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PubMed2000

Differential ion accumulation and ion fluxes in the mesophyll and epidermis of barley.

In barley (Hordeum vulgare L.) leaves, differential ion accumulation commonly results in inorganic phosphate (Pi) being confined to the mesophyll and Ca(2+) to the epidermis, with preferential epidermal accumulation of Cl(-), Na(+), and some other ions. The pattern was confirmed in this study for major inorganic anions and cations by analysis of barley leaf protoplasts. The work focused on the extent to which differences in plasma membrane ion transport processes underlie these observations. Ion transport across the plasma membrane of barley epidermal and mesophyll protoplasts was investigated electrophysiologically (by microelectrode impalement and patch clamping) and radiometrically. Data from both approaches suggested that similar types of ion-selective channels and membrane transporters, which catalyze the transport of Ca(2+), K(+), Na(+), and Pi, exist in the plasma membrane of the two cell types. In general, the simple presence or absence of ion transporters could not explain cell-type-specific differences in ion accumulation. However, patch-clamp data suggested that differential regulation of instantaneously activating ion channels in the plasma membrane could explain the preferential accumulation of Na(+) in the epidermis.

Calcium↗

PubMed2001

The outwardly rectifying chloride channel in rat peritoneal mast cells is regulated by serine/threonine kinases and phosphatases.

A slowly activating, outwardly rectifying Cl- channel (ORCC) has been described in rat peritoneal mast cells (RPMCs). This channel is activated by intracellular application of cAMP, an effect that might be mediated by a PKA-type serine/threonine protein kinase. To test this hypothesis, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed and 8-bromoadenosine 3',5'-cyclic monophosphothioate, Sp-enantiomer (Sp-8Br-cAMPS), a cell membrane-permeable activator of PKA, and three inhibitors of different serine/threonine protein phosphatases (okadaic acid, cantharidin, calyculin A), were tested. In RPMCs application of repetitive series of step hyper- and depolarizations (holding potential 0 mV, test potentials -80 to +80 mV, step size +20 mV) induced a slowly increasing, [half-maximal activation time ( t0.5) 11.0+/-1.1 min, Imax (at +80 mV) 18.7+/-3.1 pA pF-1], DIDS-sensitive, outwardly rectifying Cl- current I(Cl,OR). The activation of this current could be accelerated by Sp-8Br-cAMPS, okadaic acid or cantharidin in the extracellular solution. Co-application of Sp-8Br-cAMPS and okadaic acid increased Imax supra-additively. Calyculin A and higher concentrations of cantharidin inhibited the Cl- current via unknown mechanisms. Our findings suggest that I(Cl,OR) in RPMCs is activated by a PKA-type protein kinase, a process which is antagonized functionally by okadaic acid- and cantharidin-sensitive protein phosphatases.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta.

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.

Adenosine Triphosphate↗

PubMed2001

Evidence against the functional involvement of outwardly rectifying Cl- channels in agonist-induced mast cell exocytosis.

In isolated rat peritoneal mast cells, an outwardly rectifying Cl- channel has been described. Influx of Cl- through this Cl- channel (I(Cl-(OR)) causes hyperpolarization, which facilitates Ca2+ currents through store-operated Ca2+ channels. The exocytotic effect of nerve growth factor (NGF) in the presence of lyso-phosphatidylserine strictly depends on the presence of extracellular [Ca2+]o. The aim of the present study was to assess the importance of I(Cl-(OR)) for exocytosis induced by NGF/lyso-phosphatidylserine. Therefore, we investigated the effects on NGF/lyso-phosphatidylserine-induced exocytosis of [3H]5-hydroxytryptamine ([3H]5-HT) in rat peritoneal mast cells: (a) of two inhibitors of I(Cl-(OR)) (4,4'-diisothiocyanatostilbene2,2'-disulfonic acid [DIDS] and diethylstilbestrol), and (b) of replacement of extracellular Cl- by methylsulfate. Additionally, whole-cell patch-clamp experiments (nystatin-perforated patch) were performed. Diethylstibestrol and DIDS, in concentrations sufficient to abolish the I(Cl-(OR)) (10 microM) and the replacement of (Cl-)o by methylsulfate, were ineffective in impairing the NGF/lyso-phosphatidylserine-induced [3H]5-HT-release. These findings argue against a role of outwardly rectifying Cl- channels in exocytosis induced by NGF/lyso-phosphatidylserine in rat peritoneal mast cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Warming-activated channels of warm-sensitive neurons in rat hypothalamic slices.

In mammals, multiple thermostats in several brain areas, e.g. the preoptic area and anterior hypothalamus (POAH), may regulate core temperature. In these areas, warm-sensitive (WS) and cold-sensitive (CS) neurons have been recorded. In our previous study, we proposed that primary WS and CS neurons act as multiple thermostats having threshold temperatures as target temperatures of regulation. Here we investigated ionic basis of WS neurons in rat POAH slices with patch-clamp technique. In whole-cell current-clamp recordings, warming above threshold temperatures induced depolarization (receptor potential), leading to spike generation. Whole-cell voltage-clamp and cell-attached patch recordings indicated that warming-activated non-selective cation channels underlie the receptor potential and spike generation. We conclude that the warming-activated channel in primary WS neurons is a key factor of thermostats.

Animals↗

PubMed1999

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.

Aldosterone↗

PubMed1998

Activation of muscarinic K+ channels by extracellular ATP and UTP in rat atrial myocytes.

The effects of extracellular adenine and pyrimidine nucleotides on the acetylcholine-activated K+ channels (KACh) in rat cardiac myocytes were compared and examined by using the patch-clamp technique. In perforated-patch whole-cell recording experiments, extracellular adenosine triphosphate (ATP) reversibly caused an increase in K+ current. 8-Cyclopentyl-1,3-dipropylxanthine (CPX; 1 microM), a potent A1-adenosine-receptor antagonist, only partially antagonized the ATP-induced increase in K+ current, whereas glibenclamide (30 microM) had no effect. In cell-attached mode, adenosine and ATP activated single channels that had nearly identical conductance (29 pS) and open time (1.53 ms). These results suggest that adenosine and ATP can activate the same population of K+ channels. Uridine triphosphate (UTP; 100 microM) also caused an increase in steady-state K+ current. In cell-attached mode, the addition of UTP to the recording pipette solution (not in the bath solution) activated the channel current. The single-channel conductance and open time for UTP-induced channel current were 27 pS and 1.57 ms, respectively. These values were similar to those for the K+ channels activated by adenosine or ATP. The rank order of potency for the activation of KACh channels was adenosine = ATP > UTP. The addition of CPX (1 microM) to the pipette solution attenuated the ATP-induced channel activity by approximately 70% and fully prevented activation by AMPCPP, a less hydrolyzable ATP analog but did not cause any effect on UTP-induced channel activity. In pertussis toxin-treated cardiac myocytes, no any activity of UTP-induced KACh-channel current was observed. Our results demonstrate that extracellular ATP and UTP can directly activate KACh-channel current. This activation also was linked to pertussis toxin-sensitive G protein. The effect of extracellular ATP is mainly caused by the action on binding to A1-adenosine receptor, whereas the effect of extracellular UTP may be mediated possibly by P2u-purinergic (or 5'-nucleotide) receptor.

Acetylcholine↗

PubMed1996

Open channel block by physostigmine and procaine in embryonic-like nicotinic receptors of mouse muscle.

Embryonic-like nicotinic channels were studied in mouse myotubes. Channel currents were measured by patch-clamping outside-out excised patches to which pulses of agonists and drugs could be applied by a liquid filament switch. The holding potential of the patches was generally around 40 mV. Pulses of 10(-4) M acetylcholine elicited average channel currents which reached a peak open probability, P(o,peak,) of 0.93 within 0.5 ms and decayed with a time constant of desensitization of 20-80 ms. When physostigmine (10(-5) to 10(-3) M) or procaine (3 x 10(-5) to 10(-3) M) was added to the acetylcholine pulses, a fast decay component of the current appeared which shortened to a time constant of 0.5 ms for the maximal drug concentrations. The fast decay was followed by a slow one which declined in amplitude with increasing concentrations of the drugs. After the end of pulses of 10 M acetylcholine plus 3 x 10(-4) M physostigmine the average current rose again, reaching a peak with approximately 5 ms delay, and then decayed slowly. The amplitude of this recovery current was approximately 0.4 P(o,peak) after 5 ms pulses and decreased with increasing pulse duration due to desensitization. The results can be quantitatively modelled based on a circular reaction scheme involving desensitization. Physostigmine and procaine bind to the open state to cause channel block. Also, the blocked channel was subject to desensitization. The rate constants of block were 6 x 10(6) M(-1) s(-1) for physostigmine and 2 x 10(6) M(-1) s(-1) for procaine, and the rate of unblocking was 200 s(-1) for both blockers (at -40 mV and 20 degrees C).

Animals↗

PubMed1996

Open channel and competitive block of the embryonic form of the nicotinic receptor of mouse myotubes by (+)-tubocurarine.

1. Embryonic-like nicotinic channels were studied in mouse myotubes. Channel currents were measured by patch clamping outside-out excised patches to which pulses of agonists and drugs could be applied by a liquid filament switch. The holding potential of the patches was generally around-10 to-40 mV. 2. Pulses of 100 microM or 1 mM acetylcholine (ACh) elicited average channel currents which reached a maximum open probability of 0.93 within 0.5-1.0 ms, decayed with a time constant of desensitization of 20-80 ms, and fell rapidly to zero at the end of the pulse. When such pulses together with increasing concentrations of (+)-tubocurarine (TC) were applied to outside-out patches, the time constant of current decay, tau, decreased beginning at concentrations of TC added to the test solution of > 10 microM, and the peak amplitude of the current decreased markedly at concentrations of TC of > 30 microM due to an open channel block of nicotinic channels by TC. 3. When the outside-out patches were pre-incubated with TC, the peak current elicited by pulses of 100 microM ACh or 1 mM ACh + TC decreased markedly, beginning with concentrations of TC > 30 nM due to a competitive block. 4. The results could be quantitatively modelled by computer calculations based on a circular reaction scheme containing desensitization. TC blocked the open state as well as the unliganded closed state of the embryonic-like nicotinic receptors of mouse myotubes. Also the blocked open channel was subject to desensitization. 5. The rates of block and unblock of the open channel were 3 x 10(6) M-1 S-1 and 0.8 S-1, respectively, and those of the competitive block were 0.5 x 10(6) M-1 S-1 and 0.1 S-1, respectively (at 20 degrees C).

Acetylcholine↗

PubMed1996

Distinct functions of T- and L-type calcium channels during activation of bovine adrenal glomerulosa cells.

Calcium influx into adrenal glomerulosa cells is a key event during the stimulation of aldosterone secretion by physiological increases in extracellular potassium concentrations. Two types of voltage-operated calcium channels, T- and L-types, are present on bovine glomerulosa cells, but their respective functions are not yet clearly defined. Using the patch-clamp method in the perforated patch configuration combined with microfluorimetry of cytosolic calcium, we demonstrate that L-type channels are exclusively responsible for the sustained elevation of cytosolic calcium observed upon stimulation with extracellular potassium, even at low, physiological concentrations of this agonist. In contrast, aldosterone secretion appears closely related to T-type channel activity. Moreover, when the activity of each channel type is selectively modulated by pharmacological agents, such as dihydropyridines or zonisamide, the cytosolic calcium response can be clearly dissociated from the steroidogenic response. Similarly, modulation of T channel activation by protein kinase C results in a parallel inhibition of aldosterone secretion, without any effect on the levels of cytosolic free calcium. This direct functional link between T-type calcium channel activity and steroidogenesis suggests a model in which calcium entering the cell through these channels bypasses the cytosol to activate intramitochondrial steps of aldosterone biosynthesis.

Aldosterone↗

PubMed2000

cADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells.

The effects of cADP-ribose (cADPR), a metabolite of beta-NAD(+), on the elevation of cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) influx through voltage-activated Ca(2+) channels (VACCs) were studied in NG108-15 neuroblastomaxglioma hybrid cells. NG108-15 cells were pre-loaded with fura-2 and whole-cell patch-clamped. Application of cADPR through patch pipettes did not by itself trigger any [Ca(2+)](i) rise at the resting membrane potential. A rise in [Ca(2+)](i) was evoked upon sustained membrane depolarization, and was significantly larger in cADPR-infused cells than in non-infused cells. This potentiation in the [Ca(2+)](i) elevation was reproduced by infusion of beta-NAD(+), and was blocked by 8-bromo-cADPR and antagonized by external application of ryanodine or by pretreatment of cells with FK506. Nicotinamide inhibited beta-NAD(+)-induced, but not cADPR-elicited, potentiation. [Ca(2+)](i) increases or Ca(2+) influx, measured by Mn(2+) quenching, elicited by the same protocol of depolarization was blocked completely by nifedipine but not by omega-conotoxin. Ca(2+) influx in cADPR- or beta-NAD(+)-infused cells was steeper and greater than that in control cells, and was inhibited partly by ryanodine. In contrast, ryanodine accelerated Ca(2+) influx in non-infused cells. These results show that cADPR amplifies both depolarization-induced [Ca(2+)](i) increase and Ca(2+) influx through L-type VACCs. These results suggest that cADPR functions on ryanodine receptors as a direct agonist and also interacts with L-type VACCs as an indirect agonist, i.e. via a retrograde signal.

Adenosine Diphosphate Ribose↗

PubMed2006

[Influence of lipopolysaccharide and burn sera on ion channels in smooth muscle cells of colon of guinea pig].

OBJECTIVE: To investigate the influence of lipopolysaccharide (LPS) and burn sera on potassium channels (Kca) in smooth muscle cells of colon of guinea pig so as to elucidate the molecular mechanism of gastrointestinal motility dysfunction after severe burns. METHODS: Single smooth muscle cells were isolated from the taenia coli of guinea pig with enzyme digestion. The standard patch clamp technique was employed to record the single KCa channel currents of smooth muscle cell after challenged by LPS and burn serum. Data were recorded and analyzed by P clamp 6.04 software, and the probability of open (PO), mean open time (OT), mean close time (CT) and current amplitude (CA) were determined. Subsequently, LPS in the concentration of 20, 40, 60, 80 and 100 mg/L, respectively, and normal serum and burn serum in the concentration of 10% were respectively added into medium to examine the influence of the two clamps and sera on the KCa activity. RESULTS: In hyperkalemic solution, the KCa conductance of colonic smooth muscle cells of the guinea pig was (271 +/- 7) pS, indicating it was an ionic channel with high conductivity. Subsequent to depolarization of the membrane, inner-cellular calcium level was increased, and channel PO was also increased, which could be blocked by 1 mmol/L tetraethylammonium (TEA) outside the membrane chaff. As 40 mmol/L TEA inside the membrane chaff did not show such effect, it was proved to be KCa current. The activity of the channel as determined with two kinds of clamps was increased in a dose dependent manner with LPS challenge when the concentration of calcium was 0 mol/L. The KCa activity and PO of the channel was increased obviously when the concentration of LPS was above 40 mg/L (P < 0.05 or 0.01), and it could not be reversed after irrigation with non-LPS medium. By using the two kinds of patch clamps, the KCa were activated by burn sera, but not normal sera. CONCLUSION: Both LPS and burn sera can lead to inhibition of the gastrointestinal motility by activation of KCa channels.

Animals↗

PubMed2004

Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells.

The effect of glutamate on plant plasma membrane cation transport was studied in roots of Arabidopsis thaliana (L.) Heynh. Patch-clamp experiments using root protoplasts, (22)Na(+) unidirectional fluxes into intact roots and measurements of cytosolic Ca(2+) activity using plants expressing cytosolically-targeted aequorin in specific cell types were carried out. It was demonstrated that low-millimolar concentrations of glutamate activate within seconds both Na(+) and Ca(2+) currents in patch-clamped protoplasts derived from roots. The probability of observing glutamate-activated currents increased with increasing glutamate concentration (up to 29% at 3 mM); half-maximal activation was seen at 0.2-0.5 mM glutamate. Glutamate-activated currents were voltage-insensitive, 'instantaneous' (completely activated within 2-3 ms of a change in voltage) and non-selective for monovalent cations (Na(+), Cs(+) and K(+)). They also allowed the permeation of Ca(2+). Half-maximal Na(+) currents occurred at 20-30 mM Na(+). Glutamate-activated currents were sensitive to non-specific blockers of cation channels (quinine, La(3+), Gd(3+)). Although low-millimolar concentrations of glutamate did not usually stimulate unidirectional influx of (22)Na(+) into intact roots, they reliably caused an increase in cytosolic Ca(2+) activity in protoplasts isolated from the roots of aequorin-transformed Arabidopsis plants. The response of cytosolic Ca(2+) activity revealed a two-phase development, with a rapid large transient increase (lasting minutes) and a prolonged subsequent stage (lasting hours). Use of plants expressing aequorin in specific cell types within the root suggested that the cell types most sensitive to glutamate were in the mature epidermis and cortex. The functional significance of these glutamate-activated currents for both cation uptake into plants and cell signaling remains the subject of speculation, requiring more knowledge about the dynamics of apoplastic glutamate in plants.

Aequorin↗

PubMed2003

Voltage-gated K+ channels in rat small cerebral arteries: molecular identity of the functional channels.

Voltage-gated potassium (KV) channels represent an important dilator influence in the cerebral circulation, but the composition of these tetrameric ion channels remains unclear. The goals of the present study were to evaluate the contribution of KV1 family channels to the resting membrane potential and diameter of small rat cerebral arteries, and to identify the alpha-subunit composition of these channels using patch-clamp, molecular and immunological techniques. Initial studies indicated that 1 micromol l(-1) correolide (COR), a specific antagonist of KV1 channels, depolarized vascular smooth muscle cells (VSMCs) in pressurized (60 mmHg) cerebral arteries from -55 +/- 1 mV to -34 +/- 1 mV, and reduced the resting diameter from 152 +/- 15 microm to 103 +/- 20 microm. In patch clamped VSMCs from these arteries, COR-sensitive KV1 current accounted for 65 % of total outward KV current and was observed at physiological membrane potentials. RT-PCR identified mRNA encoding each of the six classical KV1 alpha-subunits, KV1.1-1.6, in rat cerebral arteries. However, only the KV1.2 and 1.5 proteins were detected by Western blot. The expression of these proteins in VSMCs was confirmed by immunocytochemistry and co-immunoprecipitation of KV1.2 and 1.5 from VSMC membranes suggested KV1.2/1.5 channel assembly. Subsequently, the pharmacological and voltage-sensitive properties of KV1 current in VSMCs were found to be consistent with a predominant expression of KV1.2/1.5 heterotetrameric channels. The findings of this study suggest that KV1.2/1.5 heterotetramers are preferentially expressed in rat cerebral VSMCs, and that these channels contribute to the resting membrane potential and diameter of rat small cerebral arteries.

Animals↗

PubMed1997

Inhibitory effect of baclofen on GABA-induced depolarization and GABA-activated current in primary sensory neurons.

It has been established that GABAA and GABAB receptors can exist separately and/or co-exist in the membrane of dorsal root ganglion neurons. In our previous investigation it has been shown that co-existence of these two kinds of receptors is about 80% of the neurons examined (20/25). The present study was aimed to explore whether the activation of these two kinds of receptors could interact with each other using intracellular and whole-cell patch-clamp recordings. Baclofen, a specific GABAB receptor agonist, was found to exert negative modulatory effects on the responses mediated by GABAA receptor. In experiments with intracellular recording, GABA (0.3-1000 microM)- and muscimol (100-1000 microM)-induced depolarization was attenuated markedly and reversibly by preapplication of baclofen (100 microM) (15/21 and 17/21, respectively). In whole-cell patch-clamp recordings GABA (100 microM) and two specific GABAA receptor agonists, muscimol (10 microM) and isoguvacine (50 microM), activated currents were inhibited markedly by preapplication of baclofen 30 s or more and the inhibition was concentration dependent (1-100 microM baclofen) and reversible. The possible mechanisms underlying the inhibition by baclofen of the responses mediated by GABAA receptor and the physiological significance implicated are discussed.

Animals↗

PubMed1998

Protein kinase C phosphorylation disengages human and mouse-1a P-glycoproteins from influencing the rate of activation of swelling-activated chloride currents.

1. Whole-cell, swelling-activated Cl- currents, ICl(swell), were characterized in Chinese hamster ovary (CHO) cells and found to exhibit time-dependent inactivation at depolarizing potentials, tamoxifen and dideoxyforskolin sensitivity, and an anion permeability sequence: SCN- > I- > Br- > Cl- > F- > gluconate-. 2. CHO cells permanently transfected with either the human MDR1 or mouse mdr1a cDNAs demonstrated an increased rate of activation of ICl(swell) compared with parental cells or those permanently transfected with the mouse mdr1b cDNA. However, no differences in the magnitude of the currents were observed at steady state. 3. Pretreatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) did not affect ICl(swell) in MDR1 or mdr1a permanently transfected CHO cells. In contrast, pretreatment with TPA reduced ICl(swell) in MDR1(G185V)-expressing transfected NIH3T3 fibroblasts. Subsequently, the CHO cell lines were shown to contain significantly reduced levels of protein kinase C (PKC), suggesting that PKC concentrations might be limiting in these cell lines, at least under whole-cell patch clamp conditions. 4. Addition of purified PKC to the pipette solution, followed by a pretreatment with TPA, reduced the rate of ICl(swell) activation in human Pgp- and mouse Pgp1a-expressing CHO cells to the levels observed in parental and mouse Pgp1b-expressing cells. This confirms that PKC is limiting in these cells under whole-cell, patch clamp conditions. Furthermore, these results suggest that PKC-mediated phosphorylation of human Pgp and mouse Pgp1a disengages the influence which these Pgps have on ICl(swell). 5. These studies also demonstrate a functional distinction between the two mouse homologues, Pgp1a and Pgp1b. Although both can function as drug efflux pumps, only Pgp1a can act like human Pgp to influence ICl(swell).

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Modulation of neuronal excitability by intracellular calcium buffering: from spiking to bursting.

We have investigated the detailed regulation of neuronal firing pattern by the cytosolic calcium buffering capacity using a combination of mathematical modeling and patch-clamp recording in acute slice. Theoretical results show that a high calcium buffer concentration alters the characteristic regular firing of cerebellar granule cells and that a transition to various modes of oscillations occurs, including bursting. Using bifurcation analysis, we show that this transition from spiking to bursting is a consequence of the major slowdown of calcium dynamics. Patch-clamp recordings on cerebellar granule cells loaded with a high concentration of the fast calcium buffer BAPTA (15 mM) reveal dramatic alterations in their excitability as compared to cells loaded with 0.15 mM BAPTA. In high calcium buffering conditions, granule cells exhibit all bursting behaviors predicted by the model whereas bursting is never observed in low buffering. These results suggest that cytosolic calcium buffering capacity can tightly modulate neuronal firing patterns leading to generation of complex patterns and therefore that calcium-binding proteins may play a critical role in the non-synaptic plasticity and information processing in the central nervous system.

Animals↗

PubMed2001

Multiple inhibitory effects of zatebradine (UL-FS 49) on the electrophysiological properties of retinal rod photoreceptors.

Effects of the bradycardiac agent zatebradine (UL-FS 49) were determined in intracellular and patch-clamp experiments of amphibian rod photoreceptors. Zatebradine (0.3-100 microM) greatly enhanced the bright-light-induced hyperpolarization of membrane potential in frog retinal rods and caused damped oscillations during the membrane potential recovery phase. In inner segments of the rod photoreceptor cells, the hyperpolarization-activated inward current ( Ih) contributes to the recovery of the photoresponse. Patch-clamp recordings from newt rod photoreceptor cells revealed that zatebradine caused use- and concentration-dependent (0.1-100 microM) inhibition of Ih: conductance was reduced without effects on the reversal potential or activation voltage. Our data confirmed that the pharmacological properties of Ih in rod photoreceptors were similar to those of Ih in cardiac myocytes. In addition, zatebradine inhibited voltage-gated outward K+ currents ( IK), but did not affect L-type Ca2+ currents ( ICa). These results are consistent with the inhibition of IK and Ih by zatebradine in other cells, and may explain the oscillations evoked during the recovery phase of the membrane potential. These multiple actions of zatebradine on channels in rod photoreceptors may explain its effects on the electroretinogram (ERG) in vivo and its adverse effects on vision in clinical studies.

Animals↗