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PubMed2004

pCLCA1 lacks inherent chloride channel activity in an epithelial colon carcinoma cell line.

The effects of CLCA protein expression on the regulation of Cl(-) conductance by intracellular Ca(2+) and cAMP have been studied previously in nonepithelial cell lines chosen for low backgrounds of endogenous Cl(-) conductance. However, CLCA proteins have been cloned from, and normally function in, differentiated epithelial cells. In this study, we examine the effects of differentiation of the Caco-2 epithelial colon carcinoma cell line on modulation of Cl(-) conductance by pCLCA1 protein expression. Cl(-) transport was measured as (36)Cl(-) efflux, as transepithelial short-circuit currents, and as whole cell patch-clamp current-voltage relations. The rate of (36)Cl(-) efflux and amplitude of currents in patch-clamp studies after the addition of the Ca(2+) ionophore A-23187 were increased significantly by pCLCA1 expression in freshly passaged Caco-2 cells. However, neither endogenous nor pCLCA1-dependent Ca(2+)-sensitive Cl(-) conductance could be detected in 14-day-postpassage cells. In contrast to Ca(2+)-sensitive Cl(-) conductance, endogenous cAMP-dependent Cl(-) conductance does not disappear on Caco-2 differentiation. cAMP-dependent Cl(-) conductance was modulated by pCLCA1 expression in Caco-2 cells, and this modulation was observed in freshly passaged and in mature 14-day-postpassage Caco-2 cultures. pCLCA1 mRNA expression, antigenic pCLCA1 protein epitope expression, and pCLCA1 function as a modulator of cAMP-dependent Cl(-) conductance were retained through differentiation in Caco-2 cells, whereas Ca(2+)-dependent Cl(-) conductance disappeared. We conclude that pCLCA1 expression may increase the sensitivity of preexisting endogenous Cl(-) channels to Ca(2+) and cAMP agonists but apparently lacks inherent Cl(-) channel activity under growth conditions where endogenous channels are not expressed.

Animals↗

PubMed2006

Characterizing the efficacy of calcium channel agonist-release strategies for bone tissue engineering applications.

We have previously reported on the use of Bay K8644-release strategies in combination with perfusion-compression bioreactor systems for up regulating bone formation in three-dimensional PLLA scaffolds. Here we report on the analysis of Bay activity following its release from our PLLA scaffolds over the culture period imposed in our tissue engineering protocol using UV spectroscopy in combination with whole cell patch clamping techniques. Bay was released continually from scaffolds within the physiological range required for agonist activity (1-10 microM). Patch clamping allowed for the effects of Bay released from scaffolds to be monitored directly with respect to osteoblast electrophysiology. A characteristic shift in the current-voltage (I-V) relationship of L-type VOCC currents was observed in rat osteoblast sarcoma (ROS) cells patched in a solution with Bay released from scaffolds following 14 and 28 days incubation, with statistically significant differences observed in peak currents compared to non-Bay controls. An increase in the magnitude of the peak inward currents was also noted. The electrophysiological response of osteoblasts in the presence of Bay released from scaffolds demonstrates that the released Bay is stable and maintains its bioactivity following culture of up to 28 days.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1995

Amiloride-sensitive sodium channels in confluent M-1 mouse cortical collecting duct cells.

Confluent M-1 cells show electrogenic Na+ absorption and possess an amiloride-sensitive Na(+)-conductance (Korbmacher et al., J. Gen. Physiol. 102:761-793, 1993). In the present study, we further characterized this conductance and identified the underlying single channels using conventional patch clamp technique. Moreover, we isolated poly(A)+ RNA from M-1 cells to express the channels in Xenopus laevis oocytes, and to check for the presence of transcripts related to the epithelial Na+ channel recently cloned from rat colon (Canessa et al., Nature 361:467-470, 1993). Patch clamp experiments were performed in 6-13-day-old confluent M-1 cells at 37 degrees C. In whole-cell experiments application of 10(-5) M amiloride caused a hyperpolarization of 24.9, SEM +/- 2.2 mV (n = 35) and a reduction of the inward current by 107 +/- 10 pA (n = 51) at a holding potential of -60 mV. Complete removal of bath Na+ had similar effects, indicating that the amiloride-sensitive component of the inward current is a Na+ current. The effect of amiloride was concentration-dependent with half-inhibition at 0.22 microM. The Na+ current saturated with increasing extracellular Na+ concentrations with an apparent Km of 24 mM. Na+ replacement for Li+ demonstrated a higher apical membrane conductance for Li+ than for Na+. In excised inside-out (i/o) or outside-out (o/o) patches from the apical membrane, we observed single-channels which showed slow kinetics and were reversibly inhibited by amiloride. Their average conductance for Na+ was 6.8 +/- 0.5 pS (n = 15) and for Li+ 11.2 +/- 1.0 pS (n = 14). They had no measurable conductance for K+. In o/o patches, channel activity was slightly voltage dependent with an open probability (NPo) of 0.46 +/- 0.14 and 0.16 +/- 0.05 at a holding potential of -100 and 0 mV, respectively (n = 8, P < 0.05). Using the two-microelectrode voltage-clamp technique, we assayed defolliculated stage V-VI Xenopus oocytes for an amiloride-sensitive inward current 1-6 days after injection with H2O or with 20-50 ng of M-1 poly(A)+ RNA. In poly(A)+ RNA-injected oocytes held at -60 or -100 mV application of amiloride (2 microM) reduced the Na-inward current by 25.5 +/- 4.6 nA (n = 25) while it had no effect in H2O-injected oocytes (n = 19). Northern blot analysis of M-1 poly(A+) RNA revealed the presence of transcripts related to the three known subunits of the rat colon Na+ channel (Canessa et al., Nature 367:463-467, 1994). We conclude that the channel in M-1 cells is closely related to the amiloride-sensitive epithelial Na+ channel in the rat colon and that the M-1 cell line provides a useful tool to investigate the biophysical and molecular properties of the corresponding channel in the cortical collecting duct.

Amiloride↗

PubMed1995

Whole-cell and perforated patch recordings of four distinct K+ currents in acutely dispersed coeliac-superior mesenteric ganglia neurons of adult rats.

Properties and modulation of outward membrane currents in sympathetic neurons acutely dispersed from coeliac-superior mesenteric ganglia (C-SMG) of adult rats were examined using both the whole-cell variant of the patch-clamp technique and the perforated patch approach. Under voltage-clamp, four distinct outward currents were observed: a transient outward current (IA), a voltage-dependent sustained outward current consisting of a Ca(2+)-dependent component (IKCa) and a Ca(2+)-insensitive component (IKV), and a muscarinic agonist-sensitive outward current (IM). IA was isolated by digital subtraction, and characterized by very rapid activation at potentials more positive than -60 mV and by fast and complete voltage-dependent inactivation. Half inactivation potential (Vh) and slope factor (K) were -76 mV and 8.3 mV, respectively. IA was not affected by removal of external Ca2+, 1 mM tetraethylammonium ions, muscarinic agonists, or 8-bromo-cyclic AMP, but was suppressed by 4-aminopyridine (1 mM). Depolarizing pulses from of a holding potential of -50 or -60 mV to potentials more positive than -25 mV concomitantly activated two, independent sustained outward currents which decayed slowly; one exhibited voltage-dependent activation similar to the delayed rectifier current (IKV) and the other being triggered by Ca2+ influx into the cell (IKCa). The addition of tetraethylammonium ions (1 mM) strongly reduced the amplitude of the sustained outward currents. IM was characterized as a noninactivating time- and voltage-dependent outward current which activated at membrane potentials more positive than -60 mV and slowly turned off when the membrane was hyperpolarized back to -60 mV, and was suppressed by muscarinic agonists. The rank order of potency of the agonists tested was: oxotremorine > muscarine > bethanechol.

4-Aminopyridine↗

PubMed1990

Outward sodium current in beating heart cells.

This article is a study of the fast Na current during action potentials. We have investigated the outward Na current (Mazzanti, M., and L.J. DeFelice. 1987. Biophys. J. 52:95-100) in more detail, and we have asked whether it goes through the same channels associated with the rapid depolarization phase of action potentials. We address the question by patch clamping single, spontaneously beating, embryonic chick ventricle cells, using two electrodes to record the action potential and the patch current simultaneously. The chief limitation is the capacitive current, and in this article we describe a new method to subtract it. Varying the potential and the Na concentration in the patch pipette, and fitting the corrected currents to a standard model (Ebihara, L., and E.A. Johnson. 1980. Biophys. J. 32:779-790), provides evidence that the outward current is carried by the same channels that conduct the inward current. We compare the currents in beating cells to currents in nonbeating cells using whole-cell and cell-attached patch clamp recordings. The latter tend to show more positive Na reversal potentials, with the implication that internal Na is higher in beating cells. We propose that the plateau of the action potential, which is partly due to an inward Ca current, exceeds Na action current reversal potentials, and that this driving force gives rise to an outward movement of Na ions. The existence of such a current would imply that the fast repolarization phase after the upstroke of cardiac action potentials is partly due to the Na action current.

Action Potentials↗

PubMed1990

PT pretreatment inhibits 48/80-induced activation of Ca(+)-permeable channels in rat peritoneal mast cells.

We recently reported that the secretagogue, compound 48/80, activated Ca2(+)-permeable channels of mast cells possibly via a second messenger [Kuno, Okada, and Shibata. Am. J. Physiol. 256 (Cell Physiol. 25): C560-C568, 1989]. The effects of pretreatment with pertussis toxin (PT) on compound 48/80 (48/80)-induced activation of the Ca2(+)-permeable channel have now been investigated by measuring Ca2+ signals of cell suspensions using the Ca2+ indicator fura-2 and by recording Ba2+ currents through the channel using the patch-clamp technique. In the presence of extracellular Ca2+, the fluorescence change was biphasic, with an immediate rise and a delayed peak at room temperature. The delayed peak, mainly due to Ca2+ entry through plasma membranes, was greatly reduced by pretreatment with PT. The quenching of the fluorescence by 48/80-induced Mn2+ influx was also decreased by PT, whereas the Ca2+ transients due to Ca2+ release from the intracellular stores apparently did not change. In patch-clamp recordings from cell-attached patches with pipettes containing isotonic Ba2+, the 48/80-induced Ba2+ currents were either suppressed or delayed in the PT-treated cells, under conditions where degranulation was absent. These results suggest that PT-sensitive GTP-binding protein is involved in activating the Ca2(+)-permeable channel in mast cells during stimulus-secretion coupling.

Animals↗

PubMed1987

Voltage-dependent conductances of solitary ganglion cells dissociated from the rat retina.

1. Ganglion cells were dissociated from the enzyme-treated rat retina, identified with specific fluorescent labels, and maintained in vitro. Electrophysiological properties of solitary retinal ganglion cells were investigated with both conventional intracellular and patch-clamp recordings. Although comparable results were obtained for most measurements some important differences were noted. 2. The input resistance of solitary retinal ganglion cells was considerably higher when measured with 'giga-seal' suction pipettes than with conventional intracellular electrodes. Under current-clamp conditions with both intracellular and patch pipettes, these central mammalian neurones maintained resting potentials of about -60 mV and displayed action potentials followed by an after-hyperpolarization in response to small depolarizations. The membrane currents during this activity, analysed under voltage clamp with patch pipettes, consisted of five components: Na+ current (INa), Ca2+ current (ICa), and currents with properties similar to the delayed outward, the transient (A-type), and the Ca2+-activated K+ currents (IK, IA and IK(Ca), respectively). 3. Ionic substitution, pharmacological agents, and voltage-clamp experiments revealed that the regenerative currents were carried by both Na+ and Ca2+. 100 nM-1 microM-tetradotoxin (TTX) reversibly blocked the fast spikes carried by the presumptive INa, which under voltage-clamp analysis had classical Hodgkin-Huxley-type activation and inactivation. 4. Single-channel recordings of the Na+ current (iNa) permitted comparison of these 'microscopic' events with the 'macroscopic' whole-cell current (INa). The inactivation time constant (tau h) fitted to the averaged single-channel recordings of iNa in outside-out patches was slower than the tau h obtained during whole-cell recordings of INa. 5. In the presence of 1-40 microM-TTX and 20 mM-TEA, slow action potentials appeared in intracellular recordings and were probably mediated by Ca2+. The potentials were abrogated by 3 mM-Co2+ or 200 microM-Cd2+; conversely, increasing the extracellular Ca2+ concentration from 2.5 to 10-25 mM or substitution of 1 mM-Ba2+ for 2.5 mM-Ca2+ enhanced their amplitude. ICa was measured directly in whole-cell recordings with patch pipettes after blocking INa with extracellular 1 microM-TTX and K+ currents with intracellular 120-mM Cs+ and 20 mM-TEA. 6. During whole-cell recordings with patch electrodes, extracellular 20 mM-TEA suppressed IK and, to a lesser extent, IA. Extracellular 5 mM-4-AP or a pre-pulse of the membrane potential to -40 mV prior to stronger depolarization completely blocked IA.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1988

Single-channel activity in sarcolemmal vesicles from human and other mammalian muscles.

Segments of mammalian, including human, skeletal muscle 1-2 cm long can be induced to shed vesicles by treatment with collagenase in a high-KCl solution containing no added calcium. The vesicles are encompassed by clean sarcolemma so that the gigaseal necessary for patch-clamping is readily formed. The properties of inwardly rectifying potassium channels and of calcium-activated potassium channels in patches detached from such vesicles are shown to be consistent with expectations based on earlier studies on intact muscle fibers and with patch clamp results on the same type of channels in other tissues. A chloride channel which rectifies outwardly with a conductance ranging from 15 to 50 pS is also described. The utility of sarcolemmal vesicles for the study of ion channels in human biopsy material is discussed.

Adenosine Triphosphatases↗

PubMed1991

Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones.

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2001

Electrical dimension of the nuclear envelope.

Eukaryotic chromosomes are confined to the nucleus, which is separated from the rest of the cell by two concentric membranes known as the nuclear envelope (NE). The NE is punctuated by holes known as nuclear pore complexes (NPCs), which provide the main pathway for transport of cellular material across the nuclear-cytoplasmic boundary. The single NPC is a complicated octameric structure containing more than 100 proteins called nucleoporins. NPCs function as transport machineries for inorganic ions and macromolecules. The most prominent feature of an individual NPC is a large central channel, ~7 nm in width and 50 nm in length. NPCs exhibit high morphological and functional plasticity, adjusting shape to function. Macromolecules ranging from 1 to >100 kDa travel through the central channel into (and out of) the nucleoplasm. Inorganic ions have additional pathways for communication between cytosol and nucleus. NE can turn from a simple sieve that separates two compartments by a given pore size to a smart barrier that adjusts its permeabiltiy to the metabolic demands of the cell. Early microelectrode work characterizes the NE as a membrane barrier of highly variable permeability, indicating that NPCs are under regulatory control. Electrical voltage across the NE is explained as the result of electrical charge separation due to selective barrier permeability and unequal distribution of charged macromolecules across the NE. Patch-clamp work discovers NE ion channel activity associated with NPC function. From comparison of early microelectrode work with patch-clamp data and late results obtained by the nuclear hourglass technique, it is concluded that NPCs are well-controlled supramolecular structures that mediate transport of macromolecules and small ions by separate physical pathways, the large central channel and the small peripheral channels, respectively. Electrical properties of the two pathways are still unclear but could have great impact on the understanding of signal transfer across NE and gene expression.

Aldosterone↗

PubMed1993

Expression of voltage-gated calcium channels correlates with PDGF-stimulated calcium influx and depends upon cell density in C3H 10T1/2 mouse fibroblasts.

Platelet derived growth factor (PDGF) mobilizes multiple calcium pools in the C3H 10T1/2 mouse fibroblast, including a sustained influx of extracellular calcium. We have used the whole cell patch-clamp technique to directly test for a role of plasma membrane calcium channels in this influx. Whole-cell patch-clamp recordings revealed a voltage-gated calcium channel with gating properties consistent with a 'T-type' designation. This phenotype of the C3H 10T1/2 fibroblasts was dependent upon the cell density in culture. The fraction of cells expressing calcium channels was low (< 10%) in subconfluent culture but rose to approximately 50% as the cells established a confluent monolayer. The magnitude of the PDGF-stimulated sustained calcium influx component measured using Fura-2 increased in parallel with the expression of calcium current. We interpret these results to support the hypothesis that T-type voltage-gated calcium channels contribute to the PDGF-stimulated intracellular calcium signals in these cells.

Animals↗

PubMed1996

cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

Adenylyl Cyclases↗

PubMed1989

A new type of amiloride-sensitive cationic channel in endothelial cells of brain microvessels.

Endothelial cells from brain microvessels form the blood-brain barrier. Brain microvessels and endothelial cells isolated from rat brain microvessels express an amiloride-sensitive cationic channel that was characterized using [3H]phenamil binding and patch-clamp experiments. [3H]Phenamil, a labeled amiloride analog, recognizes a single family of binding sites with a dissociation constant of 20-30 nM and a maximum binding capacity of 8-15 pmol/mg protein. The pharmacological profile of the channel (phenamil greater than benzamil greater than amiloride) is very similar to that of the epithelium Na+ channel of mammalian kidney and of frog epithelia. Long-lasting currents were observed in patch-clamp experiments using excised outside-out patches. Application of amiloride or phenamil first produced a rapid flickering of channel activity and then its complete blockade. The mean unit channel conductance at 140 mM Na+ was 23 picosiemens. The selectivity of Na+ over K+ was estimated from reversal potentials to be 1.5:1. Properties of the channel in microvessels are clearly distinct from those of the Na+ channel of the kidney, suggesting the existence of several isoforms of cationic channels that are sensitive to amiloride and its derivatives. The low selectivity cationic channel of endothelial cells in brain microvessels might be important for controlling both Na+ and K+ movements across the blood-brain barrier.

Amiloride↗

PubMed2003

Whole-cell recording of intracellular pH with silanized and oiled patch-type single or double-barreled microelectrodes.

Conventional ion-sensitive microelectrodes cannot be used in small cells, since they create too large an electrical leak at the site of penetration. Membrane potentials can be measured in such cells with the whole-cell configuration of the patch-clamp technique, after obtaining a high-resistance seal (giga-seal) to the cell membrane. Achieving such seals with patch-type microelectrodes silanized and filled with ion-sensitive cocktails has proved very difficult. Since ion-sensitive microelectrodes offer advantages over fluorescent techniques, we have developed a method which enables whole-cell recordings of membrane potential and intracellular pH to be achieved with silanized microelectrodes. We have been able to obtain high-resistance seals with silanized tips by dipping them in mineral oil. We describe the method for both single-barrel and theta-glass double-barreled microelectrodes. Double-barreled microelectrodes can be used to measure and control membrane potential in the whole-cell patch-clamp configuration while also measuring ionic activities with the adjacent barrel. We present illustrative experiments showing intracellular pH recordings in snail neurones and rat dorsal root ganglion cells, and we suggest the method can also be applied to other liquid-sensor ion-sensitive microelectrodes.

Animals↗

PubMed1994

Evidence for direct actions of general anesthetics on an ion channel protein. A new look at a unified mechanism of action.

BACKGROUND: Ion permeation through the nicotinic acetylcholine receptor channel is inhibited by general anesthetics. This inhibition could be mediated either by binding of anesthetic molecules to the channel protein itself or by the effects of anesthetics on the lipid environment of the protein. METHODS: Patch clamp recording techniques were used to investigate the effects of ether and propofol on acetylcholine receptor channels in outside-out patches from BC3H-1 cells. The kinetic and conductance properties of single channels were measured. A rapid perfusion system was used to make rapid changes in anesthetic concentration during patch clamp recording to determine the kinetics of inhibition by anesthetics. RESULTS: Ether, isoflurane (results from previous studies), and propofol produce distinct kinetic patterns of single acetylcholine receptor channel activity. Ether reduces the apparent current amplitude of channels, isoflurane induces flickering channel activity and propofol merely decreases the open time of the channel. The kinetics of inhibition are also different for these anesthetics. Ether (< 40 microseconds) is faster than isoflurane (300-600 microseconds) which is faster than propofol (> or = 2 ms). CONCLUSIONS: These diverse patterns can be interpreted in terms of a unitary mechanism in which the anesthetics interact directly with the channel protein. Each anesthetic is considered to bind to a site on the protein (perhaps, but not necessarily within the pore of the channel) and interrupt the flow of ions through the pore. Anesthetics have access to this inhibitory binding site even when the gate of the channel is closed. The pattern of channel activity induced by an anesthetic is determined by the frequency and duration of binding events.

Cells, Cultured↗

PubMed1994

Early effects of PRL on ion conductances in CHO cells expressing PRL receptor.

Chinese hamster ovary (CHO-K1) cells were stably transfected with prolactin (PRL) receptor cDNA. These cells (CHO-E32) expressed the long form of functional PRL receptor. Using microfluorimetric and patch-clamp techniques, we have investigated the effects of PRL on intracellular Ca2+ concentration ([Ca2+]i) and membrane ion conductances. Exposure of CHO-E32 cells to 5 nM PRL resulted in an increase in [Ca2+]i. Two types of response were observed: 1) a stimulation of Ca2+ entry and 2) an intracellular Ca2+ mobilization. As PRL inhibited voltage-activated Ca2+ current, the PRL-induced Ca2+ increase does not involve voltage-activated Ca2+ channels. PRL also increased a charybdotoxin-sensitive Ca(2+)-dependent K+ conductance. Simultaneous measurements showed that PRL hyperpolarized the membrane potential before increasing intracellular Ca2+ levels. In voltage clamp, hyperpolarizing voltage steps were associated with increased Ca2+ concentrations, whereas depolarizing voltage steps decreased [Ca2+]i. Cell-free patch-clamp experiments showed that PRL directly stimulates K+ channel activity. Our results suggest the existence of a regulatory complex involving a protein kinase tightly associated with the Ca(2+)-activated K+ channels and that PRL stimulates these channels by means of the activation of protein kinase. The resulting hyperpolarization stimulates Ca2+ entry, probably through voltage-insensitive nonspecific channels.

Animals↗

PubMed2002

[Effect of angiotensin II on potassium channels of ischemic ventricular myocytes of the guinea pig].

The experiments were carried out on guinea pig isolated ventricular myocytes by using whole-cell patch clamp. The effects of angiotensin II (Ang II) on potassium ion channels of acute ischemic myocytes were observed. Whole-cell patch clamp recordings showed that physiological potassium current, including delayed rectifier potassium current and inward rectifier potassium current were inhibited under the condition of simulated ischemia, and then further inhibited by treatment with Ang II. ATP-sensitive potassium currents were increased under simulated ischemia and were further enhanced by Ang II treatment.

Angiotensin II↗

PubMed1985

Rectification of single GABA-gated chloride channels in adult hippocampal neurons.

The properties of single chloride channels activated by gamma-aminobutyric acid (GABA) were investigated with hippocampal slices from adult guinea pigs. After the slices were treated with proteolytic enzymes, gigaseal recordings were made from excised patches of pyramidal or granule cell membranes. This newly developed preparation permits the application of patch-clamp techniques to the adult mammalian central nervous system. Guinea pig hippocampal slices were prepared in a conventional manner. Once prepared, the slices were treated with two different enzymes for brief periods and gently agitated. Slices generally split apart along the boundaries of the cell body regions, exposing neuronal somata. Standard patch-clamp techniques were used for the gigaseal recordings from excised patches. Solutions for both sides of the patches consisted of symmetrical concentrations of chloride, with all cation channels blocked. GABA at concentrations of 0.5-1.0 microM was added to the solution for the extracellular side of the patches. At transmembrane potentials negative to the chloride reversal potential (0 mV), the conductance through the GABA-gated chloride channels was approximately 20 pS. When the transmembrane potential was changed to positive values, the chloride conductance increased dramatically. For example, at +40 mV the conductance through the GABA-gated channels was almost 40 pS. Ramp-clamp commands were used to measure the current-voltage (I-V) relationship through single open channels. The open-channel I-V curves displayed outward rectification. The relationship between open-channel conductance and voltage could be fitted reasonably well by a single energy-barrier model for the channel, with the higher energy barrier placed near the cytoplasmic side of the membrane (at a fractional distance through the membrane of 0.73).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗