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G E Kirsch

Publications and source records attributed to G E Kirsch.

At least 55 records · Page 3Linked to original sources
PubMed1990

Toxin and kinetic profile of rat brain type III sodium channels expressed in Xenopus oocytes.

Sodium (Na+) channels are members of a multigene family and are responsible for generation and propagation of the action potential in excitable cells. We have assembled, in a transcription-competent vector, a full-length cDNA clone encoding the rat brain type III Na+ channel. Xenopus oocytes microinjected with in vitro synthesized mRNA expressed functional rat brain Na+ channels from such 'cloned' RNA transcripts. We found that type III Na+ currents in whole cell microelectrode voltage clamp and in cell-attached patch recordings decayed much more slowly than any other reported Na+ current. In addition, we saw typical and additive effects of alpha- and beta-scorpion toxins, suggesting that the Na+ channel alpha-subunit itself contains functional and distinct toxin binding sites.

Amino Acid Sequence↗
PubMed1990

Fast and slow gating of sodium channels encoded by a single mRNA.

We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.

Animals↗
PubMed1990

Inhibition of cardiac Na+ currents by isoproterenol.

The mechanism by which the beta-adrenergic agonist isoproterenol (ISO) modulates voltage-dependent cardiac Na+ currents (INa) was studied in single ventricular myocytes of neonatal rat using the gigaseal patch-clamp technique. ISO inhibited INa reversibly, making the effect readily distinguishable from the monotonic decrease of INa caused by the shift in gating that customarily occurs during whole cell patch-clamp experiments (E. Fenwick, A. Marty, and E. Neher, J. Physiol. Lond. 331: 599-635, 1982; and J. M. Fernandez, A. P. Fox, and S. Krasne, J. Physiol. Lond. 356: 565-585, 1984). The inhibition was biphasic, having fast and slow components, and was voltage-dependent, being more pronounced at depolarized potentials. In whole cell experiments the membrane-permeable adenosine 3',5'-cyclic monophosphate (cAMP) congener 8-bromo-cAMP reduced INa. In cell-free inside-out patches with ISO present in the pipette, guanosine 5'-triphosphate (GTP) applied to the inner side of the membrane patch inhibited single Na+ channel activity. This inhibition could be partly reversed by hyperpolarizing prepulses. The nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) greatly reduced the probability of single Na+ channel currents in a Mg2(+)-dependent manner. We propose that ISO inhibits cardiac Na+ channels via the guanine nucleotide binding, signal-transducing G protein that acts through both direct (membrane delimited) and indirect (cytoplasmic) pathways.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Coupling of ATP-sensitive K+ channels to A1 receptors by G proteins in rat ventricular myocytes.

ATP-sensitive K+ (K+[ATP]) current is thought to be regulated by GTP-binding proteins (G proteins), but the pathways that couple receptor, G protein, and channel have not been defined. We studied regulation of tolbutamide-sensitive K+[ATP] current in neonatal rat ventricular myocytes. Application of 0.1 mM ATP to the intracellular side of membrane patches reduced K+ [ATP] channel activity, and addition of the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at 0.1 mM restored activity. Application of 0.1 mM intracellular GTP plus 10 microM extracellular adenosine or 100 nM N6-cyclohexyladenosine had the same effect as GTP gamma S; hence K+[ATP] channels may be coupled to adenosine receptors via G proteins. To determine which G protein, we applied G alpha subunits, preactivated with GTP gamma S to the cytoplasmic side of membrane patches, and found that alpha i1, alpha i2, and alpha i3 mimicked the effect of GTP gamma S, but not alpha o or Gs, suggesting that Gi alpha acts via a membrane-delimited pathway. Adenosine receptor coupling may be important for activating K+[ATP] channels in ischemic muscle.

Adenosine↗
PubMed1989

Beta-adrenergic inhibition of cardiac sodium channels by dual G-protein pathways.

The signaling pathways by which beta-adrenergic agonists modulate voltage-dependent cardiac sodium currents are unknown, although it is likely that adenosine 3'5'-monophosphate (cAMP) is involved. Single-channel and whole-cell sodium currents were measured in cardiac myocytes and the signal transducing G protein Gs was found to couple beta-adrenergic receptors to sodium channels by both cytoplasmic (indirect) and membrane-delimited (direct) pathways. Hence, Gs can act on at least three effectors in the heart: sodium channels, calcium channels, and adenylyl cyclase. The effect on sodium currents was inhibitory and was enhanced by membrane depolarization. During myocardial ischemia the sodium currents of depolarized cells may be further inhibited by the accompanying increase in catecholamine levels.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Recombinant alpha i-3 subunit of G protein activates Gk-gated K+ channels.

G proteins, particularly those sensitive to pertussis toxin, are difficult to separate biochemically, creating uncertainty in functional assignments. For this reason the cDNAs encoding G alpha i-3 and two of the G alpha s splice variants were expressed as fusion proteins in Escherichia coli using a T7 promoter-based expression system. These proteins were denoted r alpha i-3 and r alpha s (short and long) and accumulated in bacteria to as much as 5-10% of total cellular protein, of which 5-10% was soluble in lysates. Soluble r alpha subunits were tested for stimulation of K+ channel activity in inside-out atrial membrane patches and for reconstitution of cyc- adenylyl cyclase activity. r alpha i-3, activated either by guanosine 5'-(3-thio)triphosphate (GTP gamma S) or AlF-4, stimulated in a concentration-dependent manner single channel K+ currents in isolated atrial membrane patches of three species: guinea pigs, neonatal rats, and embryonic chick. In contrast, GTP gamma S-activated r alpha s did not. In agreement with a similar study by Graziano et al. (Graziano, M. P., Casey, P. J. and Gilman, A. G. (1987) J. Biol. Chem. 262, 11375-11381), both r alpha s forms reconstituted GTP gamma S-stimulated cyc- adenylyl cyclase activity, albeit at concentrations 50-100 times higher than those needed with native Gs. The concentrations of r alpha i-3 needed to stimulate the K+ channels were also higher than needed with native human erythrocyte Gk, in this case 30-50 times. Single K+ channel currents stimulated by r alpha i-3 were indistinguishable from those stimulated by the natural effector acetylcholine. Thus, bacterial expression of G alpha subunits provided the means to demonstrate unequivocally that Gi-3 has intrinsic Gk activity.

Adenylyl Cyclases↗
PubMed1989

Modification of Na channel gating by an alpha scorpion toxin from Tityus serrulatus.

The effects of TsIV-5, a toxin isolated from the Brazilian scorpion Tityus serrulatus, on whole-cell and single-channel Na currents were determined in N18 neuroblastoma cells. In whole-cell records at a test potential of -10 mV, external application of 500 nM TsIV-5 slowed inactivation 20-fold and increased peak current by about one-third without changing time-to-peak. Both the steady-state activation and inactivation curves were shifted to more negative potentials. Other alpha scorpion toxins produce similar effects but the single-channel mechanism is not known. TsIV-5 caused a voltage-dependent prolongation of mean single-channel open time such that at a test potential of -60 mV no change was observed, whereas at -20 mV mean open time increased about threefold and prolonged bursting was observed. Macroscopic current reconstructed from summed single-channel records showed a characteristic toxin-induced potentiation of peak current and a 20-fold slowing of the decay phase. TsIV-5 does not discriminate between tissue-specific Na channel subtypes. Prolonged open times and bursting were also observed in toxin-treated Na channels from rat ventricular myocytes, rat cortical neurons, and mouse skeletal muscle. The toxin effects are shown to be consistent with a kinetic model in which TsIV-5 selectively interferes with the ability of the channel to reach the inactivated state.

Animals↗
PubMed1989

Kinetic properties of single sodium channels in rat heart and rat brain.

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

Animals↗
PubMed1989

Trypsin activation of atrial muscarinic K+ channels.

The atrial muscarinic K+ channel normally is opened by the activated G protein, Gk. Based on the assumption that an inactivating particle keeps the channel closed, several protein-modifying agents including trypsin, papain, glyoxal, and phenylglycoxal that remove Na+-channel inactivation were tested. K+ channels were studied in inside-out excised membrane patches from primary cultures of neonatal rat atrial myocytes. Of the agents tested, only trypsin activated muscarinic K+ channels, and it did so irreversibly. Trypsin was effective in the absence of muscarinic agonist or intracellular Mg2+ and guanosine 5'-triphosphate. Heat-denatured trypsin was ineffective, and trypsin inhibitor blocked the effect. Because trypsin is known to inactivate G proteins, the effect was probably on the K+ channel or a structure closely associated with it. Trypsin activation produced single-channel currents in which inward rectification, single-channel conductance, mean open time, and burst duration were indistinguishable from muscarinic activation. Trypsin cleaves proteins at lysine or arginine residues, and the arginine-specific reagents, glyoxal and phenylgloxal, did not activate K+ channels. We conclude that trypsin disrupts an inhibitory gating mechanism that normally holds the channel closed in the absence of activated Gk. The inhibitory gate is physically distinct from the gate that mediates bursting and must contain at least one trypsin cleavage point located at a lysine residue accessible from the cytoplasmic surface of the cell membrane.

Animals↗
PubMed1989

Angiotensin II modulates cardiac Na+ channels in neonatal rat.

Since chronic congestive heart failure syndromes are associated with both elevated circulating levels of angiotensin II and potentially lethal ventricular tachyarrhythmias, we investigated the effect of angiotensin II on voltage-dependent cardiac Na+ currents. Single-channel Na+ currents in neonatal rat ventricular myocytes were studied using the patch clamp method in the cell-attached mode. Angiotensin II applied outside the patch increased the frequency of opening and rates of activation and inactivation of single-channel Na+ currents within the patch. These effects were mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and were prevented by prior incubation with TPA. Therefore, we propose that angiotensin II modulates cardiac Na+ currents by a cytoplasmic second messenger, perhaps protein kinase C, and this may predispose toward arrhythmia.

Angiotensin II↗
PubMed1988

Effects of New World scorpion toxins on single-channel and whole cell cardiac sodium currents.

Purified toxins from a North American scorpion, Centruroides noxius (Cn II-10), and a South American scorpion, Tityus serrulatus (Ts-gamma), were tested on cardiac sodium channels using patch-clamp methods to record whole cell and single-channel currents. The two toxins produced similar effects on sodium currents; potassium and calcium currents were not affected. Macroscopic sodium current amplitudes, measured at test potentials greater than -20 mV where the opening probability was high, decreased in a concentration-dependent manner with a half maximum inhibitory concentration of 6 X 10(-8) M. Block was unchanged by repetitive depolarizing pulses. In the presence of scorpion toxin, the currents were rapidly blocked by tetrodotoxin (3 X 10(-5) M). Both toxins shifted the voltage dependence of sodium channel inactivation to more negative potentials. At test potentials between -50 and -70 mV, where the sodium channel opening probability is normally low, both toxins produced an increase in sodium current and slowed the rates of activation and inactivation. At intermediate potentials between -50 and -20 mV the currents in the presence of toxins crossed over the control currents. At a test potential of -20 mV, the toxins decreased single-channel activity and increased the latency to first opening. At a test potential of -60 mV, the toxins significantly prolonged channel open time. The unitary current amplitudes were unchanged at either potential. We conclude that New World scorpion toxins produce apparently complex effects on whole cell currents primarily by retarding activation gating of cardiac sodium channels.

Animals↗
PubMed1988

Alpha-subunit of Gk activates atrial K+ channels of chick, rat, and guinea pig.

A specific guanine nucleotide-binding protein, Gk, is the link by which muscarinic receptors activate atrial potassium channels (Science Wash. DC 235: 207-211, 1987). In adult guinea pigs, the alpha-subunit at picomolar concentrations mediates the holo-G protein effect (Science Wash. DC 236: 442-445, 1987), but in chick embryo it has been reported that the beta gamma-dimer at nanomolar concentrations rather than the alpha-subunit is the effective mediator (Nature Lond. 325: 321-326, 1987). This difference might have a phylogenetic or ontogenetic basis, and the present experiments tested these possibilities. Preactivated alpha k derived from human red blood cell Gk, when applied to the intracellular surface of inside-out membrane patches from the atria of embryonic chick, neonatal rat, and adult guinea pig activated single K+ channel currents. In each case, the alpha k-activated channels had the same single-channel conductance and mean open time as the muscarinic agonist-activated channels. Half-maximal activation was achieved at alpha k-concentrations of 2.4-13.8 pM. Hence, alpha k-activation of these K+ channels is independent of differences in age or species. The detergent 3-[3-cholamidopropyl)-dimethyammoniol]-1-propanesulfonate (CHAPS), which was used by Logothetis et al. (Nature Lond. 325: 321-326, 1987) at 184 microM to suspend the hydrophobic beta gamma-dimers, activated the same currents. We conclude that the effects of the beta gamma-dimer on these K+ channels is unknown and that as we had proposed earlier (Science Wash. DC 236: 442-445, 1987) it is the alpha-subunit that mediates the Gk effect.

Animals↗
PubMed1987

Temperature dependence of Na currents in rabbit and frog muscle membranes.

The effect of temperature (0-22 degrees C) on the kinetics of Na channel conductance was determined in voltage-clamped rabbit and frog skeletal muscle fibers using the triple-Vaseline-gap technique. The Hodgkin-Huxley model was used to extract kinetic parameters; the time course of the conductance change during step depolarization followed m3h kinetics. Arrhenius plots of activation time constants (tau m), determined at both moderate (-10 to -20 mV) and high (+100 mV) depolarizations, were linear in both types of muscle. In rabbit muscle, Arrhenius plots of the inactivation time constant (tau h) were markedly nonlinear at +100 mV, but much less so at -20 mV. The reverse situation was found in frog muscle. The contrast between the highly nonlinear Arrhenius plot of tau h at +100 mV in rabbit muscle, compared with that of frog muscle, was interpreted as revealing an intrinsic nonlinearity in the temperature dependence of mammalian muscle Na inactivation. These results are consistent with the notion that mammalian cell membranes undergo thermotropic membrane phase transitions that alter lipid-channel interactions in the 0-22 degrees C range. Furthermore, the observation that Na channel activation appears to be resistant to this effect suggests that the gating mechanisms that govern activation and inactivation reside in physically distinct regions of the channel.

Animals↗
PubMed1987

Expression of single calcium channels in Xenopus oocytes after injection of mRNA from rat heart.

Oocytes of Xenopus laevis, after microinjection with mRNA from rat heart, display typical high-threshold calcium (Ca) whole cell currents. To prepare to study structure-function relationships of the cardiac Ca channel molecule, we examined the fidelity of expression of biophysical and pharmacological properties at the molecular level. Cell-attached gigaseal recordings in five K-depolarized oocytes injected with adult rat heart mRNA showed single channel Ba currents with mean amplitude 1.3-1.5 pA at 0 mV, slope conductance 18-25 pS, and extrapolated reversal potential 57-68 mV. Openings were predominantly brief (mean 1.2 ms) but longer openings (mean 9 ms) were greatly enhanced in 10(-6) M BAY-K 8644, increasing the ensemble average current at 0 mV by more than fivefold. These features are typical of high-threshold cardiac Ca channels. In two patches from one injected oocyte, we saw multiple Ca channel conductances, as recently observed in other preparations. We conclude that X. laevis oocytes injected with adult rat heart mRNA produce high-threshold cardiac Ca channels with molecular properties identical to native cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Sodium channel kinetics in normal and denervated rabbit muscle membrane.

The effects of chronic denervation on sodium (Na) channels in rabbit muscle membrane were determined using intracellular microelectrodes and Vaseline gap voltage clamp techniques. The Hodgkin-Huxley model was used to describe the kinetic and steady-state parameters of channel activation and fast inactivation. Chronic (7-10 days) denervation was found to cause a decreased resting potential, lowered action potential peak, and fibrillation potentials in rabbit extensor digitorum longus (EDL) muscles. Under voltage clamp conditions, no differences were observed between denervated and normal fibers in the voltage dependence of the steady-state Na channel activation and fast inactivation curves, or in the time course of development of fast inactivation. However, in denervated fibers, the time course of recovery from fast inactivation was approximately half that measured in normal fibers. Also, whereas depolarizing holding potentials induced a long-term inactivation to varying degrees in normal EDL fibers, denervated EDL fibers and normal soleus fibers were uniformly resistant to prolonged depolarization. These results suggest that the denervation-induced development of spontaneous activity may be due in part to changes in the mechanisms that control the refractoriness of Na channels.

Animals↗
PubMed1986

Modulation of aminopyridine block of potassium currents in squid axon.

Aminopyridines are known to block potassium (K) currents in excitable membranes in a manner dependent upon membrane potential, such that the block is relieved by depolarization and restored upon repolarization. In the present study, the effects of aminopyridines on voltage-dependent potassium (K) channels were examined in internally perfused, voltage-clamped squid giant axons. The time course of block restoration after conditioning depolarization was found to be modulated by membrane electric field, K-channel gating, and external cations. Depolarized holding potentials accelerated block restoration without altering steady-state block levels, suggesting that the voltage dependence of block restoration may be related to K channel gating rather than drug binding per se. In support of this notion, low external calcium concentration, which shifts the voltage dependence of K-channel gating to more negative potentials, also accelerated block restoration. Conversely, the relationship between the rate of block restoration and membrane holding potential was shifted in the depolarizing direction by phloretin, an agent that shifts the dependence of K-channel opening on membrane potential in a similar manner. Modification of K-channel gating also was found to alter the rate of block restoration. Addition of internal zinc or internal treatment with glutaraldehyde slowed the time course of both K-channel activation and aminopyridine block restoration. Aminopyridines also were found to interact in the K channel with external Cs+, NH4+, and Rb+, each of which slowed aminopyridine block restoration. Our results suggest that aminopyridines enter and occlude K channels, and that the availability of the binding site may be modulated by channel gating such that access is limited by the probability of the channel reaching an intermediate closed state at the resting potential.

Aminopyridines↗
PubMed1983

Site of action and active form of aminopyridines in squid axon membranes.

Aminopyridines, potent potassium channel blocking agents, were studied for their site of action and active form in the nerve membrane. Voltage clamped, internally perfused squid giant axons were used. 3,4-Diaminopyridine, one of the most potent aminopyridine derivatives, blocked the potassium current much faster with internal application than with external application. When applied externally to the internally perfused axon, the onset of 3,4-diaminopyridine block was accelerated by suspending the internal flow. 4-Aminopyridine methiodide, a quaternary derivative of 4-aminopyridine, blocked the potassium current more effectively by internal application than by external application. These observations support the notion that aminopyridines act on a site more easily accessible from inside the nerve membrane than from outside. The block of the potassium current caused by internal 4-aminopyridine methiodide, similar to that caused by 4-aminopyridine or 3,4-diaminopyridine, was voltage-, time- and frequency-dependent, becoming less with longer and prolonged depolarization and with repetitive depolarizations. Low internal pH, which accelerated block re-establishment by the tertiary derivative, did not affect the quaternary derivative. Furthermore, when perfused internally at different total concentrations and different internal pH values, 2,3-diaminopyridine exhibited the same degree of block as long as the internally present cationic form concentration was kept constant. These results indicate that aminopyridines act in the cationic form.

4-Aminopyridine↗