A Xanthine-Derivative K+-Channel Opener Protects against Serotonin-Induced Cardiomyocyte Hypertrophy via the Modulation of Protein Kinases

This study investigated whether KMUP-1, a xanthine-derivative K⁺ channel opener, could prevent serotonin-induced hypertrophy in H9c2 cardiomyocytes via L-type Ca²⁺ channels (LTCCs). Rat heart-derived H9c2 cells were incubated with serotonin (10 μM) for 4 days. The cell size increased by 155.5%, and this was reversed by KMUP-1 (≥1 μM), and attenuated by the LTCC blocker verapamil (1 μM) and the 5-HT_2A antagonist ketanserin (0.1 μM), but unaffected by the 5-HT_2B antagonist SB206553. A perforated whole-cell patch-clamp technique was used to investigate Ca²⁺ currents through LTCCs in serotonin-induced H9c2 hypertrophy, in which cell capacitance and current density were increased. The LTCC current (I_Ca,L) increased ~2.9-fold in serotonin-elicited H9c2 hypertrophy, which was attenuated by verapamil and ketanserin, but not affected by SB206553 (0.1 μM). Serotonin-increased I_Ca,L was reduced by KMUP-1, PKA and PKC inhibitors (H-89, 1 μM and chelerythrine, 1 μM) while the current was enhanced by the PKC activator PMA, (1 μM) but not the PKA activator 8-Br-cAMP (100 μM), and was abolished by KMUP-1. In contrast, serotonin-increased I_Ca,L was blunted by the PKG activator 8-Br-cGMP (100 μM), but unaffected by the PKG inhibitor KT5823 (1 μM). Notably, KMUP-1 blocked serotonin-increased I_Ca,L but this was partially reversed by KT5823. In conclusion, serotonin-increased I_Ca,L could be due to activated 5-HT_2A receptor-mediated PKA and PKC cascades, and/or indirect interaction with PKG. KMUP-1 prevents serotonin-induced H9c2 cardiomyocyte hypertrophy, which can be attributed to its PKA and PKC inhibition, and/or PKG stimulation.     

15-HETE suppresses K+ channel activity and inhibits apoptosis in pulmonary artery smooth muscle cells

15-Hydroxyeicosatetraenoic acid (15-HETE) is an important hypoxic product from arachidonic acid (AA) in the wall of pulmonary vessels. Although its effects on pulmonary artery constriction are well known, it remains unclear whether 15-HETE acts on the apoptotic responses in pulmonary artery smooth muscle cells (PASMCs) and whether K⁺ channels participate in this process. These hypothesises were validated by cell viability assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, mitochondrial potentials assay, caspase activity assay and western blot. We found that 15-HETE enhanced cell survival, suppressed the expression and activity of caspase-3, upregulated bcl-2 and attenuated mitochondrial depolarization, prevented chromatin condensation and partly reversed K⁺ channel opener-induced apoptosis in PASMCs under serum-deprived conditions. Our data indicated that 15-HETE inhibits the apoptosis in PASMCs through, at least in part, inactivating K⁺ channels. Yumei Li and Qian Li contributed equally to this work.     

The potential role of Kv4.3 K+ channel in heart hypertrophy

Transient outward K⁺ current (I_to) plays a crucial role in the early phase of cardiac action potential repolarization. Kv4.3 K⁺ channel is an important component of I_to. The function and expression of Kv4.3 K⁺ channel decrease in variety of heart diseases, especially in heart hypertrophy/heart failure. In this review, we summarized the changes of cardiac Kv4.3 K⁺ channel in heart diseases and discussed the potential role of Kv4.3 K⁺ channel in heart hypertrophy/heart failure. In heart hypertrophy/heart failure of mice and rats, downregulation of Kv4.3 K⁺ channel leads to prolongation of action potential duration (APD), which is associated with increased [Ca²⁺]_i, activation of calcineurin and heart hypertrophy/heart failure. However, in canine and human, Kv4.3 K⁺ channel does not play a major role in setting cardiac APD. So, in addition to Kv4.3 K⁺ channel/APD/[Ca²⁺]_i pathway, there exits another mechanism of Kv4.3 K⁺ channel in heart hypertrophy and heart failure: downregulation of Kv4.3 K⁺ channels leads to CaMKII dissociation from Kv4.3–CaMKII complex and subsequent activation of the dissociated CaMKII, which induces heart hypertrophy/heart failure. Upregulation of Kv4.3 K⁺ channel inhibits CaMKII activation and its related harmful consequences. We put forward a new point-of-view that Kv4.3 K⁺ channel is involved in heart hypertrophy/heart failure independently of its electric function, and drugs inhibiting or upregulating Kv4.3 K⁺ channel might be potentially harmful or beneficial to hearts through CaMKII.     

Activating Effect of NO on Ca2+-Dependent K+ Channels in Smooth Muscle Cells of the Main Pulmonary Artery of the Rabbit

Using a patch-clamp technique in the whole-cell configuration, we studied the effect of a nitric oxide (NO) donor, nitroglycerin (NG), on outward transmembrane ion current in isolated smooth muscle cells (SMC) of the main pulmonary artery of the rabbit. We also studied the characteristics of unitary high-conductance Ca²⁺-dependent K⁺ channels (K_Ca channels) in the SMC membrane in the cell-attached and outside-out configurations. Nitroglycerin in a 10 M concentration increased the amplitude and intensified oscillations of outward transmembrane current induced by step depolarization. In this case, the threshold of activation of the current (–40 mV) did not change. If the potential was +70 mV, the transmembrane current in the presence of NG increased, as compared with the control, by 32.6 ± 19.4% (n = 6), on average. Simultaneous addition of 10 M NG and 1 mM tetraethylammonium chloride (TEA), a blocker of K_Ca channels, to the external solution at the potential of +70 mV decreased the amplitude of outward transmembrane current with respect to the control by 25.2 ± 11% (n = 6) and suppressed oscillations of this current. In the series of experiments carried out in the outside-out configuration (concentration of K⁺ ions in the external solution was 5.9 mM), we calculated the conductance of a single K_Ca channel, which was approximately 150 pS. In the case where the potential was equal to +40 mV, 1 mM TEA suppressed completely the current through unitary K_Ca channels. In the series of experiments performed in the cell-attached configuration, 100 M NG to a considerable extent intensified the activity of unitary high-conductance K_Ca channels by increasing the probability of the channel open state (P ₀), on average, by 80 ± 1%, as compared with the control. In this case, NG did not influence the conductance of single K_Ca channels. We concluded that the NO donor NG increases the amplitude of outward transmembrane current in SMC of the rabbit main pulmonary artery by stimulation of the activity of TEA-sensitive high-conductance K_Ca channels. Our experiments carried out on single K_Ca channels demonstrated that the activating effect of NG on K_Ca channels is realized at the expense of an increase in the P ₀ of these channels, but not of a change in the conductance of single channels.     

PKC pathway associated with the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts

Our previous study indicated that TGF-beta1 induced the expression of a transient outward K+ channel (A-type) during the phenotypic transformation of vascular fibroblasts to myofibroblasts. Here, we studied the relevant signal transduction pathway using whole cell recording and a quantitative RT-PCR technique. Results indicate that the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate (PMA, 1 microM) could mimic the effect of TGF-beta1 (20 ng/ml) on the expression of an A-type K+ channel and induced a similar A-type K+ current. Moreover, a PKC inhibitor, bisindolylmaleimide I (1 microM), could abrogate the effect of TGF-beta1 on K(V)4.2 expression. This result suggests that a PKC pathway may be involved in the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts.     

Bay K 8644 induce enhancement of K+ current in both single heart cell and smooth muscle cell

Summary The effects of the Ca²⁺ agonist Bay K 8644 on outward potassium currents have been studied in single ventricular cells of chick embryo and aortic single cells of rabbit using the whole-cell patch clamp technique. Bay K 8644 was found to increase l_K in both heart and aortic single cells. This effect of Bay K 8644 on both muscle was reversed by Mn²⁺ and blocked by 20 mM TEA. The Bay K 8644 potassium I/V curve of single heart cell had a N shape, which is Ca²⁺ dependent. These data strongly suggest that Bay K 8644 increases a g_K(ca) in both aortic and heart muscle.     

Purinergic stimulation of K+-dependent Na+/Ca2+ exchanger isoform 4 requires dual activation by PKC and CaMKII

K⁺-dependent Na⁺/Ca²⁺-exchanger isoform 4 (NCXK4) is one of the most broadly expressed members of the NCKX (K⁺-dependent Na⁺/Ca²⁺-exchanger) family. Recent data indicate that NCKX4 plays a critical role in controlling normal Ca²⁺ signal dynamics in olfactory and other neurons. Synaptic Ca²⁺ dynamics are modulated by purinergic regulation, mediated by ATP released from synaptic vesicles or from neighbouring glial cells. Previous studies have focused on modulation of Ca²⁺ entry pathways that initiate signalling. Here we have investigated purinergic regulation of NCKX4, a powerful extrusion pathway that assists in terminating Ca²⁺ signals. NCKX4 activity was stimulated by ATP through activation of the P2Y receptor signalling pathway. Stimulation required dual activation of PKC (protein kinase C) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase II). Mutating T312, a putative PKC phosphorylation site on NCKX4, partially prevented purinergic stimulation. These data illustrate how purinergic regulation can shape the dynamics of Ca²⁺ signalling by activating a signal damping and termination pathway.     

Properties of the charibdotoxin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigTaenia Coli

Using the voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In our study, we identified and studied a charibdotoxin-sensitive component of Ca²⁺-dependent K⁺ current carried through the channels of high conductance (in most publications called “big conductance,”I _BK(Ca)). This component was completely blocked by 100 nM charibdotoxin and by tetraethylammonium in concentrations as low as 1 mM.I _BK(Ca) demonstrated fast kinetics of inactivation, which nearly coincided with that of Ca²⁺ current. In addition to the dependence on Ca²⁺ concentration, this current also showed voltage-dependent properties: with a rise in the level of depolarization its amplitude increased. In many cells, depolarizing shifts in the membrane potential evoke spontaneous outward currents. Such currents probably represent the secondary effect of cyclic Ca²⁺ release from the caffeine-sensitive intracellular stores that result in short-term activation of charibdotoxin-sensitive Ca²⁺-dependent K⁺ channels.     

Neuritin Activates Insulin Receptor Pathway to Up-regulate Kv4.2-mediated Transient Outward K+ Current in Rat Cerebellar Granule Neurons

Neuritin is a new neurotrophic factor discovered in a screen to identify genes involved in activity-dependent synaptic plasticity. Neuritin also plays multiple roles in the process of neural development and synaptic plasticity. The receptors for binding neuritin and its downstream signaling effectors, however, remain unclear. Here, we report that neuritin specifically increases the densities of transient outward K⁺ currents (I_A) in rat cerebellar granule neurons (CGNs) in a time- and concentration-dependent manner. Neuritin-induced amplification of I_A is mediated by increased mRNA and protein expression of Kv4.2, the main α-subunit of I_A. Exposure of CGNs to neuritin markedly induces phosphorylation of ERK (pERK), Akt (pAkt), and mammalian target of rapamycin (pmTOR). Neuritin-induced I_A and increased expression of Kv4.2 are attenuated by ERK, Akt, or mTOR inhibitors. Unexpectedly, pharmacological blockade of insulin receptor, but not the insulin-like growth factor 1 receptor, abrogates the effect of neuritin on I_A amplification and Kv4.2 induction. Indeed, neuritin activates downstream signaling effectors of the insulin receptor in CGNs and HeLa. Our data reveal, for the first time, an unanticipated role of the insulin receptor in previously unrecognized neuritin-mediated signaling.     

The Interaction of Na+ and K+ in Voltage-gated Potassium Channels : Evidence for Cation Binding Sites of Different Affinity

Voltage-gated potassium (K⁺) channels are multi-ion pores. Recent studies suggest that, similar to calcium channels, competition between ionic species for intrapore binding sites may contribute to ionic selectivity in at least some K⁺ channels. Molecular studies suggest that a putative constricted region of the pore, which is presumably the site of selectivity, may be as short as one ionic diameter in length. Taken together, these results suggest that selectivity may occur at just a single binding site in the pore. We are studying a chimeric K⁺ channel that is highly selective for K⁺ over Na⁺ in physiological solutions, but conducts Na⁺ in the absence of K⁺. Na⁺ and K⁺ currents both display slow (C-type) inactivation, but had markedly different inactivation and deactivation kinetics; Na⁺ currents inactivated more rapidly and deactivated more slowly than K⁺ currents. Currents carried by 160 mM Na⁺ were inhibited by external K⁺ with an apparent IC₅₀ <30 μM. K⁺ also altered both inactivation and deactivation kinetics of Na⁺ currents at these low concentrations. In the complementary experiment, currents carried by 3 mM K⁺ were inhibited by external Na⁺, with an apparent IC₅₀ of ∼100 mM. In contrast to the effects of low [K⁺] on Na⁺ current kinetics, Na⁺ did not affect K⁺ current kinetics, even at concentrations that inhibited K⁺ currents by 40–50%. These data suggest that Na⁺ block of K⁺ currents did not involve displacement of K⁺ from the high affinity site involved in gating kinetics. We present a model that describes the permeation pathway as a single high affinity, cation-selective binding site, flanked by low affinity, nonselective sites. This model quantitatively predicts the anomalous mole fraction behavior observed in two different K⁺ channels, differential K⁺ and Na⁺ conductance, and the concentration dependence of K⁺ block of Na⁺ currents and Na⁺ block of K⁺ currents. Based on our results, we hypothesize that the permeation pathway contains a single high affinity binding site, where selectivity and ionic modulation of gating occur.     

Glucocorticoids stimulate the activity of large-conductance Ca2+ -activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca2+ -activated K+ current (IK(Ca)) with an EC50 value of 21 +/- 5 microM. Dexamethasone-induced increase in IK(Ca) density was suppressed by paxilline (1 microM), yet not by glibenclamide (10 microM), pandinotoxin-Kalpha (1 microM) or mifepristone (10 microM). Paxilline is a blocker of large-conductance Ca2+ -activated K+ (BKCa) channels, while glibenclamide and pandinotoxin-Kalpha are blockers of ATP-sensitive and A-type K+ channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 microM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BKCa -channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca2+ -sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 microM) also increased BKCa -channel activity. Dexamethasone-mediated stimulation of IK(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.     

Short-Term Regulation of the Proximal Tubule Na+,K+-ATPase: Increased/Decreased Na+,K+-ATPase Activity Mediated by Protein Kinase C Isoforms

In different species and tissues, a great variety of hormones modulate Na⁺,K⁺-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na⁺,K⁺-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na⁺,K⁺-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na⁺,K⁺-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.     

Kir4.1-mediated spatial buffering of K+: Experimental challenges in determination of its temporal and quantitative contribution to K+ clearance in the brain

Neuronal activity results in release of K⁺ into the extracellular space of the central nervous system. If the excess K⁺ is allowed to accumulate, neuronal firing will be compromised by the ensuing neuronal membrane depolarization. The surrounding glial cells are involved in clearing K⁺ from the extracellular space by molecular mechanism(s), the identity of which have been a matter of controversy for over half a century. Kir4.1-mediated spatial buffering of K⁺ has been promoted as a major contributor to K⁺ removal although its quantitative and temporal contribution has remained undefined. We discuss the biophysical and experimental challenges regarding determination of the contribution of Kir4.1 to extracellular K⁺ management during neuronal activity. It is concluded that 1) the geometry of the experimental preparation is crucial for detection of Kir4.1-mediated spatial buffering and 2) Kir4.1 enacts spatial buffering of K⁺ during but not after neuronal activity.     

Properties of the apamin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigtaenia coli

With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca²⁺-dependent K⁺ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I _SK(Ca)). This component did not show the temporal inactivation;I _SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI _SK(Ca) is very sensitive to changes in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ): a decrease in [Ca²⁺] _i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca²⁺ channels of L-type was not an obligatory condition for activation ofI _SK(Ca). The current-voltage relationship forI _SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Spatial,Cellular, and Intracellular Localization of Na+/K+-ATPase in the Sterically Disposed Renal Tubules of Japanese Eel

The kidney plays a crucial role in the regulation of water and ion balances in both freshwater and seawater fishes. However, the complicated structures of the kidney hamper comprehensive understanding of renal functions. In this study, to investigate the structure of sterically disposed renal tubules, we examined spatial, cellular, and intracellular localization of Na⁺/K⁺-ATPase in the kidney of the Japanese eel. The renal tubule was composed of the first (PT-I) and second (PT-II) segments of the proximal tubule and the distal tubule (DT), followed by the collecting ducts (CDs). Light microscopic immunocytochemistry detected Na⁺/K⁺-ATPase along the renal tubules and CD; however, the subcellular distribution of the Na⁺/K⁺-ATPase immunoreaction varied among different segments. Electron microscopic immunocytochemistry further revealed that Na⁺/K⁺-ATPase was distributed on the basal infoldings of PT-I, PT-II, and DT cells. Three-dimensional analyses showed that the renal tubules meandered in a random pattern through lymphoid tissues, and then merged into the CD, which was aligned linearly. Among the different segments, the DT and CD cells showed more-intense Na⁺/K⁺-ATPase immunoreaction in freshwater eel than in seawater-acclimated eel, confirming that the DT and CD segments are important in freshwater adaptation, or hyperosmoregulation. (J Histochem Cytochem 58:707–719, 2010)     

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na⁺/K⁺-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na⁺/K⁺-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na⁺/K⁺-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.     

Ultracytochemical study of Ca++-ATPase and K+-NPPase activities in retinal photoreceptors of the guinea pig

Summary Ca⁺⁺-ATPase activity was demonstrated histochemically at light- and electron-microscopic levels in inner and outer segments of retinal photoreceptor cells of the guinea pig with the use of a newly developed one-step lead-citrate method (Ando et al. 1981). The localization of ouabain-sensitive, K⁺-dependent p-nitrophenylphosphatase (K⁺-NPPase) activity, which represents the second dephosphorylative step of the Na⁺-K⁺-ATPase system, was studied by use of the one-step method newly adapted for ultracytochemistry (Mayahara et al. 1980). In retinal photoreceptor cells fixed for 15 min in 2% paraformaldehyde the electron-dense Ca⁺⁺-ATPase reaction product accumulated significantly on the inner membranes of the mitochondria but not on the plasmalemma or other cytoplasmic elements of the inner segments. The membranes of the outer segments remained unstained except the membrane arrays in close apposition to the retinal pigment epithelium. The cytochemical reaction was Ca⁺⁺- and substrate-dependent and showed sensitivity to oligomycin. When Mg⁺⁺-ions were used instead of Ca⁺⁺-ions, a distinct reaction was also found on mitochondrial inner membranes.In contrast to the localization of the Ca⁺⁺ -ATPase activity, the K⁺-NPPase activity was demonstrated only on the plasmalemma of the inner segments, but not on the mitochondria, other cytoplasmic elements or the outer segment membranes. This reaction was almost completely abolished by ouabain or by elimination of K⁺ from the incubation medium.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany     

Forskolin and phorbol esters decrease the same K+ conductance in cultured oligodendrocytes

Summary Cultured ovine oligodendrocytes (OLGs) express a number of voltage-dependent potassium currents after they attach to a substratum and as they begin to develop processes. At 24–48 hours following plating, an outward potassium current can be identified that represents a composite response of a rapidly inactivating component and a steady-state or noninactivating component. After 4–7 days in culture, OLGs also develop an inward rectifier current. We studied the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) on OLG outward currents. These compounds are known to alter the myelinogenic metabolism of OLGs. PMA, an activator of protein kinase C (PK-C), has been shown to enhance myelin basic protein phosphorylation while forskolin acting on adenylate cyclase, and thereby increasing cAMP, inhibits it. Both forskolin and PMA increase the phosphorylation of 23-cyclic nucleotide phosphodiesterase, an OLG/myelin protein. We found that forskolin decreased the steady-state outward current at 120 mV by 10% at 100nm, and by 72% at 25 m from a holding potential of –80 mV. The time course of inactivation of the peak currents was decreased, affecting both the fast and slow time constants. There was no significant change in the steady-state parameters of current activation and inactivation. The effect of forskolin was attenuated when the adenylate cyclase inhibitor adenosine (2mm) was present in the intracellular/pipette filling solution. The results of PMA experiments were similar to those obtained with forskolin. Whereas the amplitude of the currents in the presence of PMA was reduced by 28% at 1.5nm and 60% and 600nm, the decay phase of the peak currents was less affected. The PMA effect could still be seen when the intracellular Ca²⁺ was reduced to 10nm with 5mm BAPTA, but was inhibited when the cells were pre-exposed to 50 m psychosine, a PK-C inhibitor. It is postulated that the potassium currents in OLG can be physiologically modulated by two distinct second-messenger systems, perhaps converging at the level of a common phosphorylated enzyme or regulatory protein.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.