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Insulin increases the density of potassium channels in white adipocytes: possible role in adipogenesis.

We studied the potassium currents in white adipocytes obtained by culturing preadipocytes from rat epididymal tissue, both with insulin (WA(i)) and without insulin (WA(o)), in order to test the role of insulin in the development of voltage-gated potassium channels (K(v)) during adipogenesis. Occasionally, very small potassium currents (I(K,V)) were present in preadipocytes; however these currents were measured in all differentiated cells (adipocytes). WA(i) exhibited greater macroscopic potassium currents than WA(o) with no apparent differences in kinetics or voltage dependence. The current density (pA/ micro m(2)) calculated in WA(i) was higher than in WA(o). Currents were blocked by millimolar concentrations of tetrethylamonium (TEA). The effect of insulin on adipogenesis, both with and without TEA, was analysed. Four days without insulin and three days with insulin were necessary to increase the total number of cells in culture by 2.5-fold. Insulin increased the number of differentiated cells by 73.5%. Cell proliferation and differentiation were inhibited by TEA. Proliferation was affected only by high concentration of TEA. Inhibition of differentiation was dose dependent, with the concentration necessary for half-block similar to the IC(50) values to block potassium channels. These results suggest that insulin increases the density of K(v) and that these channels may be necessary for the normal growth of white adipocytes in culture.

Adipocytes↗

[The effect of nitric oxide on potassium channels of bronchial smooth muscle cells from asthmatic rats].

OBJECTIVE: To investigate the effect of nitric oxide (NO) on resting membrane potential (Em) and potassium currents of bronchial smooth muscle cells (BSMC) from asthmatic rat models. METHODS: Sixteen male SD rats were randomly divided into two groups: a control group and an asthmatic model group. Single BSMC was obtained by acute enzyme-digestion separation method. All experiments were conducted in conventional whole-cell configuration of patch clamp technique. Em currents of Ca2+ activated potassium channels (BKCa) and voltage-dependent potassium channel (Kv) of two groups were separately measured, and the changes of The resting Em and potassium currents of BSMC before and after addition of NO donor sodium nitroprusside (SNP) were also measured. RESULTS: (1) The Em of the asthmatic model group [(-29 +/- 6) mV, n = 12] was significantly lower than that of the control group [(-35 +/- 6) mV, n = 15, P < 0.05]; SNP significantly increased Em of the asthmatic model group to [(-38 +/- 7) mV, n = 12, (P < 0.0 1)], there is no significant difference of Em between normal group and that of asthmatic model group after SNP treatment (P > 0.05), which meant that SNP could repolarize BSMC to normal. (2) The mean current density of BKCa from asthmatic model group [(44 +/- 17) pA/pF, n = 8] under pulse protocol was significantly lower than that of the control group [(73 +/- 20) pA/pF, n = 10, P < 0.01], and SNP significantly increased the mean current density of the asthmatic model group to [(79 +/- 16) pA/pF, n = 10, P < 0.01], which was close to control group (P > 0.05); under ramp protocol, the current densities of control and asthmatic model group were [(75 +/- 19) pA/pF, n = 10] and [(46 +/- 16) pA/pF, n = 8] respectively, there was significant difference between two groups (P < 0.01), SNP treatment significantly increased current density of asthmatic model group to [(82 +/- 21) pA/pF, n = 8, P < 0.01]. (3) The mean current density of Kv of the asthmatic model group [(32 +/- 9) pA/pF, n = 8] under pulse protocol was significantly lower than that of the control group [(58 +/- 10) pA/pF, n = 8, P < 0.05], and SNP significantly increased the mean current density of Kv of the asthmatic model group to [(45 +/- 13) pA/pF, n = 8, P < 0.05]. Under ramp protocol, current density of Kv of asthmatic model group was [(38 +/- 11) pA/pF, n = 8], which was significantly lower than that of control group [(62 +/- 14) pA/pF, n = 8, P < 0.05], SNP treatment significantly increased Kv current density of asthmatic model group to [(53 +/- 9) pA/pF, n = 8, P < 0.05]. there was a similar change. CONCLUSIONS: SNP can improve the resting membrane potential of BSMC from rat asthmatic models and increase the potassium channel activity of BSMC, which is impaired in asthma status. The result suggests that potassium channel mediates the relaxation effect of NO on asthmatic airway smooth muscle.

Animals↗

Expression of two-pore domain potassium channels in nonhuman primate sperm.

OBJECTIVE: Two-pore domain potassium channels (K(2P)) play integral roles in cell signaling pathways by modifying cell membrane resting potential. Here we describe the expression and function of K(2P) channels in nonhuman primate sperm. DESIGN: Experimental animal study, randomized blinded concentration-response experiments. SETTING: University-affiliated primate research center. ANIMAL(S): Male nonhuman primates. INTERVENTION(S): Western blot and immunofluorescent analysis of epididymal sperm samples. Kinematic measures (curvilinear velocity and lateral head displacement) and acrosome status were studied in epididymal sperm samples exposed to K(2P) agonist (docosahexaenoic acid) and antagonist (gadolinium). MAIN OUTCOME MEASURE(S): Semiquantitative protein expression and cellular localization and quantitative changes in specific kinematic parameters and acrosome integrity. RESULT(S): Molecular analysis demonstrated expression and specific regional distribution of TRAAK, TREK-1, and TASK-2 in nonhuman primate sperm. Docosahexaenoic acid produced a concentration-dependent increase in curvilinear velocity (P<.0001) with concomitant concentration-dependent reductions in lateral head displacement (P=.005). Gadolinium reduced velocity measures (P<.01) without significantly affecting lateral head displacement. CONCLUSION(S): The results demonstrated expression and function of K(2P) potassium channels in nonhuman primate sperm for the first time. The unique, discrete distributions of K(2P) channels in nonhuman primate sperm suggest specific roles for this subfamily of ion channels in primate sperm function.

Animals↗

Functional differences between 4-aminopyridine and tetraethylammonium-sensitive potassium channels in myelinated axons.

Intracellular recordings from rat sciatic nerve fibers showed that the potassium channel blocking agents 4-aminopyridine (4-AP) and tetraethylammonium (TEA) had different effects on action potential waveform. When applied alone, TEA did not appreciably alter the waveform of an individual action potential, whereas 4-AP application resulted in action potential broadening and, in some axons, repetitive firing. A prolonged afterhyperpolarization which was blocked by TEA occurred subsequent to repetitive firing. These results indicate the presence of at least two pharmacologically defined potassium channels in mammalian peripheral nerve fibers. The 4-AP-sensitive potassium channels are important for rapid action potential repolarization and the TEA-sensitive potassium channels may serve to modulate axonal excitability during repetitive firing.

4-Aminopyridine↗

Effects of nitric oxide on the Ca2+-activated potassium channels in smooth muscle cells of the human corpus cavernosum.

Relaxation of the corpus cavernosum smooth muscle is an absolute prerequisite of penile erection. Potassium channels play a role in the physiologic regulation of corporal smooth muscle tone. Among the several subtypes of potassium channels, Ca2 +-activated potassium channel (KCa channel) subtypes are thought to be the most physiologically relevant in the regulation of corporal smooth muscle tone. The purpose of this study was to investigate the effects of nitric oxide (NO) and sildenafil on the KCa channels and elucidate the mechanisms of action on the KCa channels in smooth muscle cells of the human corpus cavernosum. The conventional patch-clamp technique was applied to short-term cultured smooth muscle cells of the human corpus cavernosum. Single-channel currents were recorded in cell-attached or inside-out patches, and whole-cell currents were recorded in perforated-patches. In cell-attached patches, sildenafil alone did not activate the KCa channels but sildenafil enhanced the NO-induced activation of KCa channels. The open probability of KCa channels was increased significantly after application of NO donor, SIN-1 (100 microM) (47 +/- 7.1%, n = 10, P=0.002). The application of sildenafil (100 nM) with SIN-1 (100 microM) markedly increased the open probability of KCa channels (148 +/- 24%, n = 8, P < 0.001). The activation by SIN-1 or sildenafil with SIN-1 was completely blocked by pretreatment of the soluble guanylate cyclase inhibitor, ODQ (10 microM). In inside-out patches. SIN-1 or sildenafil with SIN-1 failed to activate KCa channels at pCa 7.5 (n=5). SIN-1 increased the whole cell outward K+ currents in all holding potential. The increased IK by SIN-1 was inhibited by charybdotoxin (CTX) about 70%. These data provide compelling evidence consistent with the involvement of the KCa channel subtype in modulating NO-induced relaxation responses in human corporal smooth muscle. Furthermore, the activation of KCa channels is thought to be mediated by activation of soluble guanylate cyclase, leading to increased intracellular levels of cyclic GMP and the subsequent activation of protein kinase rather than direct NO effect.

Cells, Cultured↗

Formation of intermediate-conductance calcium-activated potassium channels by interaction of Slack and Slo subunits.

Large-conductance calcium-activated potassium channels (maxi-K channels) have an essential role in the control of excitability and secretion. Only one gene Slo is known to encode maxi-K channels, which are sensitive to both membrane potential and intracellular calcium. We have isolated a potassium channel gene called Slack that is abundantly expressed in the nervous system. Slack channels rectify outwardly with a unitary conductance of about 25-65 pS and are inhibited by intracellular calcium. However, when Slack is co-expressed with Slo, channels with pharmacological properties and single-channel conductances that do not match either Slack or Slo are formed. The Slack/Slo channels have intermediate conductances of about 60-180 pS and are activated by cytoplasmic calcium. Our findings indicate that some intermediate-conductance channels in the nervous system may result from an interaction between Slack and Slo channel subunits.

Amino Acid Sequence↗

Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis.

The positively charged S4 region of voltage-dependent potassium channels moves outward during depolarization, leading to channel opening, but possible movement of the negatively charged S2 region may be more complex. Here we have studied possible movement of the S2 region of the slowly activating human voltage-dependent potassium channel hKv2.1. For this, cysteine mutants in the S2 region were expressed in Xenopus oocytes by injection of cRNA. Whole-cell currents were measured using the two-electrode voltage-clamp technique, and the effect of the membrane-impermeable cysteine-binding reagent parachloromercuribenzenesulfonate (PCMBS) was studied. For mutant S223C (located just outside the membrane in the S2 region), PCMBS inhibited currents and caused faster deactivation of tail currents. The time course of reactivity of PCMBS on tail current amplitudes was faster at more negative holding potentials. There was no effect of PCMBS on potassium channel currents for mutants D225C, N226C, A230C, and V232C. These data suggest that residue S223 is exposed to the extracellular phase at normal resting potentials, making it accessible to PCMBS, but upon depolarization there is a conformational change, making it less accessible, possibly by a local rather than global movement of S2 residues into the membrane. Voltage-dependent movements of nearby residues could also explain the results.

4-Chloromercuribenzenesulfonate↗

Immunolocalization of voltage-gated potassium channel Kv3.1b subunit in the cochlea.

Immunohistochemical localization of voltage-gated potassium channel Kv3.1b subunit was studied in the cochlea. Intense Kv3.1b-like immunoreactivity was present in the type I, type III, type IV and suprastrial fibrocytes of the cochlear lateral wall. Immunostaining was also found in the interdental cells and the fibrocytes of the spiral limbus and in the supralimbal dark cells. K+ ions, which play a pivotal role in the mechanosensory transduction process in the inner ear, are recycled via gap junctional networks in the cochlea. These results suggest that the voltage-gated potassium channel, containing Kv3.1b, in the cochlear lateral wall fibrocytes may control the intracellular potential and play an important role in regulating the potassium ion recycling mechanism via gap junctions in the inner ear.

Acetic Acid↗

Nitric oxide inhibits irreversibly P815 cell proliferation: involvement of potassium channels.

Nitric oxide (NO) has been shown to inhibit both normal and cancer cell proliferation. Potassium channels are involved in cell proliferation and, as NO activates these channels, we investigated the effect of NO on the proliferation of murine mastocytoma cell lines and the putative involvement of potassium channels. NO (in the form of NO donors) caused dose-dependent inhibition of cell proliferation in the P815 cell line inducing growth arrest in the mitosis phase. Incubation with NO donor for 4 or 24 h had a similar inhibitory effect on cell proliferation, indicating that this effect is irreversible. The inhibitory effect of NO was completely prevented by the blockade of voltage- and calcium-dependent potassium channels, but not by blockade of ATP-dependent channels. NO inhibition of cell proliferation was unaffected by guanylate cyclase and by cytoskeleton disruptors. Therefore, NO inhibits cell proliferation irreversibly via a potassium channel-dependent but guanylate cyclase-independent pathway in murine mastocytoma cells.

Animals↗

Recent developments on KCNQ potassium channel openers.

During the past five years, several members of the KCNQ potassium channel gene family have been identified with a high degree of CNS specificity. Within the KCNQ family, the combination of the KCNQ2/KCNQ3 proteins, and the KCNQ5/KCNQ3 arrangement has been identified as the molecular correlates of the different M-currents. Several lines of evidence are emerging demonstrating the importance of these channels in regulating neuronal excitability; for example, determination of the excitability threshold, firing properties, and responsiveness of neurons to synaptic inputs. Recent studies have shown that KCNQ openers have potential for the treatment of several CNS disorders characterized by neuronal hyperexcitability, such as migraine, epilepsy and neuropathic pain. This article reviews the recent developments of KCNQ potassium channel openers.

Acrylamides↗

Contribution of potassium channels to the discharge properties of rat aortic baroreceptor sensory endings.

The expression of several types of membrane potassium channel at the cell body and central synaptic terminal of the rat aortic arch baroreceptor has been reported by others. It is not known if any of the same channels function at the peripheral sensory terminal of these afferent nerves. Our study examined the effect of three potassium channel blocking agents on the pressure-evoked discharge of such baroreceptors. Thirty-one single unit, regularly discharging baroreceptors were studied using an in vitro aortic arch-aortic nerve preparation. Discharge thresholds and suprathreshold pressure sensitivities were derived from responses of receptors to slowly rising ramps of pressure applied to the aortic arch. Vessel diameter was recorded along with receptor discharge to assess any drug-induced changes in vascular smooth muscle. The blocking agents tested have a range of specificities for classes of potassium channels: tetraethylammonium (TEA), 4-aminopyridine (4-AP) and charybdotoxin. TEA depressed the pressure sensitivity of all baroreceptors tested (n = 3) in a dose-dependent manner. Baroreceptor responses to 4-AP were complex (n = 22) and varied widely across individuals. Three were unaffected by 5 mM 4-AP. Most baroreceptors were generally depressed by 4-AP. Some of the 4-AP effects appeared to be related to actions at vascular smooth muscle. None of the baroreceptors tested (n = 6) was affected by charybdotoxin. The results of selective potassium channel blockade are generally consistent with what would be expected from a sustained depolarization of baroreceptor endings such as has been reported with raising extracellular potassium and probably includes effects of inactivation of other voltage-dependent channels.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Aminopyridine↗

Regulation of the cerebral circulation: role of endothelium and potassium channels.

Several new concepts have emerged in relation to mechanisms that contribute to regulation of the cerebral circulation. This review focuses on some physiological mechanisms of cerebral vasodilatation and alteration of these mechanisms by disease states. One mechanism involves release of vasoactive factors by the endothelium that affect underlying vascular muscle. These factors include endothelium-derived relaxing factor (nitric oxide), prostacyclin, and endothelium-derived hyperpolarizing factor(s). The normal vasodilator influence of endothelium is impaired by some disease states. Under pathophysiological conditions, endothelium may produce potent contracting factors such as endothelin. Another major mechanism of regulation of cerebral vascular tone relates to potassium channels. Activation of potassium channels appears to mediate relaxation of cerebral vessels to diverse stimuli including receptor-mediated agonists, intracellular second messenger, and hypoxia. Endothelial- and potassium channel-based mechanisms are related because several endothelium-derived factors produce relaxation by activation of potassium channels. The influence of potassium channels may be altered by disease states including chronic hypertension, subarachnoid hemorrhage, and diabetes.

Aging↗

Inhibitors of ATP-sensitive potassium channels stimulate intestinal cholecystokinin secretion.

Recently, a role for adenosine 5'-triphosphate(ATP)-sensitive potassium channels in the regulation of cholecystokinin (CCK) secretion has been described in STC-1 cells, an intestinal CCK-secreting cell line. To examine whether a similar mechanism might participate in the regulation of hormone secretion from native CCK cells, the effects of two established inhibitors of ATP-sensitive potassium channels (e.g. glucose, disopyramide) were examined on CCK release from dispersed murine intestinal cells. Both glucose and disopyramide were found to stimulate CCK secretion. Furthermore, CCK release induced by glucose was inhibited by the calcium channel blocker diltiazem. It is concluded that, ATP-sensitive potassium channels may play a role in the regulation of intestinal CCK secretion.

Adenosine Triphosphate↗

Protective effects of the potassium channel opener-diazoxide against injury in neonatal rat ventricular myocytes.

The mitochondrial ATP-regulated potassium channel is present in the inner membrane of heart mitochondria. Similarly to plasma membrane K(ATP), the mitochondrial channel is inhibited by antidiabetic sulfonylureas and activated by potassium channel openers, such as diazoxide. In the present work, the cytoprotective properties of diazoxide on the H9c2 cardiac myoblast cell line and neonatal rat ventricular cardiomyocytes were analysed. It was observed that 100 micromol/l diazoxide protected neonatal rat ventricular cardiomyocytes, but not H9c2 myoblasts, against injury induced by hydrogen peroxide or simulated ischemia. Moreover, diazoxide prevented hydrogen peroxide-induced mitochondrial potential depolarisation in neonatal rat ventricular cardiomyocytes. Diazoxide, at the same time, did not affect the expression level of the anti-apoptotic protein bcl-2 in these cells. The protective effects of diazoxide were suppressed by 5-hydroxydecanoic acid, a potassium channel blocker. These observations suggest that activation of the mitochondrial ATP-regulated potassium channel plays an important role in protection of neonatal cardiomyocytes against injury.

Animals↗

Mechanisms of two modulatory actions of the channel-binding protein Slob on the Drosophila Slowpoke calcium-dependent potassium channel.

Slob57 is an ion channel auxiliary protein that binds to and modulates the Drosophila Slowpoke calcium-dependent potassium channel (dSlo). We reported recently that residues 1-39 of Slob57 comprise the key domain that both causes dSlo inactivation and shifts its voltage dependence of activation to more depolarized voltages. In the present study we show that removal of residues 2-6 from Slob57 abolishes the inactivation, but the ability of Slob57 to rightward shift the voltage dependence of activation of dSlo remains. A synthetic peptide corresponding in sequence to residues 1-6 of Slob57 blocks dSlo in a voltage- and dose-dependent manner. Two Phe residues and at least one Lys residue in this peptide are required for the blocking action. These data indicate that the amino terminus of Slob57 directly blocks dSlo, thereby leading to channel inactivation. Further truncation to residue Arg(16) eliminates the modulation of voltage dependence of activation. Thus these two modulatory actions of Slob57 are independent. Mutation within the calcium bowl of dSlo greatly reduces its calcium sensitivity (Bian, S., I. Favre, and E. Moczydlowski. 2001. Proc. Natl. Acad. Sci. USA. 98:4776-4781). We found that Slob57 still causes inactivation of this mutant channel, but does not shift its voltage dependence of activation. This result confirms further the independence of the inactivation and the voltage shift produced by Slob57. It also suggests that the voltage shift requires high affinity Ca(2+) binding to an intact calcium bowl. Furthermore, Slob57 inhibits the shift in the voltage dependence of activation of dSlo evoked by Ca(2+), and this inhibition by Slob57 is greater at higher free Ca(2+) concentrations. These results implicate distinct calcium-dependent and -independent mechanisms in the modulation of dSlo by Slob.

Amino Acids↗

Molecular structure and expression of shaker type potassium channels in glial cells of trout CNS.

Two shaker-related potassium channel transcripts (tsha1, tsha2) were identified in glial cells of trout central nervous system (CNS) by polymerase chain reaction (PCR)-cloning and sequencing. While tsha1 was highly similar to the mammalian Kv1.2 subtype of potassium channels, tsha2 did not show a preferential sequence homology with a particular subtype of shaker, but exhibited uniform similarity with mammalian Kv1.1, Kv1.2, and Kv1.3, respectively. Transcripts for the shaw and shal subfamilies of voltage-gated potassium channels were detected in whole brain tissue only but not in freshly dissociated glial cells. Using mRNA extracted from different glial cell types in combination with sequence specific PCR primers, tshal was found restricted to oligodendrocytes and their progenitor cells, while tsha2 was common to astrocytes, as well.

Amino Acid Sequence↗

Influence of permeant ions on voltage sensor function in the Kv2.1 potassium channel.

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K(+)]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K(+)] modulate the rate of channel activation. Elevation of symmetrical [K(+)] or [Rb(+)] from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar [K(+)]-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

Cell Line↗

Preconditioning with potassium channel openers. A new concept for enhancing cardioplegic protection?

Ischemic preconditioning defines an adaptive endogenous mechanism in which a brief episode of reversible ischemia renders the heart more resistant to a subsequent period of sustained ischemia. Because the cardioprotective effects of ischemic preconditioning might be mediated by an activation of adenosine triphosphate-sensitive potassium channels, this study was designed to assess whether these effects could be duplicated by the preischemic administration of a potassium channel opener. Fifty isolated isovolumic buffer-perfused rat hearts underwent 45 minutes of normothermic potassium arrest followed by 1 hour of reperfusion. They were divided into five equal groups that differed with regard to the preconditioning regimen: Group 1 hearts were left untreated and served a controls; in group 2, preconditioning was achieved with 5 minutes of total global ischemia followed by 5 minutes of buffer reperfusion before cardioplegic arrest; in group 3, the preconditioning stimulus consisted of a 5-minute infusion of the potassium channel opener nicorandil (10 mumol/L) followed by 5 minutes of drug-free buffer perfusion before arrest; group 4 hearts underwent a similar protocol except that the infusion of nicorandil was preceded by that of the potassium channel blocker glibenclamide (10 mumol/L); group 5 hearts were ischemically preconditioned like those of group 2 except that the no-flow preconditioning period was also preceded by a 5-minute infusion of glibenclamide (50 mumol/L). The results demonstrate that ischemic preconditioning significantly improved contractility and reduced contracture during reperfusion, as compared with results in control hearts. These protective effects were duplicated by pretreatment with nicorandil but were abolished when the drug was antagonized by a prior infusion of glibenclamide. Likewise, the glibenclamide-induced blockade of potassium channels largely blunted the beneficial effects of ischemic preconditioning. These data suggest that opening of adenosine triphosphate-sensitive potassium channels substantially contributes to preconditioning-induced cardiac protection in a surgically relevant model of global ischemia and, consequently, that the use of potassium channel openers like nicorandil could be an effective means of enhancing cardioplegic protection.

Animals↗