SEARCH · PubMed Health

Results for “coupled patch model”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources

PubMed2001

Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathy.

There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high-threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)-induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G-protein-coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)-type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large-amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] binding, confirmed that G-protein activity was significantly diminished in STZ-induced diabetic neurons compared with age-matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G-protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes.

Analgesics, Opioid↗

PubMed2000

Dynamics of signaling between Ca(2+) sparks and Ca(2+)- activated K(+) channels studied with a novel image-based method for direct intracellular measurement of ryanodine receptor Ca(2+) current.

Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.

Animals↗

PubMed2001

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2.

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

ATP-Binding Cassette Transporters↗

PubMed2005

D2 dopamine receptors colocalize regulator of G-protein signaling 9-2 (RGS9-2) via the RGS9 DEP domain, and RGS9 knock-out mice develop dyskinesias associated with dopamine pathways.

Regulator of G-protein signaling 9-2 (RGS9-2), a member of the RGS family of G GTPase accelerating proteins, is expressed specifically in the striatum, which participates in antipsychotic-induced tardive dyskinesia and in levodopa-induced dyskinesia. We report that RGS9 knock-out mice develop abnormal involuntary movements when inhibition of dopaminergic transmission is followed by activation of D2-like dopamine receptors (DRs). These abnormal movements resemble drug-induced dyskinesia more closely than other rodent models. Recordings from striatal neurons of these mice establish that activation of D2-like DRs abnormally inhibits glutamate-elicited currents. We show that RGS9-2, via its DEP domain (for Disheveled, EGL-10, Pleckstrin homology), colocalizes with D2DRs when coexpressed in mammalian cells. Recordings from oocytes coexpressing D2DR or the m2 muscarinic receptor and G-protein-gated inward rectifier potassium channels show that RGS9-2, via its DEP domain, preferentially accelerates the termination of D2DR signals. Thus, alterations in RGS9-2 may be a key factor in the pathway leading from D2DRs to the side effects associated with the treatment both of psychoses and Parkinson's disease.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1994

Astrocyte Na+ channels are required for maintenance of Na+/K(+)-ATPase activity.

Astrocytes in vitro and in situ have been shown to express voltage-activated ion channels previously thought to be restricted to excitable cells, including voltage-activated Na+, Ca2+, and K+ channels. However, unlike neurons, astrocytes do not generate action potentials, and the functional role of voltage-activated channels in astrocytes has been an enigma. In order to study the function of Na+ channels in glial cells, we carried out ion flux measurements, patch-clamp recordings, and ratiometric imaging of [Na+]i during blockade of Na+ channels on rat spinal cord astrocytes cultured for 7-10 d. Acute blockade of astrocyte Na+ channels by TTX had multiple effects: (1) TTX reduced, in a dose-dependent manner, Na+/K(+)-ATPase activity measured as unidirectional influx of 86Rb+; (2) TTX depolarized astrocyte membrane potential at a rate of approximately 1 mV/min; (3) TTX (100 microM) reduced [Na+]i; and (4) prolonged exposure to micromolar TTX induced astrocyte death. All these effects of TTX could be mimicked by ouabain or strophanthidin, specific blockers of the Na+/K(+)-ATPase. The effects of TTX and ouabain (or strophanthidin) were not additive. These results suggest that TTX-blockable Na+ channels in glial cells serve functions that do not require their participation in action potential electrogenesis; in particular, we propose that glial Na+ channels constitute a "return" pathway for Na+/K(+)-ATPase function, which permits Na+ ions to enter the cells to maintain [Na+]i at concentrations necessary for activity of the Na+/K(+)-ATPase. Since astrocyte Na+/K(+)-ATPase is believed to participate in [K+]o homeostasis in the CNS, the coupling of Na+ flux through voltage-activated Na+ channels to ATPase activity may provide a feedback loop that participates in the regulation of K+ ion levels in the extracellular space.

Animals↗

PubMed1997

Excitation-contraction coupling in the day 15 embryonic chick heart with persistent truncus arteriosus.

Ca2+ transients were examined in embryonic chick hearts with an experimentally induced cardiac neural crest-related outflow tract defect known as persistent truncus arteriosus (PTA). In all of the animal models of neural crest-related heart defects, prenatal mortality is too high to be attributed to structural defects of the heart alone, suggesting that there is altered development of the myocardium. Earlier reports indicating reduced L-type Ca2+ current in hearts with PTA suggest that poor viability may be related to impairment of cardiac excitation-contraction coupling. To test this hypothesis, direct measurements of the systolic Ca2+ transient in fura-2-loaded myocytes from normal hearts and hearts with PTA were carried out. We found that Ca2+ transients were severely depressed in hearts with PTA and difficult to measure above background noise unless signal averaged or treated with isoproterenol (ISO). We confirmed that the reduced Ca2+ transients were due, at least partly, to a reduction in L-type Ca2+ current. In addition we found that although ISO could raise the L-type current in hearts with PTA to the level found in normal hearts in the absence of ISO, it could not fully restore the Ca2+ transient. Furthermore, caffeine-stimulated Ca2+ transients were diminished in size and the time-to-peak and the decaying phase were significantly slowed. Interestingly, these observations were not accompanied by a reduction in the number of Ca2+ release channels. These results indicated an impairment of SR function in addition to the reduction in L-type Ca2+ current. These results strongly support our hypothesis that the poor viability of embryos with PTA is due to impaired cardiac excitation-contraction coupling.

Animals↗

PubMed2004

Light signaling in scotopic conditions in the rabbit, mouse and rat retina: a physiological and anatomical study.

In the dark, light signals are conventionally routed through the following circuit: rods synapse onto rod bipolar (RB) cells, which in turn contact AII amacrine cells. AII cells segregate the light signal into the on and off pathways by making electrical synapses with on cone bipolar (CB) cells and glycinergic inhibitory chemical synapses with off CB cells. These bipolar cells synapse onto their respective ganglion cells, which transfer on and off signals to the visual centers of the brain. Two alternative pathways have recently been postulated for the signal transfer in scotopic conditions: 1) electrical coupling between rods and cones, and 2) a circuit independent of cone photoreceptors, implying direct contacts between rods and off CB cells. Anatomical evidence supports the existence of both these circuits. To investigate the contribution of these alternative pathways to scotopic vision in the mammalian retina, we have performed patch-clamp recordings from ganglion cells in the dark-adapted retina of the rabbit, mouse, and rat. Approximately one-half of the ganglion cells in the rabbit retina received off signals through a circuit that was independent of RB cells. This was shown by their persistence in the presence of the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks rod-->RB cell signaling. Consistent with this result, strychnine, a glycine receptor antagonist, was unable to abolish these off responses. In addition, we were able to show that some off cone bipolar dendrites terminate at rod spherules and make potential contacts. In the mouse retina, however, there seems to be a very low proportion of off signals carried by an APB-resistant pathway. No ganglion cells in the rat retina displayed APB- and strychnine-resistant responses. Our data support signaling through flat contacts between rods and off CB cells as the alternative route, but suggest that the significance of this pathway differs between species.

Aminobutyrates↗

PubMed2006

Carvedilol restore cardiac calcium release channel structure and function in heart failure.

BACKGROUND: The ryanodine receptor (RyR2) on cardiac sarcoplasmic reticulum is the key calcium release channel required for excitation-contraction coupling. In failing hearts, the stoichiometry and function of the RyR2 macromolecular complex is altered. AIMS: In the present study, we used a well-characterized canine model of pacing-induced HF to show that Carvedilol both restores the normal stoichiometry of the RyR2 macromolecular complex and normalizes the function of the channel. METHODS: A rapid cardiac pacing regimen that results in severe HF was used. Animals were assigned to 1 of 4 groups: (1) normal without heart instrumentation (n=6), (2) HF (n=6), (3) HF treated with Carvedilol (n=6), and (4) normal without heart instrumentation plus Carvedilol (n=6). In groups 2 and 3, after baseline measurements had been obtained, rapid left ventricular (LV) pacing was initiated at 210 bpm for 3 weeks, followed by an additional week of pacing at 240 bpm with an external pacemaker. Carvedilol was begun 2 weeks after initiation of pacing and continued for 2 weeks. Carvedilol was begun 2 weeks after baseline measurement for the control dogs with heart instrumentation. SR membranes were prepared from canine ventricular tissue, protein concentration was measured by Bradford assay. RESULTS: Here, we show that systemic oral administration of Carvedilol reverses protein kinase A hyperphosphorylation of RyR2, restores the stoichiometry of the RyR2 macromolecular complex, and normalizes single-channel function in a canine model of heart failure. CONCLUSIONS: These results may, in part, explain the improved cardiac function observed in heart failure patients treated with Carvedilol.

Administration, Oral↗

PubMed1999

Auditory collusion and a coupled couple of outer hair cells.

The discrepancies between measured frequency responses of the basilar membrane in the inner ear and the frequency tuning found in psychophysical experiments led to Bekesy's idea of lateral inhibition in the auditory nervous system. We now know that basilar membrane tuning can account for neural tuning, and that sharpening of the passive travelling wave depends on the mechanical activity of outer hair cells (OHCs)3, but the mechanism by which OHCs enhance tuning remains unclear. OHCs generate voltage-dependent length changes at acoustic rates, which deform the cochlear partition. Here we use an electrical correlate of OHC mechanical activity, the motility-related gating current, to investigate mechano-electrical interactions among adjacent OHCs. We show that the motility caused by voltage stimulation of one cell in a group evokes gating currents in adjacent OHCs. The resulting polarization in adjacent cells is opposite to that within the stimulated cell, which may be indicative of lateral inhibition. Also such interactions promote distortion and suppression in the electrical and, consequently, the mechanical activity of OHCs. Lateral interactions may provide a basis for enhanced frequency selectivity in the basilar membrane of mammals.

Animals↗

PubMed2002

Pharmacological enhancement of hemi-gap-junctional currents in Xenopus oocytes.

Hemichannels formed by expressing connexin subunits in Xenopus oocytes provide a valuable tool for revealing the gating properties of intercellular gap junctions in electrically coupled cells. We used the two electrode voltage-clamp technique to demonstrate that activation of the time-dependent outward hemichannel currents brings into play a sodium current of similar time course and opposite polarity; the interaction between these opposing currents had not been explored previously. Using the endogenous connexin (Cx38) of Xenopus oocytes as a model system, we have shown that substituting choline for sodium in the bath solution eliminates the sodium current, thereby unmasking large hemichannel currents, and enabling pharmacological studies of agents that are known to modulate gap-junctional conductances. The cinchona alkaloid quinine also effectively blocked the inward current, and in addition, enhanced significantly the Cx38 hemichannel currents in a dose-dependent fashion; the Hill coefficient of 1.9 suggests that the binding of at least two molecules of quinine is required to produce the effect. Intracellular quinine had no effect on hemichannel currents, and experiments on the displacement of quinine suggest that binding is at an external site near or within the mouth of the hemichannel. Intracellular acidification suppressed the quinine-enhanced hemichannel currents, indicating that quinine does not block the proton binding site. We found that retinoic acid (RA) and carbenoxolone, agents that block gap-junctional channels in coupled neurons and other cell types, also suppressed Cx38 hemichannel currents with an IC(50) of approximately 2 and 34 microM for RA and carbenoxolone, respectively. Raising extracellular calcium to 3 mM suppressed both the hemichannel current and the inward sodium current. These results provide a foundation upon which to further characterize the gating of hemichannel currents mediated by connexins expressed in Xenopus oocytes.

Analgesics, Non-Narcotic↗

PubMed1999

Molecular cloning and characterization of a channel-like transporter mediating intestinal calcium absorption.

Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed in Xenopus oocytes, CaT1 mediates saturable Ca(2+) uptake with a Michaelis constant of 0.44 mM. Transport of Ca(2+) by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca(2+) uptake ratio close to 2:1, indicating that CaT1-mediated Ca(2+) influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr(2+) and Ba(2+) (but not Mg(2+)), although the currents evoked by Sr(2+) and Ba(2+) are much smaller than those evoked by Ca(2+). The trivalent cations Gd(3+) and La(3+) and the divalent cations Cu(2+), Pb(2+), Cd(2+), Co(2+), and Ni(2+) (each at 100 microM) do not evoke currents themselves, but inhibit CaT1-mediated Ca(2+) transport. Fe(3+), Fe(2+), Mn(2+), and Zn(2+) have no significant effects at 100 microM on CaT1-mediated Ca(2+) transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D(3) administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca(2+) entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.

Amino Acid Sequence↗

PubMed1995

Coupling between charge movement and pore opening in voltage dependent potassium channels.

Ionic and gating currents from Shaker K+ channels were characterized with the cut-open oocyte voltage clamp (COVG) technique. Experiments were performed in normally conducting channels and in channels with the W434F mutation which completely abolished ion conduction without affecting the voltage dependence of gating charge. Subtracted and unsubtracted gating currents with the COVG technique, and gating currents recorded in cell attached macro-patches had the same properties and time course. However, OFF gating currents and ionic deactivation tails became slower after patch excision. Gating currents had the following salient properties: 1) the turn-on of the gating current shows a rising phase, 2) the more negative position of the charge-voltage curve (Q-V) vs. the conductance-voltage (G-V) curve and the charge displacement by hyperpolarizing prepulses indicate that a large fraction of the voltage-dependence occurs in the transitions between closed states, 3) the Q-V relationship showed two component with different half activation potential and effective valence; the one appearing at more negative potential had a shallower voltage dependence, while the one at more depolarized potentials had a larger effective valence and correlated with channel opening, 4) ionic and gating currents were similarly time shifted by preceding hyperpolarizing and depolarizing pulses which substantiates the relationship between charge movement and channel opening, 5) in the wild type Shaker K+ clone with fast inactivation, the OFF gating charge is partially immobilized for large depolarizing pulses, while in the mutant channel lacking inactivation the charge is recovered quickly at the end of the pulse, indicating that the channel blockade by the inactivating particle slows down charge recovery, 6) the OFF gating current rapidly returns for small depolarizations, while for larger pulses which open the channel the OFF gating current return is delayed suggesting that the closed to open transitions carry little charge and 7) in the W434F mutant with the conserved amino terminus large depolarizations that would have opened the channel induced OFF charge immobilization, indicating that although the conduction pathway was not functional, the channel can still undergo the closed-open conformation in response to voltage changes. In conclusion, the kinetic properties of gating currents discard equal and independent gating subunits with two positions. They favor the presence of interactions among the gating subunits and are consistent with a sequential model for K+ channel activation in which an early less voltage-dependent transition between very deep closed states was followed by more voltage-dependent transitions between later closed states, and finally by a much less voltage dependent transition between the last closed state and the open state.

Animals↗

PubMed2002

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis.

Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.

Amino Acid Substitution↗

PubMed1999

Nonuniform responses of transmembrane potential during electric field stimulation of single cardiac cells.

The response of cellular transmembrane potentials (V(m)) to applied electric fields is a critical factor during electrical pacing, cardioversion, and defibrillation, yet the coupling relationship of the cellular response to field intensity and polarity is not well documented. Isolated guinea pig ventricular myocytes were stained with a voltage-sensitive fluorescent dye, di-8-ANEPPS (10 microM). A green helium-neon laser was used to excite the fluorescent dye with a 15-micrometers-diameter focused spot, and subcellular V(m) were recorded optically during field stimulation directed along the long axis of the cell. The membrane response was measured at the cell end with the use of a 30-ms S1-S2 coupling interval and a 10-ms S2 pulse with strength of up to approximately 500-mV half-cell length potential (field strength x one-half the cell length). The general trends show that 1) the response of V(m) at the cell end occurs in two stages, the first being very rapid (<1 ms) and the second much slower in time scale, 2) the rapid response consists of hyperpolarization when the cell end faces the anode and depolarization when the cell end faces the cathode, 3) the rapid response varies nonlinearly with field strengths and polarity, being relatively larger for the hyperpolarizing responses, and 4) the slower, time-dependent response has a time course that varies in slope with field strength. Furthermore, the linearity of the dye response was confirmed over a voltage range of -280 to +140 mV by simultaneous measurements of optically and electrically recorded V(m). These experimental findings could not be reproduced by the updated, Luo-Rudy dynamic model but could be explained with the addition of two currents that activate outside the physiological range of voltages: a hypothetical outward current that activates strongly at positive potentials and a second current that represents electroporation of the cell membrane.

Action Potentials↗

PubMed1997

Actions of the antiarrhythmic peptide AAP10 on intercellular coupling.

Disturbances in gap junction distribution and a decrease in the connexin43 content of the heart were shown to occur after myocardial infarction and in ischemic heart disease, respectively. These changes are now thought to play an important role in the genesis of arrhythmias associated with these diseases. It is thought that agents that can increase cellular coupling might be beneficial in these situations. Recently, we presented data showing that the synthetic peptide AAP10 acts antiarrhythmically in a model of regional ischemia. The data suggested that AAP10 might act via an increase in cellular coupling. The goal of this study was to establish whether AAP10 can interact with cardiac gap junctions. Measurements of the stimulus-response-interval (SRI) in guinea pig papillary muscle showed that high concentrations of AAP10 (1 microM) can decrease the SRI by about 10% under normoxic conditions. At lower concentrations (10 nM) AAP10 had no effect on SRI under normoxic conditions but prevented the increase in the SRI induced by perfusion with hypoxic, glucose-free Tyrode's solution. Double-cell voltage-clamp experiments confirmed that AAP10 can interact with cardiac gap junctions. 10 nM AAP10 could either diminish or reverse the run-down of gap junction conductance normally observed in pairs of guinea pig ventricular myocytes. During control gap junction conductance decreased with a rate of -2.5 +/- 2.0 nS/min. After application of 10 nM AAP10 gap junction conductance increased with a rate of +1.0 +/- 0.7 nS/min (p < 0.01). After washout of AAP10 gap junction conductance decreased again with a rate not significantly different from control. Our results show that AAP10 does interact with gap junctions. Because no other effects of AAP10 on other electrophysiological parameters could be found, this action on gap junctions might be the basis of AAP10's antiarrhythmic effect seen in previous studies.

Action Potentials↗

PubMed2002

Mobility of ions, sugar, and water in the cytoplasm of Xenopus oocytes expressing Na(+)-coupled sugar transporters (SGLT1).

A model was set up to study water transport in membrane proteins expressed in Xenopus oocytes. The model was tested experimentally using human and rabbit Na+-glucose cotransporters (SGLT1), and was used to explain controversies regarding unstirred layer effects. Cotransport of Na+, sugar and water was monitored by two-electrode voltage clamp and online measurements of oocyte volume. The specific resistance of the oocyte cytoplasm was found by means of microelectrodes to be 263 +/- 91 Omega cm (S.D., n = 52), or 2.5 times that of Kulori medium, in agreement with reported values of intracellular ion concentrations and diffusion constants. Osmotically induced volume and resistance changes were compatible with a model of the oocyte in which 37 +/- 17 % (S.D., n = 66) of the intracellular volume acts as a free solution while the remainder is inert, being occupied by organelles, etc. The model explains the results of several types of experiments: rapid changes in rates of water cotransport induced by changes in clamp voltage followed by osmotic equilibration in sugar-free conditions; volume changes induced by Na+ transport via the ionophore gramicidin; and uphill water transport. Ethanol (0.5 %) induced a marked swelling of the oocytes of about 16 pl x s(-1). If the specific inhibitor of SGLT1 phlorizin is added from stock solutions in ethanol, the effect of ethanol obfuscates the effects of the inhibitor. We conclude that the transport parameters derived for water cotransport by the SGLT1 can be attributed to the protein residing in the plasma membrane with no significant influences from unstirred layer effects.

Animals↗

PubMed1997

Molecular basis of the diversity of calcium channels in cardiovascular tissues.

Voltage-dependent calcium (Ca2+) channels control a variety of physiological functions, such as excitation-contraction coupling in cardiac and smooth muscle. secretion of hormones and release of neurotransmitters. Studies on dissociated or cultured cells enabled us to compare their electrophysiological and pharmacological properties and their regulation in various tissues. Molecular genetics has provided a structural basis with which to observe the functional diversity of Ca2+ channels, which are composed of several subunits (alpha 1, alpha 2-delta, beta, gamma). Structure-function experiments, using expression in Xenopus oocytes, were designed to explain the molecular basis underlying this functional diversity. Six genes have been identified encoding the pore subunit (alpha 1) which determines the basic profile, i.e. the pharmacology of any Ca2+ channel. However, using a reconstitution model, the auxiliary subunits, but mainly beta subunits, for which four genes and several variants have been isolated, are able to modify the level of expression and the properties of a Ca2+ current directed by an alpha 1 subunit. Our structure-function studies are mainly designed to investigate the functional consequences of alpha 1-beta interaction on electrophysiological and pharmacological properties, especially in the case of cardiovascular Ca2+ channels. These studies should lead to a better understanding of the molecular basis underlying the differences between cardiac and vascular Ca2+ channels and also their implication in pathophysiology. Functional expression of the various combinations of subunit isoforms and identification of the precise oligomeric structure of voltage-dependent Ca2+ channels in specific cell types should help in the development of new therapeutic drugs.

Animals↗

PubMed2000

Physiological properties of zebrafish embryonic red and white muscle fibers during early development.

The zebrafish is a model organism for studies of vertebrate muscle differentiation and development. However, an understanding of fish muscle physiology during this period is limited. We examined the membrane, contractile, electrical coupling, and synaptic properties of embryonic red (ER) and white (EW) muscle fibers in developing zebrafish from 1 to 5 days postfertilization. Resting membrane potentials were -73 mV in 1 day ER and -78 mV in 1 day EW muscle and depolarized 17 and 7 mV, respectively, by 5 days. Neither fiber type exhibited action potentials. Current-voltage relationships were linear in EW fibers and day 1 ER fibers but were outwardly rectifying in some ER fibers at 3 to 5 days. Both ER and EW fibers were contractile at all ages examined (1 to 5 days) and could follow trains of electrical stimulation of up to 30 Hz without fatiguing for up to 5 min. Synaptic activity consisting of miniature endplate potentials (mEPPs) was observed at the earliest ages examined (1.2-1. 4 days) in both ER and EW fibers. Synaptic activity increased in frequency, and mEPP amplitudes were larger by 5 days. Miniature EPP rise times and half-widths decreased in ER fibers by 5 days, while EW fiber mEPPs showed fast kinetics as early as 1.2-1.4 days. ER and EW muscle fibers showed extensive dye coupling but not heterologous (red-white) coupling. Dye coupling decreased by 3 days yet remained at 5 days. Somites were electrically coupling, and this allowed filtered synaptic potentials to spread from myotome to myotome. It is concluded that at early developmental stages the physiological properties of ER and EW muscle are similar but not identical and are optimized to the patterns of swimming observed at these stages.

Animals↗