor P2X7 is engaged by extracellular ATP, or when microbial toxins such as nigericin, gramacidin, maitotoxin, or a-toxin form potassium channels in the plasma membrane. Several studies had suggested that the Kþ efflux acts by inducing a calcium-independent phospholipase, iPLA2, required for caspase-1 processing of cytokines. Now, the laboratories of Tschopp and Alnemri show that the efflux of Kþ directly promotes inflammasome formation by promoting the oligomerization of ASC to associate with and activate caspase-1. Petrilli et al. show that ASC oligomerization and recruitment of caspase-1 by NALP3 is inhibited by Kþ , and provide evidence that the same may also be true for NALP1, while Fernandes-Alnemri et al. show that ASC can oligomerize and activate caspase-1 independent of NLRs or other proteins at sufficiently low Kþ concentrations. The idea that ASC may be the target of Kþ regulation of caspase-1 activation is further supported by the observation that the IPAF inflammasome is not regulated by Kþ . While ASC is required for activation of caspase-1 downstream of various inflammatory triggers, it is dispensable for inflammasome activity in response to Legionella, a response that requires IPAF. Thus, under some circumstances, a requirement for Kþ efflux may be overcome by engaging IPAF and bypassing ASC. Stimuli that induce the activation of caspase-1 to process cytokines do not necessarily lead to caspase-1-dependent cell death. What determines this choice (which we have facetiously labeled as between death and taxis)? FernandesAlnemri et al. suggest that the large ASC oligomer (the ‘speck’ that earned ASC its name: apoptosis-associated speck-like protein containing a CARD), which they have dubbed the ‘ASC pyroptosome’, triggers pyroptosis via caspase-1. Simplistically, a low level of active caspase-1 may process cytokines without killing, as inhibitory mechanisms have sufficient time to counteract the potentially deadly effects of the caspase unless the latter exceeds some threshold. Since levels of activation do not affect the rates or specificities of proteases, different outcomes are likely to be a consequence of the thresholds set by endogenous inhibitors and counteracting mechanisms. While some inhibitors of the inflammasome are known (e.g., caspase-12, Iceberg), little is known about how these are engaged by different inflammasomes. It is tempting to propose that Kþ levels regulate the levels of caspase-1 activation in the following way (Figure 1). When Kþ levels are normal, the inflammasome is inhibited, unless IPAF is engaged. As Kþ levels fall, NALPs can bind to ASC and promote inflammasome formation; however, as NALPs are expressed at relatively low levels as compared to ASC and caspase-1, they limit the extent of inflammasome activity. Therefore, cytokine processing rather than death prevails. Should Kþ levels fall further, ASC can oligomerize independently of the NALPs, and therefore much higher levels of caspase-1 manifest to produce deadly results. It is intriguing that the activation of caspase-1 can trigger the repair of the plasma membrane in response to microbial toxins that cause Kþ efflux, enhancing cell survival. Such feedback effects might suggest that at low levels of Kþ efflux, cell survival strategies persist, while at higher levels the cells trigger a caspase-1 suicide pathway. One problem with this simple scenario, however attractive, is that in the cell-free systems employed, similar levels of Kþ were inhibitory to both the NALP3 inflammasome and the ASC

Heat shock proteins and p53 play a critical role in K+ channel-mediated tumor cell proliferation and apoptosis

Plasma membrane potassium (K+) channels are required for tumor cell proliferation and apoptosis. However, the signal transduction mechanisms underlying K+ channel-dependent tumor cell proliferation or apoptosis remains elusive. Using HeLa and A2780 cells as study models, we tested the hypothesis that apoptotic proteins are linked with K+ channel-dependent tumor cell cycle and apoptosis. The patch-clamping study using the whole-cell mode revealed two components of voltage-gated outward K+ currents: one is sensitive to either tetraethylammonium (TEA) or tetrandrine (Tet), a maxi-conductance Ca2+-activated K+ (BK) channel blocker, and the other is sensitive to 4-aminopyridine (4-AP), a delayed rectifier K+ channel blocker. MTT and flow cytometry assays showed that TEA, Tet, or iberiotoxin (Ibtx), a selective BK channel blocker, inhibited HeLa and A2780 cell proliferation in a dose-dependent manner with G1 phase arrest. Pretreatment with TEA or Tet also induced apoptosis in HeLa and A2780 cells. However, glibenclamide (Gli), an ATP-sensitive K+ channel blocker, did not influence K+ currents, proliferation or apoptosis. Western blot analyses showed that while pretreatment of TEA and Tet produced an increase in expressions of p53, p21, and Bax, pretreatment of these two agents led to a decrease in expressions of heat shock protein (hsp)90alpha, hsp90beta, and hsp70. Our results indicate that the blockade of BK channels results in tumor cell apoptosis and cycle arrest at G1 phase, and the transduction pathway underlying the anti-proliferative effects is linked to the increased expression of apoptotic protein p53 and the decreased expression of its chaperone proteins hsp.

In vivo assessment of safety and mechanisms underlying in vitro relaxation induced by Mikania laevigata Schultz Bip. ex Baker in the rat trachea

Mikania laevigata, popularly known in Brazil as “guaco”, is largely used in folk medicine against respiratory diseases. However, neither the assessment of the toxicity of “guaco” syrup (GS, used by humans) nor its efficacy or mechanisms of action has been properly investigated. Using in vitro procedures, we showed that the hydroalcoholic extract (HE) from Mikania laevigata induces a concentration-dependent relaxation of rat trachea which does not depend on epithelium-derived substances but involves changes in the cellular mobilization of calcium, perhaps due to a direct effect on membrane potassium channels. In addition, we assessed both oral and intraperitoneal acute toxicity, as well as the oral subchronic and chronic toxicity of GS containing controlled amounts of coumarin, the main biological marker of Mikania laevigata preparations used in humans. The calculated LD 50 of GS after intraperitoneal administration was 0.904 g/kg in mice (both sexes) and 0.967 and 0.548 g/kg in male and female rats, respectively. However, the LD 50 values of GS by the oral route were calculated to be up to 10 g/kg, in both male and female mice and rats. Repeated dose 28- or 90-day oral treatment with GS (75, 150 and 300 mg/kg) did not produce any disturbances in the hematological or biochemical parameters of either male or female rats, nor did it provide evidence of toxicity in the hepatic, renal or pancreatic systems. Besides the mechanistic findings, our results provide evidence of the safety of Mikania laevigata in rodents, even after subchronic and chronic administration, at least in relation to the evaluated parameters.

Voltage‐gated K channels support proliferation of colonic carcinoma cells

Plasma membrane potassium (K+) channels are required for cell proliferation. Evidence is growing that K+ channels play a central role in the development and growth of human cancer. Here we examine the contribution and the mechanism by which K+ channels control proliferation of T84 human colonic carcinoma cells. Numerous K+ channels are expressed in T84 cells, but only voltage-gated K+ (Kv) channels influenced proliferation. A number of Kv channel inhibitors reduced DNA synthesis and cell number, without exerting apoptotic or toxic effects. Expression of several Kv channels, such as EagI, Kv 3.4 and Kv 1.5, was detected in patch clamp experiments and in fluorescence-based assays using a voltage sensitive dye. The contribution of EagI channels to proliferation was confirmed by siRNA, which abolished EagI activity and inhibited cell growth. Inhibition of Kv channels did not interfere with the ability of T84 cells to regulate their cell vol, but it restricted intracellular pH regulation. In addition, inhibitors of Kv channels, as well as siRNA for EagI, attenuated intracellular Ca2+ signaling. The data suggest that Kv channels control proliferation of colonic cancer cells by affecting intracellular pH and Ca2+ signaling.

Ammonia and urea transport across the rumen epithelium: a review

The transport of nitrogen across the rumen epithelium is characterized by absorption of ammonia from the rumen and by an influx of urea into the rumen. The transport rates of both compounds are large and exhibit wide variation. The transport of ammonia occurs in two forms: in the lipophilic form as NH3, the magnitude of which is linearly related to the pH in the ruminal fluid at pH values above 7, while at a physiological pH of 6.5 or lower, ammonia is predominantly absorbed as NH4+ via putative potassium channels in the apical membrane. The uptake of NH4+ depends on the potential difference of the apical membrane, Pda, and shows competition with K uptake. The pathway for basolateral exit of NH4+ is unknown. Hence, the relative transport rates of NH3 or NH4+ are determined by the ruminal pH according to the Henderson-Hasselbalch equation. Transport of ammonia interacts with the transport of Na and Mg mainly via changes of the intracellular pH. Urea recycling into the rumen has been known for many years and the transport across the rumen epithelium is mediated via urea transporters in the luminal and basolateral membrane of the epithelium. Transport of urea occurs by simple diffusion, but is highly variable. A significant increase of urea influx is caused by the fermentation products CO2 and short-chain fatty acids. Conversely, there is some evidence of inhibition of urea influx by ruminal ammonia. The underlying mechanisms of this modulation of urea transport are unknown, but of considerable nutritional importance, and future research should be directed to this aspect of ruminal transport.

The good, the bad and the ugly in oxygen-sensing: ROS, cytochromes and prolyl-hydroxylases

Current concepts of cellular oxygen-sensing include an isoform of the neutrophil NADPH oxidase, different electron carrier units of the mitochondrial electron transport chain (ETC), heme oxygenase-2 (HO-2), and a subfamily of 2-oxoglutarate dependent dioxygenases termed HIF (hypoxia inducible factor) prolyl hydroxylases (PHDs) and HIF asparagyl hydroxylase FIH-1 (factor-inhibiting HIF). Different oxygen sensitivities, cell-specific distribution and subcellular localization of specific oxygen-sensing cascades involving reactive oxygen species (ROS) as second messengers may help to tailor various adaptive responses according to differences in tissue oxygen availability. Herein, we propose an integrated model for these various oxygen-sensing mechanisms that very efficiently regulate HIF-alpha activity and plasma membrane potassium-channel (PMPC) conductivity.

Voltage-dependent potassium channels are involved in glutamate-induced apoptosis of rat hippocampal neurons

The role of voltage-dependent potassium channel currents in glutamate-treated rat hippocampal neurons was investigated. Cell viability was evaluated by MTT reduction assay and morphological changes. Apoptosis was detected by Hoechst33342 staining with fluorescent microscopy and propidium iodide staining with flow cytometry. Membrane potassium channel currents were recorded with whole-cell patch clamp recordings. Results showed that after shortly exposed to glutamate, about 25 and 50% cells died in 3 h and 24 h, respectively. Meanwhile, the enhancement of I K was observed within 6 h after the glutamate insult. TEA selectively blocked I K and significantly reduced cell apoptosis. I A did not change in the insult though 4-AP, the blocker of this current, showed a protective effect against the injury. These data were in consistent with the hypothesis that K + efflux contributed to glutamate-triggered apoptosis and I K channels might have a therapeutic effect on the treatment of cerebral ischemia.

Yeast Dying to Survive

There is evidence that unicellular organisms, yeast in particular, can undergo programmed cell death through a biochemical pathway with similarities to the apoptosis machinery of multicellular organisms. However, the evolutionary advantage of programmed cell death to a unicellular organism is not obvious (to say the least), leading some investigators to question the validity of a yeast programmed cell death pathway. Ivanovska and Hardwick provide an argument in favor of the biological relevance of such a death pathway in the context of cells infected with killer viruses, where the death program may benefit the community rather than a single cell. Most yeast strains are infected with viruses. One common group of double-stranded RNA virus, the M viruses, cause death of uninfected cells but provide immunity to infected cells (through an unknown mechanism). Ivanovska and Hardwick found that such killing appears to rely on components similar to those of apoptotic pathways in higher organisms. In particular, loss of the Fis1 protein, a protein that is thought to function in control of mitochondrial permeability, as do mammalian Bcl-2 family proteins, made cells more susceptible to cell death in an assay of uninfected cells cultured with cells infected with the M1 killer virus. Other components also proposed to mediate a yeast cell death pathway--Yca1, a metacaspase with similarity to mammalian caspases, and Tok1, a plasma membrane potassium channel--were shown to mediate cell death caused by the M1 virus. The M1 virus encodes a toxin, K1. Under proper culture conditions, K1 itself could cause programmed cell death, but through a separate pathway independent of the Yca1 metacaspase. In experiments with long culture conditions designed to simulate competition for survival in the wild, killer virus-infected cells overran cultures with noninfected cells. The authors propose that a primitive mechanism that distinguishes "self" (infected) and "nonself" (uninfected) cells may be evolutionarily advantageous in providing nutrients from the dying cells that enhance survival of the population of cells harboring the killer virus. I. Ivanovska, J. M. Hardwick, Viruses activate a genetically conserved cell death pathway in a unicellular organism. J. Cell Biol. 170 , 391-399 (2005). [Abstract] [Full Text]

An interactive two-state model for cell membrane potassium and sodium ion channels in electric fields using the pair approximation

An interactive two-state model for cell membrane potassium and sodium ion channels in the presence of electric fields is proposed and studied using the pair approximation. In the model, ion channels which can either be in an open or closed states are regarded as residing on a two-dimensional surface with a regular array of sites and the interactions between the neighbouring channels and the external electric field are incorporated. The minimization of the free energy is performed with respect to the pair or bond variables and maximum fractions of open potassium and sodium channels are obtained. Using known parameters for the squid giant axon the model gives qualitative agreement with the mean-field results and experimental measurements for potassium and sodium trans-membrane conductance.

Inotropic effect of Citrus sinensis (L.) Osbeck leaf extracts on the guinea pig atrium

The objective of the present investigation was to determine the contractile effect of crude and acetone leaf extracts of Citrus sinensis (L.) Osb. on mammalian myocardium. Crude leaf extracts have been used in folk medicine to treat neurological disorders. Some flavonoids isolated from this plant presented a positive inotropic effect on myocardium. This motivated us to test the extracts on the atria of guinea pigs of both sexes (300-500 g) and surprisingly we observed inotropic depression instead of an increase in force. The maximum effect of the crude extract was 79.4 +/- 8.1% of the control force amplitude (N = 5 hearts, 10 trials, 27 +/- 0.1 degrees C, stimulus: 2 Hz, 400 V, 0.5 ms). The EC50 for crude, ethanol, acetic, aqueous, and acetone extracts was 300, 300, 600, 1000, and 140 microg/ml, respectively, with a Hill constant of 1.8, 2.0, 2.5, 2.0, and 1.4, respectively. Blockade of cholinergic, beta-adrenergic, or opioid membrane receptors with 1.5 microM atropine sulfate, 1 microM propranolol, and 10 microM naloxone, respectively, did not change the effect of the crude extract. The acetone extract abolished the Bowditch positive staircase phenomenon (N = 5 hearts, 10 trials, 27 +/- 0.1 degrees C), suggesting a possible reduction of the calcium inward current, and also promoted the so-called Woodworth phenomenon. The effect was concentration-dependent and indicated the existence of another inhibitory contractile mechanism such as the simultaneous activation of some of the membrane potassium channels reducing the myocardial action potential duration and further decreasing the cellular calcium entry.

Fatal Hypoglycemia Associated with Levofloxacin

Fluoroquinolones are generally regarded as safe antimicrobial agents with relatively few adverse effects or drug interactions. Thus, they enjoy widespread use for treatment of community- and hospital-acquired infections. Although uncommon, hypoglycemia has been reported with all the fluoroquinolones and appears to occur most frequently in elderly patients with type 2 diabetes mellitus who are receiving therapy with oral hypoglycemics. The exact mechanism of this effect is unknown but is postulated to be a result of blockage of adenosine 5'-triphosphate-sensitive potassium channels in pancreatic beta-cell membranes. We report a case of fatal hypoglycemia related to levofloxacin administration in an elderly patient with diabetes. As with other fluoroquinolones, levofloxacin can cause profound and prolonged hypoglycemia. Clinicians should be cognizant of this potential adverse effect in patients with diabetes who are receiving levofloxacin therapy.