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Results for “coupled patch model”

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At least 217 records · Page 12Linked to original sources

PubMed2001

GABA(A) receptor beta 2 Tyr97 and Leu99 line the GABA-binding site. Insights into mechanisms of agonist and antagonist actions.

The identification of residues that line neurotransmitter-binding sites and catalyze allosteric transitions that result in channel gating is crucial for understanding ligand-gated ion channel function. In this study, we used the substituted cysteine accessibility method and two-electrode voltage clamp to identify novel gamma-aminobutyric acid (GABA)-binding site residues and to elucidate the secondary structure of the Trp(92)-Asp(101) region of the beta(2) subunit. Each residue was mutated individually to cysteine and expressed with wild-type alpha(1) subunits in Xenopus oocytes. GABA-gated currents (I(GABA)) were measured before and after exposure to the sulfhydryl reagent, N-biotinylaminoethyl methanethiosulfonate (MTS). V93C, D95C, Y97C, and L99C are accessible to derivatization. This pattern of accessibility is consistent with beta(2)Val(93)-Leu(99) adopting a beta-strand conformation. Both GABA and SR95531 protect Y97C and L99C from modification, indicating that these two residues line the GABA-binding site. In D95C-containing receptors, application of MTS in the presence of SR95531 causes a greater effect on I(GABA) than MTS alone, suggesting that binding of a competitive antagonist can cause movements in the binding site. In addition, we present evidence that beta(2)L99C homomers form spontaneously open channels. Thus, mutation of a binding site residue can alter channel gating, which implies that Leu(99) may be important for coupling agonist binding to channel gating.

Amino Acid Sequence↗

PubMed2002

Dual and opposing roles of presynaptic Ca2+ influx for spontaneous GABA release from rat medial preoptic nerve terminals.

Calcium influx into the presynaptic nerve terminal is well established as a trigger signal for transmitter release by exocytosis. By studying dissociated preoptic neurons with functional adhering nerve terminals, we here show that presynaptic Ca2+ influx plays dual and opposing roles in the control of spontaneous transmitter release. Thus, application of various Ca2+ channel blockers paradoxically increased the frequency of spontaneous (miniature) inhibitory GABA-mediated postsynaptic currents (mIPSCs). Similar effects on mIPSC frequency were recorded upon washout of Cd2+ or EGTA from the external solution. The results are explained by a model with parallel Ca2+ influx through channels coupled to the exocytotic machinery and through channels coupled to Ca2+-activated K+ channels at a distance from the release site.

Animals↗

PubMed2003

Modulation of Ca2+ channel currents by a novel antidementia drug N-(4-Acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate (FK960) in rat hippocampal neurons.

N-(4-Acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate (FK960), a novel antidementia drug, has been demonstrated to ameliorate memory deficits in various experimental models of dementia. This drug selectively increases somatostatin release from hippocampal slices and augments long-term potentiation (LTP) in the CA3 area of the hippocampus. In the present study, the effects of FK960 on voltage-activated Ca2+ channels were investigated in acutely isolated rat hippocampal neurons, using whole-cell patch-clamp technique to clarify the cellular mode of action of FK960. Application of somatostatin significantly reduced Ca2+ currents via G protein-coupled signaling pathways. This inhibitory effect was significantly abolished by FK960 when applied in combination. In contrast, FK960 showed only modest inhibition on the reduction in Ca2+ currents produced by baclofen, an agonist of GABAB receptor. Intracellular application of the protein kinase inhibitor H-7 did not alter somatostatin-induced inhibition and had no significant effect on blockade by FK960. In addition, application of FK960 alone produced modest but apparent increases in Ca2+ currents without significant changes in the activation kinetics of the channels. The dose-response relationship on calcium current enhancement was bell-shaped with a maximum effect at 0.1 microM FK960, the same concentration as that for increasing on somatostatin release and CA3-LTP. These results show that FK960 reverses G protein-dependent inhibition of Ca2+ currents by somatostatin in hippocampal neurons. Enhancement of Ca2+ currents by FK960 may be due to its modulatory actions on Ca2+ channels, rather than removal of G protein-inhibited tonic currents. Together, these mechanisms may be involved in the selective effects of FK960 on somatostatin release, excitatory transmission, and synaptic plasticity in the hippocampus.

Animals↗

PubMed2003

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Animals↗

PubMed2003

Synergistic activation of mitogen-activated protein kinase by insulin and adenosine triphosphate in liver cells: permissive role of Ca2+.

We have previously demonstrated that insulin and G(q)-coupled receptor agonists individually activate mitogen-activated protein kinase (MAPK) in liver cells and both effects involve an influx of extracellular Ca(2+). Yet, these agonists have opposing physiological actions on hepatocyte glucose metabolism. We thus investigated the interaction between insulin and the P2Y(2) purinergic agonist adenosine triphosphate (ATP) on MAPK in HTC cells, a model hepatocyte cell line, and determined the involvement of cytosolic Ca(2+). Insulin and ATP each induced a dose-dependent phosphorylation of p44/42 MAPK that was partially inhibited by EGTA. However, pretreatment with insulin markedly increased the MAPK phosphorylation response to ATP. This potentiation was canceled by chelation of extracellular Ca(2+) with EGTA. We used patch clamp electrophysiology and fluorescence microscopy to understand the role of intracellular Ca(2+) in this effect. Insulin and ATP, respectively, induced monophasic and multiphasic changes in membrane potential and intracellular Ca(2+) as expected. Pretreatment with 10 nmol/L insulin significantly decreased the initial rapid depolarization (inward nonselective cation current [NSCC]), as well as the compounded Ca(2+) response induced by 100 micro mol/L ATP. However, in Ca(2+)-free conditions, insulin did not modify the Ca(2+) mobilized from internal pools after stimulation with ATP. Upon Ca(2+) readmission, internal store depletion by ATP or thapsigargin doubled the rate of capacitative Ca(2+) influx, whereas insulin increased this influx 1.32-fold. On the other hand, insulin pretreatment counteracted the increased rate of Ca(2+) influx induced by ATP but not by thapsigargin. In summary, insulin counteracts the membrane potential and Ca(2+) responses to ATP in HTC cells. However, insulin and ATP effects on MAPK activation are synergistic and Ca(2+) influx plays a permissive role. Therefore, the opposing metabolic actions of insulin and G(q)-coupled receptor agonists involve an interaction in signaling pathways that resides downstream of Ca(2+) influx.

Adenosine Triphosphate↗

PubMed2000

Cellular and functional defects in a mouse model of heart failure.

Heart failure and dilated cardiomyopathy develop in mice that lack the muscle LIM protein (MLP) gene (MLP(-/-)). The character and extent of the heart failure that occurs in MLP(-/-) mice were investigated using echocardiography and in vivo pressure-volume (P-V) loop measurements. P-V loop data were obtained with a new method for mice (sonomicrometry) using two pairs of orthogonal piezoelectric crystals implanted in the endocardial wall. Sonomicrometry revealed right-shifted P-V loops in MLP(-/-) mice, depressed systolic contractility, and additional evidence of heart failure. Cellular changes in MLP(-/-) mice were examined in isolated single cells using patch-clamp and confocal Ca(2+) concentration ([Ca(2+)]) imaging techniques. This cellular investigation revealed unchanged Ca(2+) currents and Ca(2+) spark characteristics but decreased intracellular [Ca(2+)] transients and contractile responses and a defect in excitation-contraction coupling. Normal cellular and whole heart function was restored in MLP(-/-) mice that express a cardiac-targeted transgene, which blocks the function of beta-adrenergic receptor (beta-AR) kinase-1 (betaARK1). These data suggest that, despite the persistent stimulus to develop heart failure in MLP(-/-) mice (i.e., loss of the structural protein MLP), downregulation and desensitization of the beta-ARs may play a pivotal role in the pathogenesis. Furthermore, this work suggests that the inhibition of betaARK1 action may prove an effective therapy for heart failure.

Animals↗

PubMed2005

Beyond two-cell networks: experimental measurement of neuronal responses to multiple synaptic inputs.

Oscillations of large populations of neurons are thought to be important in the normal functioning of the brain. We have used phase response curve (PRC) methods to characterize the dynamics of single neurons and predict population dynamics. Our past experimental work was limited to special circumstances (e.g., 2-cell networks of periodically firing neurons). Here, we explore the feasibility of extending our methods to predict the synchronization properties of stellate cells (SCs) in the rat entorhinal cortex under broader conditions. In particular, we test the hypothesis that PRCs in SCs scale linearly with changes in synaptic amplitude, and measure how well responses to Poisson process-driven inputs can be predicted in terms of PRCs. Although we see nonlinear responses to excitatory and inhibitory inputs, we find that models based on weak coupling account for scaling and Poisson process-driven inputs reasonably accurately.

Animals↗

PubMed1995

Opioid inhibition in locus coeruleus.

1. Inhibition of locus coeruleus (LC) neurons by opioids is mediated by hyperpolarization associated with an increase in potassium conductance. However, opioids caused an outward current even at potentials more negative than the potassium equilibrium potential in the majority of LC neurons recorded in brain slices cut in the horizontal plane. 2. Whole cell and intracellular recording were made from LC neurons in the slice preparation and currents were measured with a switch clamp amplifier. 3. Local application of [Met]5enkephalin (ME) by iontophoresis resulted in two different effects depending on the site of application. Iontophoresis of ME close to the cell caused an outward current that reversed polarity near the potassium equilibrium potential. Whereas application at a distance > 200 microns resulted in an outward current that did not reverse polarity even at strongly negative potentials. 4. When potassium conductances were blocked with internal Cs and/or external Ba, the opioid current was reduced to < 15% of control. 5. Low sodium solutions were used to reduce sodium-dependent conductances. These solutions hyperpolarized LC neurons themselves, decreased outward currents caused by ME, and shifted the reversal potential of the ME induced current to less negative values. 6. Electrotonic coupling of LC neurons in neonatal animals is thought to synchronize spontaneous activity. In this study, synchronous activity in cells from adult animals also was observed, suggesting that electrotonic coupling in the LC persists into adulthood. 7. Carbenoxolone, an agent thought to block electrotonic coupling, had no effect on the action potential, spontaneous activity, or the resting conductance but blocked electrotonic coupling between LC neurons in neonates, blocked synchronous activity in LC neurons from adult animals, and shifted the reversal potential of the opioid current to the potassium equilibrium potential. 8. All results are consistent with the hypothesis that LC cells are electrotonically coupled. Such electrotonic coupling at the level of the nucleus locus coeruleus would mediate synchronous regulation of noradrenaline in widespread areas of the brain.

Animals↗

PubMed2001

Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel.

Sequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K(+) channel (KcsA), support the tenet that voltage-gated K(+) channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface.

Animals↗

PubMed2004

Simultaneous contribution of two rod pathways to AII amacrine and cone bipolar cell light responses.

Rod signals traverse several synapses en route to cone bipolar cells. In one pathway, rods communicate directly with cones via gap junctions. In a second pathway, signals flow rods-rod bipolars-AII amacrines-cone bipolars. The relative contribution of each pathway to retinal function is not well understood. Here we have examined this question from the perspective of the AII amacrine. AIIs form bidirectional electrical synapses with on cone bipolars. Consequently, as on cone bipolars are activated by outer plexiform inputs, they too should contribute to the AII response. Rod bipolar inputs to AIIs were blocked by AMPA receptor antagonists, revealing a smaller, non-AMPA component of the light response. This small residual response did not reverse between -70 and +70 mV and was blocked by carbenoxolone, suggesting that the current arose in on cone bipolars and was transmitted to AIIs via gap junctions. The residual component was evident for stimuli 2 log units below cone threshold and was prolonged for bright stimuli, demonstrating that it was rod driven. Because the rod bipolar-AII pathway was blocked, the rod-driven residual current likely was generated via the rod-cone pathway activation of on cone bipolars. Thus for a large range of intensities, rod signals reach the inner retina by both rod bipolar-AII and rod-cone coupling pathways.

Amacrine Cells↗

PubMed1986

Some limitations of the cell-attached patch clamp technique: a two-electrode analysis.

With two independent patch electrodes sealed to small clusters of electrically coupled chick embryo cardiac cells, we have measured four parameters: true seal and patch resistance, channel conductance, and membrane potential. One electrode was in the cell-attached mode, and recorded current flowing in parallel through the membrane patch and seal. The second electrode, sealed on a different cell in the cluster, was in the whole cell recording configuration, and served to record or control the membrane potential of the cluster. We fit the four measured parameters to a simple electrical model to reveal errors not usually recognized in the patch-clamp technique. Among these are the following: (1) The apparent seal resistance, determined by changing the potential in a patch electrode, may be a poor estimate of true seal resistance, since it includes the parallel combination of seal- and patch-resistance. (2) Patch resistance may be influenced by the electrode filling solution, and is often much lower than is usually assumed. (3) With a small cell preparation that has an input resistance in the gigaohm range, measurements of single-channel conductance using a cell-attached patch electrode may be inaccurate because cell membrane potential does not remain constant as electrode potential is varied.

Animals↗

PubMed2004

Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment.

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

Animals↗

PubMed1999

Cellular basis for the acute inhibitory effects of IL-6 and TNF- alpha on excitation-contraction coupling.

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF- alpha). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic actions of interleukin-6 (IL-6) and TNF- alpha in the same model. We studied the effects of cytokines on [Ca(2+)](i)transients (using indo-1), cell shortening (CS) (using a video motion detector) and the L-type Ca(2+)channel current (I(Ca)) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF- alpha (500 U/ml) decreased both peak systolic [Ca(2+)](i)(IL-6: 0.43+/-0.01 to 0.40+/-0.01, n=5, P<0.05; TNF- alpha : 0.42+/-0.02 to 0.39+/-0.02, n=5, P<0.05) and the amplitude of CS (IL-6: 7.5+/-0.9 to 6.2+/-0.5 micrometers, n=5, P<0.05; TNF- alpha : 6.7+/-0.7 to 5.8+/-0.7 micrometers, n=5, P<0.05) without detectable reductions in I(Ca)(IL-6: 0.9+/-0.1 to 0.9+/-0.1 nA, n=4, N.S.; TNF- alpha : 1.1+/-0.3 to 1.1+/-0.2 nA, n=4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl- L arginine (300 micromol/l), blocked the effects of IL-6 but not of TNF- alpha. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both I(Ca)(2.8+/-0.5 to 2. 0+/-0.3 nA) and the amplitude of CS (10.4+/-2.4 to 7.5+/-1.9 micrometer) within 5 min. TNF- alpha also clearly depressed I(Ca)(2.9+/-0.9 to 2.3+/-0.7 nA) and the amplitude of CS (7.0+/-1.4 to 5.5+/-1.3 micrometer) in beta -adrenergic stimulated cells. TNF- alpha significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 micromol/l) mimicked those of TNF- alpha on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF- alpha did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.

Animals↗

PubMed2002

ENaC-CFTR interactions: the role of electrical coupling of ion fluxes explored in an epithelial cell model.

CFTR and the epithelial Na channel (ENaC) are two membrane proteins expressed in the apical membrane of several epithelia. They have been shown to influence each other's function but the mechanisms of this interaction are not fully understood. We have explored the role of electrical coupling between the ion fluxes carried by these two electrogenic transporters as one among other possible mechanisms through which these two proteins could interact. For this purpose, we have built a mathematical model of an epithelial cell monolayer comprising variable apical Cl(-) and Na(+) conductances, a basolateral Na,K-pump, basolateral Cl(-) and K(+) conductances and a paracellular "shunt" conductance. The model allows us to simulate ion transport under current- or voltage-clamp conditions and to monitor individual ionic membrane currents, membrane potentials and intracellular ion concentrations. Using a set of transport parameters extrapolated from published work, we can show, first, that the model reproduces the expected effects of conductance inhibition on transepithelial transport, and transepithelial and single membrane potentials or conductances. Second, concerning the influence of CFTR on ENaC, the simulations show that: (1) under current-clamp conditions, the amplitude of the apical membrane Cl(-) conductance has a strong influence on the transepithelial depolarization induced by inhibition of the apical Na(+) conductance, (2) under voltage-clamp conditions, the decrease of short-circuit current induced by inhibition of the apical Na(+) conductance is also significantly decreased by the presence of a large apical membrane Cl(-) conductance. Our results show that when ENaC and CFTR are expressed in the same apical membrane, the electrical coupling of these two ion channels can explain at least part of the effect of CFTR activation on the Na(+) current flowing through ENaC.

Amiloride↗

PubMed1997

Low-amplitude oscillations in the inferior olive: a model based on electrical coupling of neurons with heterogeneous channel densities.

The mechanism underlying subthreshold oscillations in inferior olivary cells is not known. To study this question, we developed a single-compartment, two-variable, Hodgkin-Huxley-like model for inferior olive neurons. The model consists of a leakage current and a low-threshold calcium current, whose kinetics were experimentally measured in slices. Depending on the maximal calcium and leak conductances, we found that a neuron model's response to current injection could be of four qualitatively different types: always stable, spontaneously oscillating, oscillating with injection of current, and bistable with injection of current. By the use of phase plane techniques, numerical integration, and bifurcation analysis, we subdivided the two-parameter space of channel densities into four regions corresponding to these behavioral types. We further developed, with the use of such techniques, an empirical rule of thumb that characterizes whether two cells when coupled electrically can generate sustained, synchronized oscillations like those observed in inferior olivary cells in slices, of low amplitude (0.1-10 mV) in the frequency range 4-10 Hz. We found that it is not necessary for either cell to be a spontaneous oscillator to obtain a sustained oscillation. On the other hand, two spontaneous oscillators always form an oscillating network when electrically coupled with any arbitrary coupling conductance. In the case of an oscillating pair of electrically coupled nonidentical cells, the coupling current varies periodically and is nonzero even for very large coupling values. The coupling current acts as an equalizing current to reconcile the differences between the two cells' ionic currents. It transiently depolarizes one cell and/or hyperpolarizes the other cell to obtain the regenerative response(s) required for the synchronized oscillation. We suggest that the subthreshold oscillations observed in the inferior olive can emerge from the electrical coupling between neurons with different channel densities, even if the inferior olive nucleus contains no or just a small proportion of spontaneously oscillating neurons.

Animals↗

PubMed1998

Alcohols potentiate the function of 5-HT3 receptor-channels on NCB-20 neuroblastoma cells by favouring and stabilizing the open channel state.

1. 5-HT3 receptor-mediated ion current was recorded from NCB-20 neuroblastoma cells using the whole-cell patch-clamp technique. Rapid drug superfusion was used to study the mechanism of alcohol potentiation of 5-HT3 receptor function and to analyse effects of alcohols on receptor-channel kinetics in detail. 2. Trichloroethanol (TCEt) increased in a dose-dependent way the initial slope, 20-80% rise time and measured desensitization rate of the current induced by low concentrations (1-2 microM) of 5-HT. Ethanol (EtOH) and butanol (ButOH) had similar effects on the 5-HT3 receptor-induced current. 3. TCEt and ButOH decreased the measured desensitization rate of current induced by 10 microM 5-HT, a maximally effective concentration of agonist. These alcohols also increased the relative amplitude of steady state to peak current induced by 2 or 10 microM 5-HT, indicating a possible decrease in the intrinsic rate of desensitization. 4. TCEt also decreased the deactivation rate of the current activated by 2 microM 5-HT after a short pulse of agonist application. 5. Current sweeps generated by 1 microM 5-HT in the presence or absence of 10 mM TCEt or 100 mM EtOH were well fitted using a modified standard kinetic model derived from the nicotinic acetylcholine receptor. This analysis indicated that potentiation by alcohols could be accounted for by increases in the association rate constant coupled with decreases in the dissociation and desensitization rate constants. 6. This study suggests that alcohols potentiate 5-HT3 receptor-mediated current by both increasing the rate of channel activation and stabilizing the open state by decreasing the rates of channel deactivation and desensitization.

Alcohols↗

PubMed1998

Activation of protein kinase Calpha couples cell volume to membrane Cl- permeability in HTC hepatoma and Mz-ChA-1 cholangiocarcinoma cells.

Physiological increases in liver cell volume lead to an adaptive response that includes opening of membrane Cl- channels, which is critical for volume recovery. The purpose of these studies was to assess the potential role for protein kinase C (PKC) as a signal involved in cell volume homeostasis. Studies were performed in HTC rat hepatoma and Mz-ChA-1 human cholangiocarcinoma cells, which were used as model hepatocytes and cholangiocytes, respectively. In each cell type, cell volume increases were followed by: 1) translocation of PKC from cytosolic to particulate (membrane) fractions; 2) a 10- to 40-fold increase in whole-cell membrane Cl- current density; and 3) partial recovery of cell volume. In HTC cells, the volume-dependent Cl- current response (-46 +/- 5 pA/pF) was inhibited by down-regulation of PKC (100 nmol/L phorbol 12-myristate 13-acetate for 18 hours [PMA]; -1.97 +/- 1.5 pA/pF), chelation of cytosolic Ca2+ (2 mmol/L EGTA; -5.3 +/- 4.0 pA/pF), depletion of cytosolic adenosine triphosphate (ATP) (3 U/mL apyrase; -12.58 +/- 1. 45 pA/pF), and by the putative PKC inhibitor, chelerythrine (25 micromol/L; -7 +/- 3 pA/pF). In addition, PKC inhibition by chelerythrine and calphostin C (500 nmol/L) prevented cell volume recovery from swelling. Similar results were obtained in Mz-ChA-1 biliary cells. These findings indicate that swelling-induced activation of PKC represents an important signal coupling cell volume to membrane Cl- permeability in both hepatic and biliary cell models.

Alkaloids↗

PubMed1992

Analysis of drug action at single-channel level.

Many drugs interact directly with ion channel proteins to alter gating and permeation functions. Single-channel recording affords resolution of drug-induced functional changes in channel behavior at the molecular level. Drug and toxin molecules that block ion channels are useful probes of channel mechanisms because blocking sites are often coupled to other pharmacologically relevant binding sites. Simple kinetic schemes describing fast block, slow block, and binding competition between two blocking molecules provide useful models of drug-induced blocking processes. From a careful perspective, a single channel is best approached as the analog of a purified enzyme preparation in the hands of an enzymologist. The confidence gained by knowing that one is viewing a single subtype must be weighed against the possibility that the channel could have been altered in the process of patch isolation or bilayer reconstitution. As in all kinetic studies, a curve fit to a two-state scheme is contingent on the possibility that a more complex multi-state system can masquerade as the simple cartoon one would like to put forward.

Binding, Competitive↗