Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line

Previously, we showed that the peakdensity of the transient outward K⁺ current(I_to) expressed in GH3 cells was different inthe S phase than in other phases of the cell cycle. Using cellsynchronization, we show here that I_to dropsprecisely at the quiescent (G₀ phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I_to, but rather to a modificationin its inactivation kinetics. Molecular determination of K⁺channel subunits showed that I_to required theexpression of Kv1.4, Kv4.1, and Kv4.3. We found that the increase inI_to density during the quiescent state wasaccompanied by an increase in Kv1.4 protein expression, whereas Kv4.3expression remained unchanged. We further demonstrate that the linkbetween I_to expression and cell proliferation isnot mediated by variations in cell excitability. These results providenew evidence for the cell cycle dependence ofI_to expression, which could be relevant inunderstanding the mechanisms leading to pituitary adenomas.

     

Differential modulation of voltage-dependent K+ currents in colonic smooth muscle by oxidants

The effect of oxidants on voltage-dependent K⁺ currents was examined in mouse colonic smooth muscle cells. Exposure to either chloramine-T (Ch-T), an agent known to oxidize both cysteine and methionine residues, or the colon-specific oxidant monochloramine (NH₂Cl) completely suppressed the transient outward K⁺ current (I_to) while simultaneously enhancing the sustained delayed rectifier K⁺ current (I_dr). In contrast, the cysteine-specific oxidants hydrogen peroxide (H₂O₂) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) exhibited partial and slow suppression of I_to by inducing a shift in channel availability of -18 mV without affecting I_dr. After enhancement by NH₂Cl or Ch-T, I_dr was sensitive to 10 mM tetraethylammonium but not to other K⁺ channel blockers, suggesting that it represented activation of the resting I_dr and not a separate K⁺ conductance. Extracellular dithiothreitol (DTT) partially reversed the effect of H₂O₂ and DTNB on I_to but not the actions of NH₂Cl and Ch-T on either I_dr or I_to. Dialysis of myocytes with GSH (5 mM) or DTT (5 mM) prevented suppression of I_to by H₂O₂ and DTNB but did not alter the effects of NH₂Cl or Ch-T on either I_dr or I_to. Ch-T and NH₂Cl completely blocked I_to generated by murine K_v4.1, 4.2, and 4.3 in Xenopus oocytes, an effect not reversible by intracellular DTT. In contrast, intracellular DTT reversed the effect of H₂O₂ and DTNB on the cloned channels. These results suggest that I_to is suppressed via modification of both methionine and cysteine residues, whereas enhancement of I_dr likely results from methionine oxidation alone. colon; colitis; redox; ion channel     

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

K+ current inhibition by amphiphilic fatty acid metabolites in rat ventricular myocytes

Fatty acid metabolites accumulate in the heart underpathophysiological conditions that affect -oxidation and can elicit marked electrophysiological changes that are arrhythmogenic. The purpose of the present study was to determine the impact of amphiphilic fatty acid metabolites on K⁺currents that control cardiac refractoriness and excitability. Transient outward(I_to) andinward rectifier(I_K1)K⁺ currents were recorded by thewhole cell voltage-clamp technique in rat ventricular myocytes, and theeffects of two major fatty acid metabolites were examined:palmitoylcarnitine and palmitoyl-coenzyme A (palmitoyl-CoA).Palmitoylcarnitine (0.5-10 µM) caused a concentration-dependent decrease in I_todensity in myocytes internally dialyzed with the amphiphile; 10 µMreduced mean I_todensity at +60 mV by 62% compared with control(P < 0.05). In contrast, externalpalmitoylcarnitine at the same concentrations had no effect, nor didinternal dialysis significantly alterI_K1. Dialysiswith palmitoyl-CoA (1-10 µM) produced a smaller decrease inI_to densitycompared with that produced by palmitoylcarnitine; 10 µM reduced meanI_to density at+60 mV by 37% compared with control(P < 0.05). Both metabolites delayedrecovery of I_tofrom inactivation but did not affect voltage-dependent properties.Moreover, the effects of palmitoylcarnitine were relatively specific,as neither palmitate (10 µM) nor carnitine (10 µM) alone significantly influencedI_to when added tothe pipette solution. These data therefore suggest that amphiphilicfatty acid metabolites downregulateI_to channels by amechanism confined to the cytoplasmic side of the membrane. Thisdecrease in cardiac K⁺ channelactivity may delay repolarization under pathophysiological conditionsin which amphiphile accumulation is postulated to occur, such asdiabetes mellitus or myocardial infarction.

     

Mechanism of shortened action potential duration in Na+-Ca2+ exchanger knockout mice

In cardiac-specific Na⁺-Ca²⁺ exchanger (NCX) knockout (KO) mice, the ventricular action potential (AP) is shortened. The shortening of the AP, as well as a decrease of the L-type Ca²⁺ current (I_Ca), provides a critical mechanism for the maintenance of Ca²⁺ homeostasis and contractility in the absence of NCX (Pott C, Philipson KD, Goldhaber JI. Excitation-contraction coupling in Na⁺-Ca²⁺ exchanger knockout mice: reduced transsarcolemmal Ca²⁺ flux. Circ Res 97: 1288–1295, 2005). To investigate the mechanism that underlies the accelerated AP repolarization, we recorded the transient outward current (I_to) in patch-clamped myocytes isolated from wild-type (WT) and NCX KO mice. Peak I_to was increased by 78% and decay kinetics were slowed in KO vs. WT. Consistent with increased I_to, ECGs from KO mice exhibited shortened QT intervals. Expression of the I_to-generating K⁺ channel subunit Kv4.2 and the K⁺ channel interacting protein was increased in KO. We used a computer model of the murine AP (Bondarenko VE, Szigeti GP, Bett GC, Kim SJ, and Rasmusson RL. Computer model of action potential of mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 287: 1378–1403, 2004) to determine the relative contributions of increased I_to, reduced I_Ca, and reduced NCX current (I_NCX) on the shape and kinetics of the AP. Reduction of I_Ca and elimination of I_NCX had relatively small effects on the duration of the AP in the computer model. In contrast, AP repolarization was substantially accelerated when I_to was increased in the computer model. Thus, the increase in I_to, and not the reduction of I_Ca or I_NCX, is likely to be the major mechanism of AP shortening in KO myocytes. The upregulation of I_to may comprise an important regulatory mechanism to limit Ca²⁺ influx via a reduction of AP duration, thus preventing Ca²⁺ overload in situations of reduced myocyte Ca²⁺ extrusion capacity. genetically altered mice; cardiac myocytes; short QT interval; transient outward current     

Properties of ATP-dependent K(+) channels in adrenocortical cells

Bovine adrenocortical zona fasciculata (AZF)cells express a novel ATP-dependent K⁺-permeable channel(I_AC). Whole cell and single-channel recordings were used to characterize I_AC channels withrespect to ionic selectivity, conductance, and modulation bynucleotides, inorganic phosphates, and angiotensin II (ANG II). Inoutside-out patch recordings, the activity of unitaryI_AC channels is enhanced by ATP in the patchpipette. These channels were K⁺ selective with nomeasurable Na⁺ or Ca²⁺ conductance. Insymmetrical K⁺ solutions with physiological concentrationsof divalent cations (M²⁺), I_ACchannels were outwardly rectifying with outward and inward chordconductances of 94.5 and 27.0 pS, respectively. In the absence ofM²⁺, conductance was nearly ohmic. Hydrolysis-resistantnucleotides including AMP-PNP and NaUTP were more potent than MgATP asactivators of whole cell I_AC currents. Inorganicpolytriphosphate (PPP_i) dramatically enhancedI_AC activity. In current-clamp recordings, nucleotides and PPP_i produced resting potentials in AZFcells that correlated with their effectiveness in activatingI_AC. ANG II (10 nM) inhibited whole cellI_AC currents when patch pipettes contained 5 mMMgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP.Inhibition by ANG II was not reduced by selective kinase antagonists.These results demonstrate that I_AC is adistinctive K⁺-selective channel whose activity isincreased by nucleotide triphosphates and PPP_i.Furthermore, they suggest a model for I_AC gatingthat is controlled through a cycle of ATP binding and hydrolysis.

     

Characteristics of hyperpolarization-activated cation currents in portal vein smooth muscle cells

Voltage-clamp studies offreshly isolated smooth muscle cells from rabbit portal veinrevealed the existence of a time-dependent cation current evoked bymembrane hyperpolarization (termed I_h). Both therate of activation and the amplitude of I_h wereenhanced by membrane hyperpolarization. Half-maximal activation ofI_h was about 105 mV with conventional wholecell and 80 mV when the perforated patch technique was used. Incurrent clamp, injection of hyperpolarizing current produced a markeddepolarizing "sag" followed by rebound depolarization. Activationof I_h was augmented by an increase in theextracellular K⁺ concentration and was blocked rapidly byexternally applied Cs⁺ (1-5 mM). The bradycardic agentZD-7288 (10 µM), a selective inhibitor of I_h,produced a characteristically slow inhibition of the portal veinI_h. The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs⁺.Cs⁺ significantly decreased the frequency of spontaneouscontractions in both whole rat portal vein and rabbit portal veinsegments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clonescorresponding to HCN2, 3, and 4. The present study shows that portalvein myocytes contain genes shown to encode forhyperpolarization-activated channels and exhibit an endogenous currentwith characteristics similar to I_h in other celltypes. This conductance appears to determine, in part, the rhythmicityof this vessel.

     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

Modulation of cardiac Ca2+ channels by isoproterenol studied in transgenic mice with altered SR Ca2+ content

Phospholamban(PLB) ablation is associated with enhanced sarcoplasmic reticulum (SR)Ca²⁺ uptake and attenuation of thecardiac contractile responses to -adrenergic agonists. In thepresent study, we compared the effects of isoproterenol (Iso) on theCa²⁺ currents(I_Ca) ofventricular myocytes isolated from wild-type (WT) and PLB knockout(PLB-KO) mice. Current density and voltage dependence ofI_Ca were similarbetween WT and PLB-KO cells. However, I_Ca recorded fromPLB-KO myocytes had significantly faster decay kinetics. Iso increasedI_Ca amplitude inboth groups in a dose-dependent manner (50% effective concentration,57.1 nM). Iso did not alter the rate ofI_Ca inactivationin WT cells but significantly prolonged the rate of inactivation inPLB-KO cells. When Ba²⁺ was usedas the charge carrier, Iso slowed the decay of the current in both WTand PLB-KO cells. Depletion of SRCa²⁺ by ryanodine also slowed therate of inactivation ofI_Ca, and subsequent application of Iso further reduced the inactivation rate ofboth groups. These results suggest that enhancedCa²⁺ release from the SR offsetsthe slowing effects of -adrenergic receptor stimulation on the rateof inactivation ofI_Ca.

     

Cell cycle-related changes in transient K(+) current density in the GH3 pituitary cell line

Our aim was to determinewhether the expression of K⁺ currents is related to thecell cycle in the excitable GH3 pituitary cell line. K⁺currents were studied by electrophysiology, and bromodeoxyuridine (BrdU) labeling was used to compare their expression in cells thereafter identified as being in the S or non-S phase of the cellcycle. We show that the peak density of the transient outward K⁺ current (I_to) was 33% lower incells in S phase (BrdU+) than in cells in other phases of the cellcycle (BrdU). The voltage-dependence of I_towas not modified. However, of the two kinetic components ofI_to inactivation, the characteristics of thefast component differed significantly between BrdU+ and BrdU cells.Recovery from inactivation of I_to showedbiexponential and monoexponential function in BrdU and BrdU+ cells,respectively. This suggests that the molecular basis of this currentvaries during the cell cycle. We further demonstrated that4-aminopyridine, which blocks I_to, inhibited GH3cell proliferation without altering the membrane potential. These datasuggest that I_to may play a role in GH3 cellproliferation processes.

     

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+

Treatment of rabbits with angiotensin-converting enzyme (ACE)inhibitors increases the apparent affinity of theNa⁺-K⁺pump for Na⁺. To explore themechanism, we voltage clamped myocytes from control rabbits and rabbitstreated with captopril with patch pipettes containing 10 mMNa⁺. When pipette solutions wereK⁺ free, pump current(I_p) formyocytes from captopril-treated rabbits was nearly identical to thatfor myocytes from controls. However, treatment caused a significantincrease in I_pmeasured with pipettes containingK⁺. A similar difference wasobserved when myocytes from rabbits treated with the ANG II receptorantagonist losartan and myocytes from controls were compared.Treatment-induced differences in I_p wereeliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed ofamino acids 530-558 in pipette solutions. Treatmentwith captopril had no effect on the voltage dependence ofI_p. We concludethat ANG II regulates the pump's selectivity for intracellularNa⁺ at sites near the cytoplasmicsurface. Protein kinase C is implicated in the messenger cascade.

     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Nucleotide regulation of the voltage-dependent nonselective cation conductance in murine colonic myocytes

ATP is proposed to be a major inhibitory neurotransmitter in the gastrointestinal (GI) tract, causing hyperpolarization and smooth muscle relaxation. ATP activates small-conductance Ca²⁺-activated K⁺ channels that are involved in setting the resting membrane potential and causing inhibitory junction potentials. No reports are available examining the effects of ATP on voltage-dependent inward currents in GI smooth muscle cells. We previously reported two types of voltage-dependent inward currents in murine proximal colonic myocytes: a low-threshold voltage-activated, nonselective cation current (I_VNSCC) and a relatively high-threshold voltage-activated (L-type) Ca²⁺ current (I_L). Here we have investigated the effects of ATP on these currents. External application of ATP (1 mM) did not affect I_VNSCC or I_L in dialyzed cells. ATP (1 mM) increased I_VNSCC and decreased I_L in the perforated whole-cell configuration. UTP and UDP (1 mM) were more potent than ATP on I_VNSCC. ADP decreased I_L but had no effect on I_VNSCC. The order of effectiveness was UTP = UDP > ATP > ADP. These effects were not blocked by pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS), but the phospholipase C inhibitor U-73122 reversed the effects of ATP on I_VNSCC. ATP stimulation of I_VNSCC was also reversed by protein kinase C (PKC) inhibitors chelerythrine chloride or bisindolylmaleimide I. Phorbol 12,13-dibutyrate mimicked the effects of ATP. RT-PCR showed that P2Y₄ is expressed by murine colonic myocytes, and this receptor is relatively insensitive to PPADS. Our data suggest that ATP activates I_VNSCC and depresses I_L via binding of P2Y₄ receptors and stimulation of the phospholipase C/PKC pathway. inhibitory junction potentials; smooth muscle; enteric nervous system     

Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells

The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K⁺ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K⁺ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K⁺ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 µM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min (P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT_2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl-sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT_2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K⁺ efflux at the apical membrane of the choroid plexus. delayed-rectifying potassium channel; serotonin     

Functional characterization of voltage-gated K+ channels in mouse pulmonary artery smooth muscle cells

Mice are useful animal models to study pathogenic mechanisms involved in pulmonary vascular disease. Altered expression and function of voltage-gated K⁺ (K_V) channels in pulmonary artery smooth muscle cells (PASMCs) have been implicated in the development of pulmonary arterial hypertension. K_V currents (I_K(V)) in mouse PASMCs have not been comprehensively characterized. The main focus of this study was to determine the biophysical and pharmacological properties of I_K(V) in freshly dissociated mouse PASMCs with the patch-clamp technique. Three distinct whole cell I_K(V) were identified based on the kinetics of activation and inactivation: rapidly activating and noninactivating currents (in 58% of the cells tested), rapidly activating and slowly inactivating currents (23%), and slowly activating and noninactivating currents (17%). Of the cells that demonstrated the rapidly activating noninactivating current, 69% showed I_K(V) inhibition with 4-aminopyridine (4-AP), while 31% were unaffected. Whole cell I_K(V) were very sensitive to tetraethylammonium (TEA), as 1 mM TEA decreased the current amplitude by 32% while it took 10 mM 4-AP to decrease I_K(V) by a similar amount (37%). Contribution of Ca²⁺-activated K⁺ (K_Ca) channels to whole cell I_K(V) was minimal, as neither pharmacological inhibition with charybdotoxin or iberiotoxin nor perfusion with Ca²⁺-free solution had an effect on the whole cell I_K(V). Steady-state activation and inactivation curves revealed a window K⁺ current between –40 and –10 mV with a peak at –31.5 mV. Single-channel recordings revealed large-, intermediate-, and small-amplitude currents, with an averaged slope conductance of 119.4 ± 2.7, 79.8 ± 2.8, 46.0 ± 2.2, and 23.6 ± 0.6 pS, respectively. These studies provide detailed electrophysiological and pharmacological profiles of the native K_V currents in mouse PASMCs. K_V channels     

Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling

We have previously demonstrated that the sarcolemmalNa⁺-K⁺pump current(I_p) in cardiacmyocytes is stimulated by cell swelling induced by exposure tohyposmolar solutions. However, the underlying mechanism has not beenexamined. Because cell swelling activates stretch-sensitive ionchannels and intracellular messenger pathways, we examined their rolein mediating I_pstimulation during exposure of rabbit ventricular myocytes to ahyposmolar solution.I_p was measuredby the whole cell patch-clamp technique. Swelling-induced pumpstimulation altered the voltage dependence ofI_p. Pumpstimulation persisted in the absence of extracellularNa⁺ and under conditions designedto minimize changes in intracellular Ca²⁺, excluding an indirectinfluence on I_pmediated via fluxes through stretch-activated channels. Pumpstimulation was protein kinase C independent. The tyrosine kinaseinhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitorLY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acidabolished I_pstimulation. Our findings suggest that swelling-induced pumpstimulation involves the activation of tyrosine kinase,phosphatidylinositol 3-kinase, and a serine/threonine proteinphosphatase. Activation of this messenger cascade maycause activation by the dephosphorylation of pump units.     

Sequential and opposite regulation of two outward K(+) currents by ET-1 in cultured striatal astrocytes

In the brain,astrocytes represent a major target for endothelins (ETs), a family ofpeptides that can be released by several cell types and that havepotent and multiple effects on astrocytic functions. Four types ofK⁺ currents (I_K) were detected invarious proportions by patch-clamp recordings of cultured striatalastrocytes, including the A-type I_K, theinwardly rectifying I_K IR, theCa²⁺-dependent I_K(I_K Ca), and the delayed-rectifiedI_K (I_K DR). Variationsin the shape of current-voltage relationships were related mainly todifferences in the proportion of these currents. ET-1 was found toregulate with opposite effects the two more frequently recorded outwardK⁺ currents in striatal astrocytes. Indeed, this peptideinduced an initial activation of I_K Ca(composed of SK and BK channels) and a delayed long-lasting inhibitionof I_K DR. In current-clamp recordings, theactivation of I_K Ca correlated with a transient hyperpolarization, whereas the inhibition ofI_K DR correlated with a sustaineddepolarization. These ET-1-induced sequential changes inmembrane potential in astrocytes may be important for the regulation ofvoltage gradients in astrocytic networks and the maintenance ofK⁺ homeostasis in the brain microenvironment.

     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Pulmonary vasoconstriction and vascularmedial hypertrophy greatly contribute to the elevated pulmonaryvascular resistance in patients with pulmonary hypertension. A rise incytosolic free Ca²⁺ ([Ca²⁺]_cyt)in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (E_m) regulates[Ca²⁺]_cyt by governing Ca²⁺influx through voltage-dependent Ca²⁺ channels. Thusintracellular Ca²⁺ may serve as a shared signaltransduction element that leads to pulmonary vasoconstriction andvascular remodeling. In PASMC, activity of voltage-gated K⁺(Kv) channels regulates resting E_m. In thisstudy, we investigated whether changes of Kv currents[I_K(V)], E_m, and[Ca²⁺]_cyt affect cell growth by comparingthese parameters in proliferating and growth-arrested PASMC. Serumdeprivation induced growth arrest of PASMC, whereas chelation ofextracellular Ca²⁺ abolished PASMC growth. Resting[Ca²⁺]_cyt was significantly higher, andresting E_m was more depolarized, inproliferating PASMC than in growth-arrested cells. Consistently, wholecell I_K(V) was significantly attenuated in PASMCduring proliferation. Furthermore, E_mdepolarization significantly increased resting[Ca²⁺]_cyt and augmented agonist-mediatedrises in [Ca²⁺]_cyt in the absence ofextracellular Ca²⁺. These results demonstrate that reducedI_K(V), depolarized E_m, and elevated [Ca²⁺]_cyt may play a criticalrole in stimulating PASMC proliferation. Pulmonary vascular medialhypertrophy in patients with pulmonary hypertension may be partlycaused by a membrane depolarization-mediated increase in[Ca²⁺]_cyt in PASMC.

     

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

The action of cytochalasins, actin-disrupting agents on human Kv1.5 channel (hKv1.5) stably expressed in Ltk^– cells was investigated using the whole cell patch-clamp technique. Cytochalasin B inhibited hKv1.5 currents rapidly and reversibly at +60 mV in a concentration-dependent manner with an IC₅₀ of 4.2 µM. Cytochalasin A, which has a structure very similar to cytochalasin B, inhibited hKv1.5 (IC₅₀ of 1.4 µM at +60 mV). Pretreatment with other actin filament disruptors cytochalasin D and cytochalasin J, and an actin filament stabilizing agent phalloidin had no effect on the cytochalasin B-induced inhibition of hKv1.5 currents. Cytochalasin B accelerated the decay rate of inactivation for the hKv1.5 currents. Cytochalasin B-induced inhibition of the hKv1.5 channels was voltage dependent with a steep increase over the voltage range of the channel's opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. Cytochalasin B produced no significant effect on the steady-state activation or inactivation curves. The rate constants for association and dissociation of cytochalasin B were 3.7 µM/s and 7.5 s^–1, respectively. Cytochalasin B produced a use-dependent inhibition of hKv1.5 current that was consistent with the slow recovery from inactivation in the presence of the drug. Cytochalasin B (10 µM) also inhibited an ultrarapid delayed rectifier K⁺ current (I_K,ur) in human atrial myocytes. These results indicate that cytochalasin B primarily blocks activated hKv1.5 channels and endogenous I_K,ur in a cytoskeleton-independent manner as an open-channel blocker. voltage-gated K⁺ channel; heart; open channel block