Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Electrical maturation of the murine oocyte: an increase in calcium current coincides with acquisition of meiotic competence

We have used the whole-cell recording technique to compare three stages of primary and secondary oocytes from F1 hybrid mice (C57BL/6J x SJL/J): neonatal germinal vesicle (NGV) stage primary oocytes from 10- to 20-day-old, prepubescent mice; mature germinal vesicle (MGV) stage primary oocytes from 12-week-old, post-pubescent, superovulated mice; first polar body (FPB) stage secondary oocytes from 12-week-old, post-pubescent mice during the normal oestrus cycle or following superovulation. NGV, MGV and FPB oocytes all exhibit two voltage-dependent currents: an inward, rapidly activating/inactivating current, and an outward, slowly activating/non-inactivating current. In 1.5 mmol/l external Ca the average peak inward current is -2.9, -12.4 and -13.8 microA/cm2 in NGV, MGV and FPB oocytes, respectively. In 20 mmol/l Ca these currents increase and the reversal potential shifts to the right. The outward current decreases slightly with growth and development: at 40 mV test potentials, NGV oocytes have average outward currents of 8.9 microA/cm2, and MGV and FPB oocytes have currents of 5.0 and 5.5 microA/cm2, respectively. Thus, MGV oocytes express FPB current patterns. The reversal potentials, kinetics and pharmacology of the currents indicate that Ca channels carry the inward current and K channels carry the outward current. During growth in vivo a gradual depolarisation accompanies maturation. Resting potentials ranged from -45 to -30 mV in NGV oocytes to -35 to -17 mV in MGV oocytes to -20 mV to -3 mV in FPB oocytes. These data suggest that a selective increase occurs in the number of Ca channels during oocyte growth. This increase precedes nuclear maturation and coincides with the acquisition of meiotic competence.     

Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates

Human embryonic kidney cells (HEK 293) are widely used as an expression system in studies of ion channels. However, their endogenous ionic currents remain largely unidentified. To characterize these currents, we performed patch clamp experiments on this expression system. In whole-cell voltage clamp mode, the HEK 293 cells showed mainly outward currents using physiological concentrations of Na+ and K+ and symmetric concentrations of Cl- (150 mM) across the plasma membranes. K+ currents contributed to a small portion of these outward currents, since a shift of the reversal potentials of only approximately 20 mV was seen with a change of extracellular K+ concentration from 3 to 150 mM. In contrast, the reversal potential shifted approximately 25 mV when extracellular Cl- was reduced to 50 mM, indicating that most of the outward currents are carried by Cl-. In inside-out patches, several distinct Cl- currents were identified. They were: (1) 350 pS Cl- current, which was voltage-activated and had a moderate outward rectification; (2) 240 pS Cl- current with a weak outward rectification; and (3) 55 pS Cl- current, which was voltage-activated, sensitive to DIDS, and showed a strong outward rectification. Activation of these Cl- currents did not require an elevation of free Ca2+ level in the cytosol. Besides these three currents, we observed two other Cl- currents with much smaller conductances (25 and 16 pS, respectively). Two different K+ currents were seen in the HEK 293 cells, with one of them (125 pS) showing inward rectification and the other (70 pS) outward rectification. Moreover, a 50 pS cation channel was recorded in these cells. The presence of a variety of ion channels in the HEK 293 cells suggests that a great precaution needs to be taken when this expression system is used in studies of several similar ion channels.     

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.     

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

The electrophysiological properties of the Na+/I- symporter (NIS) were examined in a cloned rat thyroid cell line (FRTL-5) using the whole-cell patch-clamp technique. When the holding potential was between -40 mV and -80 mV, 1 mM NaI and NaSCN induced an immediate inward current which was greater with SCN- than with I-. The reversal potential for I- and SCN- induced membrane currents was +50 mV. This is close to the value of +55 mV calculated by the Nernst equation for Na+. These results are consistent with I- and SCN- translocation via the NIS that is energized by the electrochemical gradient of Na+ and coupled to the transport of two or more Na+. There was no change in the membrane current recording with ClO-4 indicating that ClO-4 was either not transported into the cell, or the translocation was electroneutral. ClO-4 addition, however, did reverse the inward currents induced by I- or SCN-. These effects of I-, SCN- and ClO-4 on membrane currents reflect endogenous NIS activity since the responses duplicated those seen in CHO cells transfected with NIS. There were additional currents elicited by SCN- in FRTL-5 cells under certain conditions. For example at holding potentials of 0 and +30 mV, 1 mM SCN- produced an increasingly greater outward current. This outward current was transient. In addition, when SCN- was washed off the cells a transient inward current was detected. Unlike SCN-, 1-10 mM I- had no observable effect on the membrane current at holding potentials of 0 and +30 mV. The results indicate FRTL-5 cells may have a specific SCN- translocation system in addition to the SCN- translocation by the I- porter. Differences demonstrated in current response may explain some of the complicated influx and efflux properties of I-, SCN- and ClO-4 in thyroid cells.     

Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.     

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.     

Potentiation and inhibition of nicotinic acetylcholine receptors by spermine in the TE671 human muscle cell line

Nicotinic acetylcholine receptors (nAChR) of the TE671 cell line were investigated using whole-cell and membrane patch recording techniques. At negative holding potentials (VH), pulses of acetylcholine (ACh) elicited whole-cell inward currents that rapidly desensitized. The EC50 value for ACh at VH = -60 mV was 7.8 microM. The ACh-induced current reversed at approximately 0 mV. Desensitization of nAChR by ACh was biphasic and reversible within approximately 20 sec. Spermine (1-100 microM) potentiated responses to ACh (10 microM - 1 mM) by reducing the rate of onset of desensitization; potentiation was inhibited by arcaine (10-100 microM). Spermine (1 mM) noncompetitively antagonized the AChinduced current. Antagonism by 1 to 5 mM spermine was voltage-dependent, increasing with negative VH. In 100 microM arcaine, this antagonism was shown to contain a voltage-independent component. Spermine (10 mM) increased the EC50 values for ACh, suggesting that at this concentration the polyamine is also a competitive antagonist. Single channel openings elicited during application of ACh to outside-out patches had a conductance of 47 pS at VH = -60 mV. At 10 and 100 microM, spermine increased channel open probability (po), but at 1 mM spermine, po was not significantly different from controls. The single channel conductance for ACh was unaffected by 10 and 100 microM spermine, but was decreased by 1 mM spermine. Spermine promoted the occurrence of approximately 27 pS openings. It is proposed that spermine acts at an excitatory modulatory site similar to that present on N-methyl-D-aspartate receptors and at least three inhibitory sites on nAChR of TE671 cells.     

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder

1. A whole-cell voltage clamp technique was used to examine the effects of purinoceptor and muscarinic receptor agonists on voltage-sensitive Ca2+ channels in guinea-pig isolated urinary bladder cells. 2. When the cell membrane was clamped at the holding potential, rapid application of ATP elicited a large inward current in normal solution containing 2.5 mM Ca2+, and reduced the subsequent Ca2+ channel current evoked by a depolarizing pulse (0 mV). Carbachol (CCh) elicited little membrane current, but similarly reduced the Ca2+ current. 3. When purinoceptor agonists were rapidly applied during conditioning depolarizations at +80 mV, an outward current was elicited, and the Ca2+ channel current evoked by the subsequent test potential of 0 mV was not affected. Application of CCh at +80 mV also elicited an outward current, but it reduced the subsequently evoked Ca2+ current. 4. The inhibitory effect of muscarinic agonists on the Ca2+ channel current was attenuated by caffeine (10 mM). 5. In Ca(2+)-free, low-Mg2+ solution, a Na+ current flowing through voltage-dependent Ca2+ channels was evoked by depolarization. This current was not reduced by bath application of purinoceptor agonists (ATP and alpha,beta-methylene ATP). 6. These results suggest that the main effect of purinoceptor stimulation is opening of non-selective cation channels, and that muscarinic stimulation triggers Ca2+ release from intracellular stores. Voltage-sensitive Ca2+ channels are inactivated through an increase in intracellular Ca2+ induced by either activation of purinoceptor or muscarinic receptors.     

Membrane currents influencing action potential latency in granule neurons of the rat cochlear nucleus

Granule cells are the most numerous neurons in the cochlear nucleus, but, because of their small size, little information on their membrane properties and ionic currents is available. We used an in vitro slice preparation of the rat ventral cochlear nucleus to make whole-cell recordings from these cells. Under current clamp, some granule neurons fired spontaneous action potentials and all generated a train of action potentials on depolarization (threshold current, 10-35 pA). Hyperpolarization increased the latency to the first action potential evoked during a subsequent depolarization. We examined which voltage-gated currents might underlie this latency shift. In addition to a fast inward Na+ current, depolarization activated two outward potassium currents. A transient current was rapidly inactivated by membrane potentials positive to -60 mV, while a second, more slowly inactivating current was observed following the decay of the transient current. No hyperpolarization-activated conductances were observed in these cells. Modelling of the currents suggests that removal of inactivation on hyperpolarization accounts for the increased action potential latency in granule cells. Such a mechanism could account for the 'pauser'-type firing patterns of the fusiform cells which receive a prominent projection from the granule cells in the dorsal cochlear nucleus.     

When management works: an organizational culture that facilitates learning to self-manage type 2 diabetes

Endogenous voltage-gated potassium currents were investigated in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells using whole-cell voltage clamp recording. Depolarizing voltage steps from -70 mV triggered an outwardly rectified current in nontransfected HEK293 cells. This current had an amplitude of 296 pA at +40 mV and a current density of 19.2 pA/pF. The outward current was eliminated by replacing internal K+ with Cs+ and suppressed by the K+ channel blockers tetraethylammonium and 4-aminopyridine. Raising external K+ attenuated the outward current and shifted the reversal potential towards positive potentials as predicted by the Nernst equation. The current had a fast activation phase but inactivated slowly. These features implicate delayed rectifier (I(K))-like channels as mediators of the observed current, which was comparable in size to I(K) currents in many other cells. A small native inward rectifier current but no transient outward current I(A), the M current I(M), or Ca2+-dependent K+ currents were detected in HEK293 cells. In contrast to these findings in HEK293 cells, little or no I(K)-like current was detected in CHO cells. The difference in endogenous voltage-activated currents in HEK293 and CHO cells suggest that CHO cell lines are a preferred system for exogenous K+ channel expression.     

Should unipolar leads be implanted in the atrium? A Holter electrocardiographic comparison of threshold adapted unipolar and high sensitive bipolar sensing

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches). 2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba2+ currents at - 50 mV (Ki=30.6 nM). 3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (> or =30 microM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately -82 mV by application of 100 microM levcromakalim. 4. The 100 microM levcromakalim-induced inward current (symmetrical 140 mM K+ conditions, -50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 microM). In contrast, Bay K 8644 (1 microM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current. 5. When 100 microM nifedipine was included in the pipette solution during conventional whole-cell recording at -50 mV, application of levcromakalim (100 microM) caused a significant inward membrane current which was suppressed by 5 microM glibenclamide. On the other hand, inclusion of 5 microM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current. 6. The levcromakalim-induced K+ channel openings in cell-attached configuration were suppressed by subsequent application of 5 microM glibenclamide but not of 100 microM nifedipine. 7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K+ channel activity mainly through extracellular blocking actions on the K+ channel itself.     

Calcium current and inactivation in identified neurons in Hermissenda crassicornis

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human brain activity related to speed discrimination tasks

This study examined the ionic mechanism of ibutilide, a class III antiarrhythmic in clinical use, on freshly isolated human atrial cells. Cells had resting potentials of -71.4 +/- 2.4 mV, action potentials with overshoot of 36.8 +/- 1.8 mV, duration of 265 +/- 89 msec at 90% repolarization and slow repolarization (n = 16). Ibutilide, at 10(-7) M, markedly increased action potential duration. Four types of outward currents were detected: Ito, Iso, a delayed rectifier and IK1. Ibutilide had no inhibitory effect on these outward currents at 10(-7) M (n = 28). In K(+)-free solutions and -40 mV holding potential, mean peak inward current at 20 mV was -1478 +/- 103 pA (n = 12). Ibutilide increased this current to -2347 +/- 75 pA at 10(-7) M, with half maximal effect (Kd) of 0.1 to 0.9 nM between -10 and +40 mV (n = 21). At similar concentrations, the drug increased APD, with Kd of 0.7 and 0.23 nM at 70 and 90% repolarization, respectively (n = 8). Ibutilide shifted the mid-point of the steady-state inactivation curve from -21 to -12.2 mV (n = 6), and reduced current decline during repetitive depolarization (n = 5). The drug induced inward current was carried by Na+o through a nifedipine inhibited inward channel because Na+o removal eliminated the effect, and nifedipine abolished the inward current and the drug induced APD prolongation. We propose that a Na+ current through the L-type Ca++ channel mediates ibutilide's potent clinical class III antiarrhythmic action.     

Polyribosome dynamics: size-distribution as a function of attachment, translocation and release of ribosomes

A Ca(2+)-activated Cl- conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mM ATP and 1 microM free Ca2+ and bathed in N-methyl-D-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nM or less than 1 nM free Ca2+ strongly reduced the Cl- currents, indicating the currents were Ca(2+)-dependent. Relaxation analysis of the "on" currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl- channels was: NO3- (2.00) > I- (1.85) > or = Br- (1.69) > Cl- (1.00) > bicarbonate (0.77) > or = acetate (0.70) > propionate (0.41) > > glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mM, the Ca(2+)-dependency of the Cl- current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-gamma S (2 mM) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mM). The addition of the calmodulin inhibitors trifluoperazine (100 microM) or calmidazolium (25 microM) to the bath solution and the inclusion of KN-62 (1 microM), a specific inhibitor of calmodulin kinase, or staurosporin (10 nM), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca(2+)-activated Cl- currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca(2+)-activated Cl- currents. The outward Cl- currents at +69 mV were inhibited by NPPB (100 microM), IAA-94 (100 microM), DIDS (0.03-1 mM), 9-AC (300 microM and 1 mM) and DPC (1 mM), whereas the inward currents at -101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl- conductance controlled by cytosolic Ca2+ and ATP levels in rat submandibular acinar cells.     

Inwardly rectifying potassium (IRK) currents are correlated with IRK subunit expression in rat nucleus accumbens medium spiny neurons

Inwardly rectifying K+ (IRK) channels are critical for shaping cell excitability. Whole-cell patch-clamp and single-cell RT-PCR techniques were used to characterize the inwardly rectifying K+ currents found in projection neurons of the rat nucleus accumbens. Inwardly rectifying currents were highly selective for K+ and blocked by low millimolar concentrations of Cs+ or Ba2+. In a subset of neurons, the inwardly rectifying current appeared to inactivate at hyperpolarized membrane potentials. In an attempt to identify this subset, neurons were profiled using single-cell RT-PCR. Neurons expressing substance P mRNA exhibited noninactivating inward rectifier currents, whereas neurons expressing enkephalin mRNA exhibited inactivating inward rectifier currents. The inactivation of the inward rectifier was correlated with the expression of IRK1 mRNA. These results demonstrate a clear physiological difference in the properties of medium spiny neurons and suggest that this difference could influence active state transitions driven by cortical and hippocampal excitatory input.