Specific block of cloned Herg channels by clofilium and its tertiary analog LY97241

The class III antiarrhythmic drug clofilium is known to block diverse delayed rectifier K+ channels at micromolar concentrations. In the present study we investigated the potency of clofilium and its tertiary analog LY97241 to inhibit K+ channels, encoded by the human ether-a-go-go related gene (HERG). Clofilium blocked HERG channels in a voltage-dependent fashion with an IC50 of 250 nM and 150 nM at 0 and +40 mV, respectively. LY97241 was almost 10-fold more potent (IC50 of 19 nM at +40 mV). Other cloned K+ channels which are also expressed in cardiac tissue, Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv4.2, Kir2.1, or I(Ks), were not affected by 100-fold higher concentrations. Block of HERG channels by LY97241 was voltage dependent and the rate of HERG inactivation was increased by LY97241. A rise of [K+]0 decreased both, rate of HERG inactivation and LY97241 affinity. The HERG S631A and S620T mutant channels which have a strongly reduced degree of inactivation were 7-fold and 33-fold less sensitive to LY97241 blockade, indicating that LY97241 binding is affected by HERG channel inactivation. In summary, the antiarrhythmic action of clofilium and its analog LY97241 appears to be caused by their potent, but distinct ability for blocking HERG channels.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

Histochemistry of the surface mucous gel layer of the human colon

Two characteristic features of the rapid component of the cardiac delayed rectifier current (IKr) are prominent inward rectification and an unexpected reduction in activating current with decreased [K+]o. Similar features are observed with heterologous expression of HERG, the gene thought to encode the channel carrying IKr, moreover, recent studies indicate that the mechanism underlying rectification of HERG current is the inactivation that channels rapidly undergo during depolarizing pulses. The present studies were designed to determine the mechanism of IKr rectification and [K+]o sensitivity in the mouse atrial myocyte cell line, AT-1 cells. Reducing [Mg2+]i to 0, which reverses inward rectification of some K+ channels, did not alter IKr current-voltage relationships, although it did decrease sensitivity to the IKr blockers dofetilide and quinidine 2- to 5-fold. To determine the presence and extent of fast inactivation of IKr in AT-1 cells, a brief hyperpolarizing pulse (20 ms to -120 mV) was applied during long depolarizations. Immediately after this pulse, a very large outward current that decayed rapidly to the previous activating current baseline was observed. This outward current component was blocked by the IKr-specific inhibitor dofetilide, indicating that it represented recovery from fast inactivation during the hyperpolarizing step, with fast reinactivation during the return to depolarized potential. With removal of inactivation using this approach, current-voltage relationships for IKr ([K+]o, 1 to 20 mmol/L) were linar and reversed close to the predicted Nernst potential for K+. In addition, decreased [K+]o decreased the time constants for open-->inactivated and inactivated-->open transitions. Thus, in these cardiac myocytes, as with heterologously expressed HERG, IKr undergoes fast inactivation that determines its characteristic inward rectification. These studies demonstrate that the mechanism underlying decreased activating current observed at low [K+]o is more extensive fast inactivation.     

Immunohistochemical detection of vascular growth factors in angiomatous and atypical meningiomas, as well as hemangiopericytomas

Nonselective cation channels have been identified and linked to important cell functions in rat hepatocytes. In this study, we characterized inward rectifying nonselective cation channels in detail by the patch clamp technique in human HepG2 cells. Channel properties were studied with high resistance borosilicate pipettes in cell-attached and inside-out configurations. With Ringer's solution and KCl as pipette solutions, the conductances were 19.7 +/- 2.1 and 22.2 +/- 0.0 picosiemens (pS), and reversal potentials were 30.9 +/- 3.5 and 31.3 +/- 4.6 mV, respectively. The channel was permeable to Ba2+, and the sequence of permeability ratios was Na+ > K+ > Cs+ > Ba2+. In the cell-attached configuration, the channel had a higher opening probability at depolarizing potential than at hyperpolarizing. In the inside-out patches with symmetric Ringer's solution, the current voltage curve was linear with conductance of 19.8 +/- 0.9 pS. Reversal potential shifted from -0.2 +/- 1.0 mV to 23.2 +/- 1.0 mV when the bath solution was replaced by dilute Ringer's solution. In the inside-out configuration, the gating was Ca(2+)-dependent, and the opening probability increased with increasing intracellular calcium concentration ([Ca2+]i). An outward rectifying channel appeared when [Ca2+]i was less than 1 mumol/L. The nonselective channel was reversibly blocked by 10 mumol/L internal flufenamic acid. We conclude that Ca(2+)- and voltage-dependent nonselective cation channels are present in human HepG2 cells. The channels might be involved in the regulation of Ca2+ influx and are associated with activation of other ion channels.     

Ca2+-dependent inactivation of a store-operated Ca2+ current in human submandibular gland cells. Role of a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump

Stimulation of human submandibular gland cells with carbachol, inositol trisphosphate (IP3), thapsigargin, or tert-butylhydroxyquinone induced an inward current that was sensitive to external Ca2+ concentration ([Ca2+]e) and was also carried by external Na+ or Ba2+ (in a Ca2+-free medium) with amplitudes in the order Ca2+ > Ba2+ > Na+. All cation currents were blocked by La3+ and Gd3+ but not by Zn2+. The IP3-stimulated current with 10 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-triphosphate and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette solution, showed 50% inactivation in <5 min and >5 min with 10 and 1 mM [Ca2+]e, respectively. The Na+ current was not inactivated, whereas the Ba2+ current inactivated at a slower rate. The protein kinase inhibitor, staurosporine, delayed the inactivation and increased the amplitude of the current, whereas the protein Ser/Thr phosphatase inhibitor, calyculin A, reduced the current. Thapsigargin- and tert-butylhydroxyquinone-stimulated Ca2+ currents inactivated faster. Importantly, these agents accelerated the inactivation of the IP3-stimulated current. The data demonstrate that internal Ca2+ store depletion-activated Ca2+ current (ISOC) in this salivary cell line is regulated by a Ca2+-dependent feedback mechanism involving a staurosporine-sensitive protein kinase and the intracellular Ca2+ pump. We suggest that the Ca2+ pump modulates ISOC by regulating [Ca2+]i in the region of Ca2+ influx.     

K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1)

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

Hypoxia induced by Na2S2O4 increases [Na+]i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na+-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na+]i) and resting potential (Em) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na+-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO2 122 Torr), [Na+]i was 12-13 mM corresponding to a Na+ equilibrium potential (ENa) of about 58 mV. Em was about -42 mV. Hypoxia, induced by Na2S2O4 1 mM (PO2 10 Torr), depolarized the cells by about 20 mV, [Na+]i increased by 21 mM and ENa dropped to about 35 mV. One millimolar of CoCl2 depressed, or blocked, the effects of Na2S2O4 on [Na+]i but did not affect hypoxic depolarization. Voltage-clamping at -70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba2+. All currents were blocked by 1 mM CoCl2 suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na+]i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na+ increases (inflow) probably occurred by disturbing a Na+/K+ exchange mechanism and not by activation of voltage-gated channels.     

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33-CD34+ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33-CD34+ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (Kir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31% of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCF. Kir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs+ or Ba2+ Antisense oligodeoxynucleotides directed against Kir blocked both mRNA and functional expression of Kir channels. Kir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33-CD34+ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs+ or Ba2+ induced a similar degree of inhibition (40-60%) of progenitor cell generation. These findings strongly suggest an essential role for Kir in the process of cytokine-induced primitive progenitor cell growth and differentiation.     

Inhibitory effects of berberine on voltage- and calcium-activated potassium currents in human myeloma cells

The effects of berberine, an isoquinoline alkaloid, were investigated in human myeloma cells. In cells with intracellular Ca2+ concentration ([Ca2+]i) = 10 nM, the depolarizing square pulses from -80 mV elicited an instantaneous outward current with an inactivation. This outward current was voltage dependent, activating at -30 mV and showed inactivation with repetitive depolarization, and was hence believed to be n type voltage-activated K+ current (IK(V)). Berberine (30 microM) produced a prolongation in the recovery of IK(V) inactivation. In cells with [Ca2+]i = 1 microM, berberine also inhibited A23187-induced IK(Ca). Berberine (1-300 microM) caused the inhibition of IK(V) and IK(Ca) in the concentration-dependent manners. The IC50 values of berberine-induced inhibition of IK(V) and IK(Ca) were approximately 15 microM and 50 microM, respectively. In inside-out configurations, berberine inside the pipette suppressed the activity of K(Ca) channels without changing the single channel conductance. Berberine also inhibited the proliferation of this cell line and the IC50 value of berberine-induced inhibition of cell proliferation was 5 microM. Thus, the cytotoxic effect of berberine in cancer cells may be partially explained by its direct blockade of these K+ channels.     

Natriuretic peptides increase a K+ conductance in rat mesangial cells

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3',5'-cyclicmonophosphate) peptides on membrane voltages (Vm) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/l. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on Vm these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodalatin) hyperpolarized Vm significantly by 1.6 +/- 0.4 mV (BNP, n=8), 3.7 +/- 0.3 mV (CDD/ANP-99-126, n=25) and 2.8 +/- 0.4 mV (urodilatin, n=9), respectively. The half-maximally effective concentration (EC50) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/l 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/l Ba2+. The K+ channel blocker 293 B (100 micromol/l) depolarized basal Vm by 4.3 +/- 0.4 mV (n=8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/l) (n=8). The K+ channel opener cromakalim (10 micromol/l) neither influenced basal Vm nor altered the hyperpolarization induced by 160 nmol/l CDD/ANP-99-126 (n=8). Adenosine (100 micromol/l) hyperpolarized Vm by 13.4 +/- 1.3 mV (n=16). At 100 micromol/l, 293 B did not inhibit the adenosine-induced hyperpolarization (n=6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 +/- 0.6 mV (n=10). CDD/ANP-99-126 (160 nmol/l) failed to modulate the value to which Vm depolarized in the presence of 1 nmol/l angiotensin II (n=10), but accelerated the repolarization to basal Vm by 49 +/- 20% (n=8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K+ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba2+, but not by 293 B and cannot be activated by cromakalim. This increase in the K+ conductance seems to be additive to that inducable by adenosine, indicating that different K+ channels are activated by these hormones.     

Kinetics of Ca2+ release evoked by photolysis of caged InsP3 in rat submandibular cells

Quantitative time-resolved measurements of cytosolic Ca2+ release by photolysis of caged InsP3 have been made in single rat submandibular cells using patch clamp whole-cell recording to measure the Ca2+-activated Cl- and K+ currents. Photolytic release of InsP3 from caged InsP3 at 100 Joules caused transient inward (V(H) = 60 mV) and outward (V(H) = 0 mV) currents, which were nearly symmetric in their time course. The inward current was reduced when pipette Cl- concentration was decreased, and the outward current was suppressed by K+ channel blockers, indicating that they were carried by Cl- and K+, respectively. Intracellular pre-loading of the InsP3 receptor antagonist heparin or the Ca2+ chelator EGTA clearly prevented both inward and outward currents, indicating that activation of Ca2+-dependent Cl- and K+ currents underlies the inward and the outward currents. At low flash intensities, InsP3 caused Ca2+ release which normally activated the K+ and Cl- currents in a mono-transient manner. At higher intensities, however, InsP3 induced an additional delayed outward K+ current (I[K,(delay)]). I[K(delay)] was independent of the initial K+ current, independent of extracellular Ca2+, inhibited by TEA, and gradually prolongated by repeated flashes. The photolytic release of Ca2+ from caged Ca2+ did not mimic the I[K(delay)]. It is suggested that Ca2+ releases from the InsP3-sensitive pools in an InsP3 concentration-dependent manner. Low concentrations of InsP3 induce the transient Ca2+-dependent Cl- and K+ currents, which reflects the local Ca2+ release, whereas high concentrations of InsP3 induce a delayed Ca2+-dependent K+ current, which may reflect the Ca2+ wave propagation.     

Profiles of the fatty acids in the plasma membrane of human brain tumors

The ionic channels and signal transduction pathways underlying the 5-hydroxytryptamine (5-HT)-induced hyperpolarization in neurons of the rat dorsolateral septal nucleus (DLSN) were examined by using intracellular and voltage-clamp recording techniques. Application of 5-HT (1-50 microM) caused a hyperpolarizing response associated with a decreased membrane resistance in DLSN neurons. The hyperpolarization induced by 5-HT was blocked by Ba2+ (1 mM) but not by tetraethylammonium (TEA, 3 mM), glibenclamide (100 microM) and extracellular Cs+ (2 mM). 8-Hydroxy-di-n-propylamino tetralin (8-OH-DPAT; 3 microM), a selective agonist for the 5-HT1A receptor, mimicked 5-HT in producing the hyperpolarization. The 5-HT hyperpolarization was blocked by NAN-190 (5 microM), a 5-HT1A receptor antagonist. CP93129 (100 microM), a 5-HT1B receptor agonist, and L-694-247 (100 microM), a 5-HT1B/1D receptor agonist, also produced hyperpolarizing responses. The order of agonist potency was 8-OH-DPAT > CP93129 > or = L-694-247. (+/-)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (DOI, 100 microM), a 5-HT2 receptor agonist, and RS67333 (100 microM), a 5-HT4 receptor agonist, caused no hyperpolarizing response. The voltage-clamp study showed that 5-HT caused an outward current (I5-HT) in a concentration-dependent manner. I5-HT was associated with an increased membrane conductance. I5-HT reversed the polarity at the equilibrium potential for K+ calculated by the Nernst equation. I5-HT showed inward rectification at membrane potentials more negative than-70 mV. Ba2+ (100 microM) blocked the inward rectifier K+ current induced by 5-HT. I5-HT was irreversibly depressed by intracellular application of guanosine 5'-O-(3-thiotriphosphate)(GTP-gamma S) but not by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). These results suggest that in rat DLSN neurons activation of 5-HT1A receptors causes a hyperpolarizing response by activating mainly the inward rectifier K+ channels through a GTP-binding protein.     

Somatostatin increases a voltage-insensitive K+ conductance in rat CA1 hippocampal neurons

Somatostatin (SST) is a neuropeptide involved in several central processes. In hippocampus, SST hyperpolarizes CA1 pyramidal neurons and augments the K+ M current (IM). However, the limited involvement of IM at resting potential in these cells suggests that the peptide also may modulate another channel to hyperpolarize hippocampal pyramidal neurons (HPNs). We studied the effect of SST on noninactivating conductances of rat CA1 HPNs in a slice preparation. Using MK886, a specific inhibitor of the enzymatic pathway that leads to the augmentation of IM by SST, we have uncovered and characterized a second conductance activated by the peptide. SST did not affect IM when applied with MK886 or the amplitudes of the slow Ca2+-dependent K+ afterhyperpolarization-current and the cationic Q current but still caused an outward current, indicating that SST acts upon another conductance. In the presence of MK886, SST elicited an outward current that reversed around -100 mV and that displayed a linear current-voltage relationship. Reversal potentials obtained in different external K+ concentrations are consistent with a conductance carried solely by K+ ions. The slope of the current-voltage relationship increased proportionately with the extracellular K+ concentration and remained linear. This suggests that SST opens a voltage-insensitive leak current (IK(L)) in HPNs not an inwardly rectifying K+ current as reported in other neuron types. A low concentration of extracellular Ba2+ (150 M) only slightly decreased the SST-induced effect in a voltage-independent manner, whereas a high concentration of Ba2+ (2 mM) completely blocked it. Extracellular Cs+ (2 mM) did not affect the outward SST current but inhibited the inward component. We conclude that SST inhibits HPNs by activating two different K+ conductances: the voltage-insensitive IK(L) and the voltage-dependent IM. The hyperpolarizing effect of SST at resting membrane potential appears to be mainly carried by IK(L), whereas IM dominates at slightly depolarized potentials.     

Modulation of GABAC response by Ca2+ and other divalent cations in horizontal cells of the catfish retina

GABAC responses were recorded in cultured cone-driven horizontal cells from the catfish retina using the patch clamp technique. At a holding potential of -49 mV, a bicuculline-resistant inward current (IGABA) was observed when 10 microM GABA was applied. The amplitude of IGABA increased as the extracellular Ca2+ ([Ca2+]o) was increased. Concentration-response curves of IGABA at 2.5 and 10 mM -Ca2+-o had similar EC50 (3.0 and 3.1 microM) and Hill coefficients (1.54 and 1. 24). However, the maximal response estimated at 10 mM [Ca2+]o was larger than the maximal response at 2.5 mM [Ca2+]o. Increasing Ca influx through voltage-gated Ca channels and the resulting rise in the intracellular Ca2+ concentration had no effects on IGABA. However, IGABA was inhibited by extracellular divalent cations, with the following order of the inhibitory potency: Zn2+ > Ni2+ > Cd2+ > Co2+. The inhibitory action of Zn2+ on the [Ca2+]o-dependent IGABA increase was noncompetitive. The action of [Ca2+]o on IGABA was mimicked by Ba2+ or Sr2+. These results demonstrate that the extracellular domain of GABAC receptors has two functionally distinct binding sites represented by Ca2+ (facilitation) and Zn2+ (inhibition). Since [Ca2+]o and [Zn2+]o change into the opposite direction by light, it seems likely that they modify cooperatively the efficacy of the positive feedback consisting of the GABAC receptor.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K+ channel have been described in vascular smooth muscle: Voltage-activated K+ channels (Kv) are encoded by the Kv gene family, Ca(2+)-activated K+ channels (BKCa) are encoded by the slo gene, inward rectifiers (KIR) by Kir2.0, and ATP-sensitive K+ channels (KATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4-1.6, Kv2.1, Kv9.3, Kv beta 1-beta 4, slo alpha and beta, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K+ channels are modulated by physiological vasodilators, which increase K+ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate KATP channels. These include adenosine, calcitonin gene-related peptide, and beta-adrenoceptor agonists. beta-adrenoceptors can also activate BKCa and Kv channels. Several vasoconstrictors that activate protein kinase C inhibit KATP channels, and inhibition of BKCa and Kv channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BKCa channels, and possibly KATP channels. Hypoxia leads to activation of KATP channels, and activation of BKCa channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of Kv channels. Vasodilation to increased external K+ involves KIR channels. Endothelium-derived hyperpolarizing factor activates K+ channels that are not yet clearly defined. Such K+ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Time-dependent outward currents through the inward rectifier potassium channel IRK1. The role of weak blocking molecules

Outward currents through the inward rectifier K+ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg2+ (0.44-1.1 mM) or putrescine (approximately 0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K+. Without internal Mg2+, small outward currents flowed only at potentials between -80 (the reversal potential) and approximately -40 mV during voltage steps applied from -110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg2+, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between -40 and 0 mV were concurrent with the contribution of Mg2+ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to -50 mV after short depolarizing steps (> 0 mV), a transient increase appeared in outward currents at -50 mV. Since the peak amplitude depended on the fraction of Mg(2+)-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg2+ block, followed by a re-block of channels by spermine. Shift in the holding potential (-110 to -80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg(2+)-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg2+. When both spermine (1 microM, an estimated free spermine level during whole-cell recordings) and putrescine (300 microM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg2+ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

HERG-like K+ channels in microglia

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.     

Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells

The interaction of large depolarization and dihydropyridine Ca2+ agonists, both of which are known to enhance L-type Ca2+ channel current, was examined using a conventional whole-cell clamp technique. In guinea pig detrusor cells, only L-type Ca2+ channels occur. A second open state (long open state: O2) of the Ca2+ channels develops during large depolarization (at +80 mV, without Ca2+ agonists). This was judged from lack of inactivation of the Ca2+ channel current during the large depolarizing steps (5 s) and slowly deactivating inward tail currents (= 10-15 ms) upon repolarization of the cell membrane to the holding potential (-60 mV). Application of Bay K 8644 (in 2.4 mM Ca(2+)-containing solutions) increased the amplitude of the Ca2+ currents evoked by simple depolarizations, and made it possible to observe inward tail currents (= 2.5-5 ms at -60 mV). The open state induced by large depolarization (O2*) in the Bay K 8644 also seemed hardly to inactivate. After preconditioning with large depolarizing steps, the decay time course of the inward tail currents upon repolarization to the holding potential (-60 mV) was significantly slowed, and could be fitted reasonably with two exponentials. The fast and slow time constants were 10 and 45 ms, respectively, after 2 s preconditioning depolarizations. Qualitatively the same results were obtained using Ba2+ as a charge carrier. Although the amplitudes of the inward currents observed in the test step and the subsequent repolarization to the holding potential were decreased in the same manner by additional application of nifedipine (in the presence of Bay K 8644), the very slow deactivation time course of the tail current was little changed. The additive enhancement by large depolarization and Ca2+ agonists of the inward tail current implies that two mechanisms separately induce long opening of the Ca2+ channels: i.e., that there are four open states.     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...