A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K(+) homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca(2+)-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K(+). Membrane potentials for half-maximal open probability (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P(o)=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca(2+)]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 microM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 ms, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26+/-0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLFs and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K(+) recycling dynamics in the mammalian cochlea.     

Analysis of single K(ATP) channels in mammalian dentate gyrus granule cells

ATP-sensitive potassium (K(ATP)) channels are heteromultimer complexes of subunits from members of the inwardly rectifying K(+) channel and the ATP-binding cassette protein superfamilies. K(ATP) channels couple metabolic state to membrane excitability, are distributed widely, and participate in a variety of physiological functions. Understood best in pancreatic beta cells, where their activation inhibits insulin release, K(ATP) channels have been implicated also in postischemia cardio- and neuroprotection. The dentate gyrus (DG) is a brain region with a high density of K(ATP) channels and is relatively resistant to ischemia/reperfusion-induced cell death. Therefore we were interested in describing the characteristics of single K(ATP) channels in DG granule cells. We recorded single K(ATP) channels in 59/105 cell-attached patches from DG granule cells in acutely prepared hippocampal slices. Single-channel openings had an E(K) close to 0 mV (symmetrical K(+)) and were organized in bursts with a duration of 19.3 +/- 1.6 (SE) ms and a frequency of 3.5 +/- 0.8 Hz, a unitary slope conductance of 27 pS, and a low, voltage-independent, probability of opening (P(open), 0.04 +/- 0.01). Open and closed dwell-time histograms were fitted best with one (tau(open) = 1.3 +/- 0.2 ms) and the sum of two (tau(closed,fast) = 2.6 +/- 0.9 ms, tau(closed,slow) = 302.7 +/- 67. 7 ms) exponentials, respectively, consistent with a kinetic model having at least a single open and two closed states. The P(open) was reduced ostensibly to zero by the sulfonylureas, glybenclamide (500 nM, 2/6; 10 microM,11/14 patches) and tolbutamide (20 microM, 4/6; 100 microM, 4/4 patches). The blocking dynamics for glybenclamide included transition to a subconductance state (43.3 +/- 2.6% of control I(open channel)). Unlike glybenclamide, the blockade produced by tolbutamide was reversible. In 5/5 patches, application of diazoxide (100 microM) increased significantly P(open) (0.12 +/- 0.02), which was attributable to a twofold increase in the frequency of bursts (8.3 +/- 2.0 Hz). Diazoxide was without effect on tau(open) and tau(closed,fast) but decreased significantly tau(closed,slow) (24.4 +/- 2.6 ms). We observed similar effects in 6/7 patches after exposure to hypoxia/hypoglycemia, which increased significantly P(open) (0.09 +/- 0.03) and the frequency of bursts (7.1 +/- 1.7 Hz) and decreased significantly tau(closed,slow) (29.5 +/- 1.8 ms). We have presented convergent evidence consistent with single K(ATP) channel activity in DG granule cells. The subunit composition of K(ATP) channels native to DG granule cells is not known; however, the characteristics of the channel activity we recorded are representative of Kir6.1/SUR1, SUR2B-based channels.     

Characterization of GABA(A) and glycine receptors in neurons of the developing rat inferior colliculus

GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) expressed in developing neurons (P3-8) of the rat inferior colliculus (IC) were investigated using fast application of transmitters on nucleated patches and whole-cell patch-clamp recordings. At a holding potential of -60 mV and an E(Cl) close to 0 mV GABA and glycine activated inward currents with a half-maximal activation concentration of 127 microM and 353 microM, respectively. GABA(A)Rs activated and deactivated significantly faster than GlyRs (20-80% rise-time: 0.71 ms for GABA(A)Rs and 1.31 ms for GlyRs; amplitude-weighted decay time constant, tau(w), 22.4+/-1.3 ms for GABA(A)Rs and 108.7+/-32.9 ms for GlyRs). The tau(w) values for receptor desensitization were 26.8+/-1.9 ms for GABA(A)Rs and 177.9+/-47.2 ms for GlyRs. Recovery from desensitization was significantly faster for GlyRs (tau=334 ms) than for GABA(A)Rs (tau1=82 ms; tau2=3783 ms). The tau(w) values of GABA(A)R-mediated spontaneous and miniature inhibitory postsynaptic currents were not significantly different from those of GABA(A)Rs activated by fast application, but significantly different to the tau(w) of GlyRs activated by fast application. The different properties of GABA(A)Rs and GlyRs expressed in the same IC neuron suggest a distinct functional contribution to the development of inhibitory synapses in the IC.     

Identification of a novel voltage-gated Na+ channel rNav1.5a in the rat hippocampal progenitor stem cell line HiB5

A conditionally immortalised cell line, HiB5, derived from embryonic hippocampal precursor cells expressed a voltage-gated Na+ channel with electrophysiological characteristics shifted to more negative voltages and a lower sensitivity to tetrodotoxin [concentration required for half-maximal inhibition (IC50) 0.9 microM] compared with endogenous neuronal Na+ channels. The channel activation and steady-state inactivation occurred at very negative potentials with the threshold for activation at -55 mV and half-maximal inactivation at -78.7 mV. The channel was blocked by lamotrigine and sipatrigine voltage and state dependently, with potencies 5-20 times higher (IC50 12 and 1.8 microM at -80 mV respectively) than the corresponding block of endogenous Na+ channels from neurones and cloned rNa(v)1.2a (rBIIA) alpha-subunits. Both compounds slowed the channel's recovery from inactivation. Whereas lamotrigine and sipatrigine had similar effects on the fast inactivated state, the effect of sipatrigine on the slow inactivation state was more pronounced, rendering this compound overall the more effective. The molecular subtype mainly expressed by HiB5 cells was identified using RT-PCR and was a novel splice variant of rNa(v)1.5 (rNa(v)1.5a). It differs from the known rNa(v)1.5 version in that it lacks 53 amino acids in the intracellular loop between domains II and III. rNa(v)1.5a was also detected in mRNA derived from rat whole brain.     

Serotonin inhibits the peptide FMRFamide response through a cyclic AMP-independent pathway in Aplysia.

1. The S-K+ conductance was isolated by voltage-clamping near the resting potential pleural mechanosensory neurons of Aplysia in culture. This background conductance is modulated in opposite directions by two distinct, transmitter-controlled second-messenger cascades: it is enhanced by the peptide FMRFamide through the 12-lipoxygenase pathway of arachidonic acid, and it is decreased by serotonin (5-HT) through adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphorylation. 2. The dose-dependent activating effect of FMRFamide (0.01-500 microM) on the S-K+ conductance was measured in the presence and the absence either of 1-100 microM 8-bromo-cAMP (8b-cAMP, a membrane-permeable and hydrolysis-resistant analogue of cAMP), or of 0.01-0.1 microM 5-HT. 3. When 8b-cAMP was applied, it produced a slow inward current response due to closure of the S-K+ conductance. This response was antagonized by FMRFamide in a dose-dependent mode. Application of 100 microM FMRFamide, in the presence of 1-10 microM 8b-cAMP, produced an outward current response larger than the control FMRFa response and equal to the sum of the responses to FMRFamide alone and to 8b-cAMP alone. Similarly, at 500 microM, FMRFamide completely antagonized the closing action of maximal 8b-cAMP levels (100 microM). This observation confirms previous work that indicated that FMRFamide can reopen S-K+ channels closed by FMRFamide. 4. In contrast, in the presence of moderate concentrations of 5-HT (0.01 microM), which produce a slow inward current due to the closing of the S-K+ conductance, FMRFamide elicited a response that only partially antagonized this 5-HT action. Under maximal 5-HT concentrations (0.1 microM), the 5-HT response was not antagonized by any FMRFamide concentration: instead, the FMRFamide response was smaller than the control response without 5-HT. This experiment suggests that 5-HT, with an action independent from cAMP, inhibits the effect of FMRFamide on the S-K+ channel. 5. The dose-dependent inhibitory effect of 5-HT (0.001-10 microM) on the S-K+ conductance was measured in the presence and the absence either of FMRFamide (1-50 microM), which stimulates the release and metabolism of arachidonic acid in Aplysia sensory neurons or of arachidonic acid (25 microM). 6. Under these conditions, supramaximal concentrations of 5-HT could not completely suppress the slow outward current evoked by FMRFamide or by arachidonic acid, indicating that a component of the arachidonic-mediated response to FMRFamide is resistant to actions that maximally increase the S-K+ channel phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ventilatory response to hypoxia in the anesthetized rat

Inhibitory postsynaptic membrane channels which are activated by glycine were investigated by means of the noise analysis technique. Dose-response curves were obtained for gamma-aminobutyric acid (GABA) in the presence and in the absence of glycine, and it was concluded that GABA and glycine are likely to activate the same receptors. However, glycine proved to have a very low affinity for the inhibitory postsynaptic receptors; this affinity was smaller than that of GABA by a factor of 1 . 10(3)-2 . 10(3). The mean open time tau of the postsynaptic Cl- channels activated by glycine at E = -100 mV and E = -60 mV membrane potentials were tau = 6.1 ms +/- 1.5 ms and tau = 17.7 ms +/- 2.2 ms, respectively. These values are in agreement with the tau obtained by activation with GABA (Dudel et al. 1980); however, on activation by glycine the potential dependence of tau was larger by a factor of 1.35. At E = -100 mV the conductance gamma of glycine-operated channels was about 3 pS which is a third of the respective conductance elicited by GABA. In the presence of high concentrations of glycine (0.1-0.5 mol/l) spontaneous inhibitory postsynaptic currents (sIPSCs) and 'giant' spontaneous inhibitory postsynaptic currents (gsIPSCs) were observed. Furthermore at high concentrations of glycine an additional glycine-induced noise component was found in the power spectra of current fluctuations at higher frequencies. It was concluded that this spectral component resulted from the closing of otherwise open K+ channels, as has been observed already on application of GABA (Dudel and Finger 1980). The mean duration of the low conductance state was tau- = 2.2 ms +/- 0.9 ms and the conductance decrease gamma- coupled to this process was estimated to be about 3 pS. In Na+ free- and Ca2+-enriched bathing solutions the glycine-induced conductances gamma and gamma- were reduced by a factor of about 1.7 while tau and tau- remained unchanged. The decrease in gamma and gamma- was most likely effected by the increase in concentration of divalent cations.     

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons.The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.     

Electrophysiological characteristics of rat gustatory cyclic nucleotide--gated channel expressed in Xenopus oocytes

The complementary DNA encoding gustatory cyclic nucleotide--gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na(+). Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability (P(o)) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na(+) approximately K(+) > Rb(+) > Li(+) > Cs(+) with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na(+) currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants (K(i)s) of intracellular Mg(2+) were determined as 360 +/- 40 microM at 70 mV and 8.2 +/- 1.5 mM at -70 mV with z delta value of 1.04, while K(i)s of extracellular Mg(2+) were as 1.1 +/- 0.3 mM at 70 mV and 20.0 +/- 0.1 microM at -70 mV with z delta of 0.94. Although 100 microM l-cis-diltiazem blocked significant portions of outward Na(+) currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.     

Alteration of the membrane lipid environment by L-palmitoylcarnitine modulates KATP channels in guinea-pig ventricular myocytes

Sarcolemmal adenosine 5'-triphosphate-sensitive K+ channels (K(ATP)) are dramatically up-regulated by a membrane phospholipid, phosphatidyl-inositol-4,5-bisphosphate (PIP2). During ischaemia, L-palmitoylcarnitine (L-PC), a fatty acid metabolite, accumulates in the sarcolemma and deranges the membrane lipid environment. We therefore investigated whether alteration of the membrane lipid environment by L-PC modulates the K(ATP) channel activity in inside-out patches from guinea-pig ventricular myocytes. L-PC (1 microM) inhibited KATP channel activity, without affecting the single channel conductance, through interaction with Kir6.2. L-PC simultaneously enhanced the ATP sensitivity of the channel [concentration for half-maximal inhibition (IC50) fell from 62.0+/-2.7 to 30.3+/-5.5 microM]. In contrast, PIP2 attenuated the ATP sensitivity (IC50 343.6+/-54.4 microM) and restored Ca2+-induced inactivation of KATP channels (94.1+/-13.7% of the control current immediately before the Ca2+-induced inactivation). Pretreatment of the patch membrane with 1 microM L-PC, however, reduced the magnitude of the PIP2-induced recovery to 22.7+/-6.3% of the control (P<0.01 vs. 94.1+/-13.7% in the absence of L-PC). Conversely, after the PIP2-induced recovery, L-PC's inhibitory action was attenuated, but L-PC partly reversed the PIP2-mediated decrease in the ATP sensitivity (IC50 fell from 310+/-19.2 to 93.1+/-9.8 microM). Thus, interaction between L-PC and PIP2 in the plasma membrane appears to regulate K(ATP) channels.     

Modulation of a cloned human A-type voltage-gated potassium channel (hKv1.4) by the protein tyrosine kinase inhibitor genistein

A cloned, human, A-type, voltage-gated potassium channel (hKv1.4) was expressed transiently in Chinese hamster ovary cells and the effects of the broad-spectrum tyrosine kinase inhibitor genistein on hKv1.4 were studied using the whole-cell patch-clamp recording method. Genistein (up to 50 microM) reversibly reduced the peak currents of hKv1.4 by 44.9+/-12%. In addition, genistein markedly slowed the activation kinetics (time constant tau(a)) of hKv1.4. At +50 mV, tau(a) increased from 1.8+/-0.3 to 5.0+/-0.6 ms (P<0.01). The effect of genistein on the channel inactivation kinetics (time constant tau(i)) was more complex, in that tau(i) was increased significantly at lower step potentials but unaltered at +50 mV or more depolarized potentials. Tail current analysis showed that genistein had no effect on the kinetics of deactivation (time constant tau(d)), but shifted the steady-state activation curve significantly to the right by about 15 mV (potential for half-maximal activation, V1/2, changed from -7.4+/-4.4 to +7.7+/-2.7 mV) with a moderate change in the slope (k) of the curve (from 17.4+/-2.2 to 23+/-1.0 mV, P<0.05). Genistein slightly altered the slope of the steady-state inactivation curve from -5.5+/-0.4 to -7.5+/-0.4 mV (P<0.01). The recovery rate from inactivation was not altered by genistein. The tyrosine phosphatase inhibitor orthovanadate (1 mM) alone had little impact on current amplitude or channel kinetics. However, orthovanadate significantly, but not completely, blocked the effect of genistein on current amplitude (by 25.5%) and kinetics (by 67.1%). Daidzein (up to 50 microM), an inactive analogue of genistein, had no effect on current amplitude or kinetics. In contrast to genistein, another tyrosine kinase inhibitor, herbimycin A, had little effect on the channel peak amplitude or kinetics. In addition, genistein had a similar impact on the channel peak current amplitude and kinetics in cells with or without pre-treatment with herbimycin A (10 microM). The data suggest that genistein-induced inhibition of tyrosine phosphorylation may not be the exclusive mechanism by which hKv1.4 is down-regulated and channel gating affected. Genistein may produce a non-catalytic blockade of this channel.     

The properties of the Kir6.1–6.2 tandem channel co-expressed with SUR2A

Functional ATP-sensitive K (KATP) channels have an octameric subunit structure with four pore-forming subunits (Kir6.x) and four sulfonylurea receptors (SURx). In the present study, the properties of the heteromeric KATP channel whose pore subunits are composed of Kir6.1 and Kir6.2 were examined using a heterologous expression system. In COS7 cells co-transfected with Kir6.1, Kir6.2 and SUR2A at a ratio of 1:1:2, KATP channels showed various unitary conductances between those of Kir6.1/SUR2A (33.6+/-4.2 pS) and Kir6.2/ SUR2A (67.1+/-1.6 pS). Kir6.1-6.2 tandem protein, constructed by fusing the C-terminus of Kir6.1 to the N-terminus of Kir6.2 with a ten glutamine linker sequence, also formed a channel with an intermediate conductance (58.9+/-1.5 pS). Kir6.2 and Kir6.1-6.2 showed similar sensitivity to ATP4-: half-maximal inhibition (IC50) was obtained at 14.1+/-12.8 microM and 17.6+/-9.6 microM, respectively. In the presence of Mg2+, Kir6. 1-6.2 was significantly less sensitive than Kir6.2 to MgATP (IC50=95.5+/-49.6 microM versus 18.9+/-5.0 microM). These results suggest that Kir6.1 and Kir6.2 are endowed with the potential to form a heteromeric KATP channel, which has a low sensitivity to MgATP.     

Thermogenesis in brown adipose tissue: increase by 5-HT2A receptor activation and decrease by 5-HT1A receptor activation in conscious rats

Body temperature is decreased by 5-hydroxytryptamine 1A (5-HT1A) agonists and increased by 5-HT2A agonists. The present study determined whether changes in interscapular brown adipose tissue (iBAT) thermogenesis contribute to these effects in conscious unrestrained animals. Male Sprague-Dawley rats were pre-instrumented for measurement of iBAT and core temperature and tail artery blood flow one week before experiments. In the first series of experiments, rats were transferred from warm (25-28 degrees C) to cold (5-10 degrees C) environments. This increased iBAT temperature (+1.3 +/- 0.2 degrees C, P<0.01, n = 7) and reduced tail artery flow. Injection of the 5-HT1A agonist, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin, 0.5 mg/kg, s.c.) reversed the increase in iBAT thermogenesis (-1.5 +/- 0.4 degrees C, P<0.01, n = 6), and decreased core temperature (-1.5 +/- 0.4 degrees C, P<0.01, n = 6). Pre-treatment with WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride), a 5-HT1A antagonist, prevented effects of 8-OH-DPAT. In the second series of experiments, injection of a 5-HT2A agonist, DOI (R(-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride, 0.1 mg/kg, s.c.) increased both iBAT (+1.9 +/- 0.1 degrees C, P<0.01, n = 7) and core temperatures (+1.4+/-0.2 degrees C, P<0.01, n=7), and decreased tail artery blood flow. Subsequent injection of SR 46349B (trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl) propen-1-yl)-phenol, hemifumarate, 0.5 mg/kg, s.c.), a 5-HT2A antagonist, reduced all these changes. Results indicate that activation of 5-HT1A receptors reduces sympathetic outflow to BAT and that activation of 5-HT2A receptors increases this outflow. Changes in core temperature mediated by brain/spinal pathways regulated by 5-HT1A and 5-HT2A receptors reflect coordinated changes in BAT-mediated heat production as well as changes in heat dissipation via the thermoregulatory cutaneous vascular beds.     

Membrane potential and conductance of frog skin gland acinar cells in resting conditions and during stimulation with agonists of macroscopic secretion

Frog skin glands were stripped of connective tissue and investigated using the nystatin-permeabilized whole-cell patch-clamp configuration. The membrane potential in unstimulated acinar cells was -69.5+/-0.7 mV, and the conductance was dominated by K+, based on ion substitution experiments. The cells were electrically coupled through heptanol- and halothane-sensitive gap junctions. During application of gap junction blockers, the whole-cell current/voltage relationship displayed strong outward rectification. Outward currents were blocked by barium. Stimulation by agonists known to cause increases in either cytosolic cAMP ([cAMP]c) (isoproterenol, prostaglandin E2, both at 2 microM) or free cellular Ca2+ concentration ([Ca2+]c) (noradrenaline, 10 microM, added with propranolol, 5 microM; carbachol, 100 microM) in the frog skin glands caused reversible depolarization: by 34+/-3 mV, 36+/-3 mV, 25+/-3 mV (plateau-phase), and 20+/-3 mV, respectively. Ion substitution experiments showed that stimulation through either pathway (cAMP or Ca2+) resulted in the activation of a Cl- conductance. Application of noradrenaline or adrenaline resulted in a faster depolarization (rates 22 mV/s, 26 mV/s) than stimulation by isoproterenol or prostaglandin E2 (5.6-5.7 mV/s). Cells that were depolarized by exposure to isoproterenol or prostaglandin E2 partially repolarized when stimulated by noradrenaline. The repolarization was blocked by Ba2+ (5 mM) or prazosine (1 microM), consistent with the activation of Ca(2+)-dependent K+ channels via alpha1-adrenergic receptors. We conclude that in the frog skin gland both Ca(2+)-dependent and cAMP-dependent Cl- channels are present in the apical membrane. Increases in free [Ca2+]c in the cAMP-stimulated gland results in the activation of K+ channels, thereby increasing the driving force for Cl- exit.     

Activation of wild-type and ΔF508-CFTR by phosphodiesterase inhibitors through cAMP-dependent and -independent mechanisms

 The cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) and its modulation through inhibition of phosphodiesterases (PDE) were studied with the cell-attached patch-clamp technique in Calu-3 cells (expressing endogenous CFTR) and NIH3T3 cells [expressing either wild-type (Wt)-CFTR or ΔF508-CFTR]. In Calu-3 cells, CFTR current was augmented by increasing concentrations of 8-(4-chlorophenylthio)-adenosine 3′,5′-cyclic monophosphate (CPT–cAMP) and reached a saturating level at ≥60 μM. Varying the forskolin concentration also modulated CFTR activity; 10 μM was maximally effective since supplemental application of 200 μM CPT–cAMP had no additional effect. Activation of CFTR by increasing the cAMP concentration occurs through an increase of the NP _o (product of the number of functional channels and the open probability) since the single-channel amplitude remains unchanged. In Calu-3 and NIH3T3-Wt cells, PDE inhibitors, milrinone (100 μM), 8-cyclopentyl-1,3-dipropylxanthine (CPX, 25 μM), and 3-isobutyl-1-methylxanthine (IBMX, 200 μM), did not enhance CFTR current initially activated with 10 μM forskolin, but each potentiated CFTR activity elicited with a submaximal forskolin concentration (e.g., 100 nM) and prolonged the deactivation of CFTR channel current upon removal of forskolin. Millimolar IBMX increased the NP _o of both Wt- and ΔF508-CFTR even under maximal cAMP stimulation. Quantitatively, these effects of millimolar IBMX on NP _o approximate those of genistein, which potentiates the cAMP-dependent CFTR activity via a mechanism that does not involve increases in cellular cAMP. Thus, depending on the concentration, PDE inhibitors may affect CFTR through different mechanisms. Received: 27 October 1998 / Accepted: 10 November 1998     

Nicotinic acetylcholine receptors are directly affected by agents used to study protein phosphorylation.

1. Messenger RNAs for the subunits of the muscle nicotinic acetylcholine receptor (nAChR) were expressed in Xenopus oocytes. A two-electrode voltage clamp was used to measure the acetylcholine (ACh)-induced macroscopic currents. In addition, patch-clamp techniques were used to study nAChR channels in whole cells and in outside-out patches excised from BC3H-1 cells and in patches from oocytes. The single-channel and macroscopic currents were modified by compounds that are usually used to study protein phosphorylation. 2. IBMX (3-isobutyl-1-methylxanthine) is a phosphodiesterase inhibitor. Because it elevates the intracellular concentration of adenosine 3',5'-cyclic monophosphate (cAMP), IBMX is often used to indirectly activate cAMP-dependent protein kinase. H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] is mainly used as a rather nonspecific inhibitor of protein kinase activity. Both IBMX and H-7 directly inhibit ACh-induced currents independent of their action on phosphorylation. This direct effect of these compounds is similar to the previously reported inhibition of nAChRs and K+ channels by forskolin, which is commonly used to elevate intracellular cAMP. 3. Macroscopic currents induced in the oocytes by 50 microM ACh had an average peak current of 605 nA, and the currents decayed biexponentially with tau of 15 and 225 s. When 300 microM H-7 was added simultaneously with the ACh, the average peak current was 228 nA and the tau were 1 and 108 s. When 500 microM IBMX was added simultaneously with the ACh, the average peak current was 308 nA and the tau were 9 and 237 s. H-7 and IBMX decreased the peak current induced by ACh, and the compounds increased the decay rate of the current. Under these experimental conditions, the IC50 for reduction of peak amplitude at -30 mV was 160 microM for H-7 and 475 microM for IBMX. 4. H-7 preferentially inhibits the open conformation of the nAChR channel, but there is also some inhibition of the closed channel. The inhibition is voltage dependent: inhibition decreases e-fold per 34 mV depolarization. H-7 does not become trapped within the closed channel and does not significantly alter desensitization under our experimental conditions. 5. H-7 and IBMX interrupt or terminate single-channel openings in membrane patches excised from oocytes or BC3H-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane channels activated by taurine in cultured mouse spinal cord neurons

Patch-clamp methods were used to compare biophysical properties of anion channels activated by taurine and gamma-aminobutyric acid in the membrane of cultured mouse spinal neurons. Outside-out patches were voltage clamped at -80 mV at a temperature of 21-23 degrees C. Bath application of GABA (1.5-2 microM) or taurine (5-40 microM) induced chloride-dependent single-channel currents in 14/20 patches tested. Amplitude distributions of these currents showed peaks corresponding to conductance levels of 8, 16, 27 and 46 pS. Only a few percent of GABA-induced events reached the 46 pS level, while 30% of taurine-induced currents were of this size. The average lifetime of taurine-activated channels in the open state was 1.0 +/- 0.07 ms, significantly shorter than the corresponding value for GABA (1.6 +/- 0.08 ms). Taurine-induced currents were abolished by 10 microM strychnine, but persisted in the presence of 50 microM bicuculline.     

Characterization of two distinct 5-HT receptors coupled to adenylate cyclase activation and ion current generation in NCB-20 cells.

The level of cyclic AMP in NCB-20 cells was increased by serotonin (5-HT), 5-methoxytryptamine and 2-methyl-5-HT with EC50 of 0.5 +/- 0.1, 1.0 +/- 0.1, 10 +/- 0.1 microM, respectively. The 5-HT-mediated increase of cyclic AMP content was completely blocked by metergoline but unaffected by 5-HT3 antagonists, ICS 205-930, MDL 72222, quipazine and 5-HT2 antagonist, ketanserin. Putative 5-HT1A agonists (8-OH-DPAT, ipsapirone, and buspirone) and 5-HT1B agonists (TFMPP and m-CPP) affected neither basal nor forskolin-dependent cyclic AMP accumulation. Receptor binding studies suggest that NCB-20 cells are devoid of 5-HT1A and 5-HT1B receptor sites. Application of 5-HT onto NCB-20 cells resulted in membrane depolarization by an evoked inward current which displayed rapid desensitization. 5-HT-mediated current had a reversal potential around 0 mV and was potently and reversibly inhibited by ICS 205-930. Our data suggest that in NCB-20 cells the 5-HT3 receptor is involved in the generation of inward currents, while the 5-HT receptor coupled to adenylate cyclase does not seem to correspond to any of the known receptor subtypes.     

Activation properties of chemically skinned fibres from human isolated bronchial smooth muscle.

1. The contractile activation properties of human isolated bronchial smooth muscle were investigated using chemically (beta-escin) skinned strips. 2. Concentration-dependent contractions were induced by free ion concentrations of Ca2+ (0.5-3 microM), Sr2+ (2-200 microM) and Ba2+ (50-1000 microM). The resulting -log[cation]-tension relationships were fitted by sigmoidal curves with EC50 values (cation concentration required to produce half-maximal tension) and co-operativity factors (Hill coefficient, nH) of, respectively, 0.25 microM and 3.4 for Ca2+, 12 microM and 2.64 for Sr2+ and 100 microM and 1.73 for Ba2+. Maximal responses to Sr2+ and Ba2+ were 125.5 +/- 15.4 and 96 +/- 8.1% (n = 5) respectively of the maximum tension induced by Ca2+. 3. Trifluoperazine (5-100 microM), cyclic AMP (50-300 microM) and cyclic GMP (50-100 microM) each antagonized Ca2+ in a concentration-dependent manner. On the other hand, okadaic acid (OA, 0.2-1 microM) potentiated Ca2+ and increased the maximum response to Ca2+ (+25 +/- 5.4%, n = 5, for 1 microM OA). 4. This study has demonstrated the high Ca2+ sensitivity of the activation mechanism of human isolated bronchial smooth muscle. It also suggests that control of the contractile machinery in the human bronchus involves processes of phosphorylation and dephosphorylation. The beta-escin-treated human bronchus may be a useful model for investigating the cellular basis of some pathophysiological processes such as bronchial hyper-responsiveness.     

Effect of lamotrigine on the Ca(2+)-sensing cation current in cultured hippocampal neurons.

Concentrations of extracellular calcium ([Ca(2+)](e)) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca(2+)](e) activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of -60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca(2+)](e) from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC(50) of 171 +/- 25.8 (SE) microM. The effect of LTG was independent of membrane potential. In the presence of 300 microM LTG, the amplitude of csNSC current was decreased by 31 +/- 3% at -60 mV and 29 +/- 2.9% at +40 mV (P > 0.05). LTG depressed csNSC current without affecting the potency of Ca(2+) block of the current (IC(50) for Ca(2+) block of csNSC currents in the absence of LTG: 145 +/- 18 microM; in the presence of 300 microM LTG: 136 +/- 10 microM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca(2+)](e) caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 microM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca(2+) imaging experiment showed that LTG also inhibited the increase in intracellular Ca(2+) concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.