Range Of Membrane Potentials Research Articles

Voltage- and activity-dependent chloride conductance controls the resting status of the intact rat sympathetic neuron.

Remarkable activity dependence was uncovered in the chloride conductance that operates in the subthreshold region of membrane potential, by using the two-microelectrode voltage-clamp technique in the mature and intact rat sympathetic neuron. Both direct and synaptic neuron tetanization (15 Hz, 10-s duration to saturate the response) resulted in a long-lasting (not less than 15 min) increase of cell input conductance (+70-150% 10 min after tetanus), accompanied by the onset of an inward current with the same time course. Both processes developed with similar properties in the postganglionic neuron when presynaptic stimulation was performed under current- or voltage-clamp conditions and were unaffected by external calcium on direct stimulation. The posttetanic effects were sustained by gCl increase because both conductance and current modifications were blocked by 0.5 mM Anthracene-9-carboxylic acid (a chloride channel blocker) but were unaffected by TEACl or cesium chloride treatments. The chloride channel properties were modified by stimulation: their voltage sensitivity and rate of closure in response to hyperpolarization strongly increased. The voltage dependence of the three major conductances governing the cell subthreshold status (gCl, gK, and gL) was evaluated over the -40/-110 mV membrane potential range in unstimulated neurons and compared with previous results in stimulated neurons. A drastic difference between the voltage-conductance profiles was observed, exclusively sustained by gCl increase. The chloride channel thus hosts an intrinsic mechanism, a memory of previous neuron activity, which makes the chloride current a likely candidate for natural controller of the balance between opposite resting currents and thus of membrane potential level.

Spironolactone and Its Main Metabolite, Canrenoic Acid, Block Human Ether-a-Go-Go–Related Gene Channels

It has been demonstrated that spironolactone (SP) decreases the QT dispersion in chronic heart failure. In this study, the effects of SP and its metabolite, canrenoic acid (CA), on human ether-a-go-go-related gene (HERG) currents were analyzed. HERG currents elicited in stably transfected Chinese hamster ovary cells were measured with the whole-cell patch-clamp technique. SP decreased HERG currents in a concentration-dependent manner (IC50=23.0+/-1.5 micromol/L) and shifted the midpoint of the activation curve to more negative potentials (Vh=-13.1+/-3.4 versus -18.9+/-3.6 mV, P<0.05) without modifying the activation and deactivation kinetics. SP-induced block (1 micromol/L) appeared at the range of membrane potentials coinciding with that of channel activation, and thereafter, it remained constant, reaching 24.7+/-3.8% at +60 mV (n=6, P<0.05). CA (0.01 nmol/L to 500 micromol/L) blocked HERG channels in a voltage- and frequency-independent manner. CA at 1 nmol/L shifted the midpoint of the activation curve to -19.9+/-1.8 mV and accelerated the time course of channel activation (tau=1064+/-125 versus 820+/-93 ms, n=11, P<0.01). The envelope of the tail test demonstrated that at the very beginning of the pulses to +40 mV (25 ms), a certain amount of block was apparent (31.3+/-9.9%). CA did not modify the voltage-dependence of HERG channel inactivation (Vh=-60.8+/-5.6 versus -62.9+/-3.1 mV, n=6, P>0.05) or the kinetics of the reactivation process at any potential tested. CA and aldosterone also blocked the native I(Kr) in guinea-pig ventricular myocytes. At concentrations reached after administration of therapeutic doses of SP, CA blocked the HERG channels by binding to both the closed and open states of the channel.

Control of outer vestibule dynamics and current magnitude in the Kv2.1 potassium channel.

In Kv2.1 potassium channels, changes in external [K+] modulate current magnitude as a result of a K+-dependent interconversion between two outer vestibule conformations. Previous evidence indicated that outer vestibule conformation (and thus current magnitude) is regulated by the occupancy of a selectivity filter binding site by K+. In this paper, we used the change in current magnitude as an assay to study how the interconversion between outer vestibule conformations is controlled. With 100 mM internal K+, rapid elevation of external [K+] from 0 to 10 mM while channels were activated produced no change in current magnitude (outer vestibule conformation did not change). When channels were subsequently closed and reopened in the presence of elevated [K+], current magnitude was increased (outer vestibule conformation had changed). When channels were activated in the presence of low internal [K+], or when K+ flow into conducting channels was transiently interrupted by an internal channel blocker, increasing external [K+] during activation did increase current magnitude (channel conformation did change). These data indicate that, when channels are in the activated state under physiological conditions, the outer vestibule conformation remains fixed despite changes in external [K+]. In contrast, when channel occupancy is lowered, (by channel closing, an internal blocker or low internal [K+]), the outer vestibule can interconvert between the two conformations. We discuss evidence that the ability of the outer vestibule conformation to change is regulated by the occupancy of a nonselectivity filter site by K+. Independent of the outer vestibule-based potentiation mechanism, Kv2.1 was remarkably insensitive to K+-dependent processes that influence current magnitude (current magnitude changed by <7% at membrane potentials between −20 and 30 mV). Replacement of two outer vestibule lysines in Kv2.1 by smaller neutral amino acids made current magnitude dramatically more sensitive to the reduction in K+ driving force (current magnitude changed by as much as 40%). When combined, these outer vestibule properties (fixed conformation during activation and the presence of lysines) all but prevent variation in Kv2.1 current magnitude when [K+] changes during activation. Moreover, the insensitivity of Kv2.1 current magnitude to changes in K+ driving force promotes a more uniform modulation of current over a wide range of membrane potentials by the K+-dependent regulation of outer vestibule conformation.

Single ionic channels of two Caenorhabditis elegans chemosensory neurons in native membrane.

The genome of Caenorhabditis elegans contains representatives of the channel families found in both vertebrate and invertebrate nervous systems. However, it lacks the ubiquitous Hodgkin-Huxley Na+ channel that is integral to long-distance signaling in other animals. Nematode neurons are presumed to communicate by electrotonic conduction and graded depolarizations. This fundamental difference in operating principle may require different channel populations to regulate transmission and transmitter release. We have sampled ionic channels from the somata of two chemosensory neurons (AWA and AWC) of C. elegans. A Ca2+-activated, outwardly rectifying channel has a conductance of 67 pS and a reversal potential indicating selectivity for K+. An inwardly rectifying channel is active at potentials more negative than -50 mV. The inward channel is notably flickery even in the absence of divalent cations; this prevented determination of its conductance and reversal potential. Both of these channels were inactive over a range of membrane potentials near the likely cell resting potential; this would account for the region of very high membrane resistance observed in whole-cell recordings. A very-large-conductance (> 100 pS), inwardly rectifying channel may account for channel-like fluctuations seen in whole-cell recordings.

The functional properties of the human ether-à-go-go-like (HELK2) K+ channel.

The voltage-dependent K+ channels belonging to the ether-à-go-go family (eag, erg, elk) are widely expressed in the mammalian CNS. Their neuronal function, however, is poorly understood. Among the elk clones, elk2 is the most abundantly expressed in the brain. We have characterized the human ELK2 channel (HELK2) expressed in mammalian cell lines. Moreover, we have detected helk2 mRNA and ELK2-like currents in freshly dissociated human astrocytoma cells. HELK2 was inhibited by Cs+ in a voltage-dependent way (Kd was 0.7 mm, at -120 mV). It was not affected by Way 123398 (5 micro m), dofetilide (10 micro m), quinidine (10 micro m), verapamil (20 micro m), haloperidol (2 micro m), astemizole (1 micro m), terfenadine (1 micro m) and hydroxyzine (30 micro m), compounds known to inhibit the biophysically related HERG channel. The crossover of the activation and inactivation curves produced a steady state 'window' current with a peak around -20 mV and considerably broader than it usually is in voltage-dependent channels, including HERG. Similar features were observed in the ELK2 clone from rat, in the same experimental conditions. Thus, ELK2 channels are active within a wide range of membrane potentials, both sub- and suprathreshold. Moreover, the kinetics of channel deactivation and removal of inactivation was about one order of magnitude quicker in HELK2, compared to HERG. Overall, these properties suggest that ELK2 channels are very effective at dampening the neuronal excitability, but less so at producing adaptation of action potential firing frequency. In addition, we suggest experimental ways to recognize HELK2 currents in vivo and raise the issue of the possible function of these channels in astrocytoma.

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors.

Gating of voltage-dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage-dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation-activated cyclic nucleotide-gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation-activated currents (I(h)) can be measured only from the cell regions where HCN1 is expressed. Half-activation voltage (-75.0 +/- 0.3 mV) and kinetics (t(1/2) of 101 +/- 8 ms at -110 mV and 20 degrees C) of the I(h) in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 +/- 0.8 mV) in the half-activation voltage of I(h). In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current-to-voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.

Preferential loss of EC coupling gradation at positive membrane potentials in rat ventricular myocytes.

Rat ventricular myocytes switched from a solution containing 1 mM extracellular Ca to one containing 5 mM extracellular Ca exhibited larger Ca currents that inactivated more rapidly. Their Ca transients exhibited a slower decay. In some cells the amplitude of the Ca transients increased markedly, but not in others. Strikingly, in about half the cells examined, the graded nature of the Ca transients over the range of positive membrane potentials was lost, as were Ca transients elicited upon repolarization. These changes were manifest without equivalent loss of gradation over the range of negative membrane potentials (-35 to 0 mV). Such preferential loss of gradation at positive membrane potentials also occurred in solutions devoid of extracellular and/or intracellular Na, and was abolished by nifedipine. The results suggest that Ca-induced Ca release from the SR at positive potentials can saturate or become regenerative if the Ca entry trigger is increased. The lack of a similar effect at negative potentials indicates that large numbers of activated L-type channels are critical for induction of regenerative behavior.

Enhancement of N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials in the neostriatum after methamphetamine sensitization. An in vitro slice study.

It has been suggested that behavioral methamphetamine sensitization involves changes in cortical excitatory synaptic inputs to neostriatal (Str) projection neurons. To test this, we performed blind whole-cell recording of medium spiny neurons in Str slice preparations. In Str neurons of naive rats, the amplitude of the subcortical white matter stimulation-induced N-methyl- D-aspartate receptor-mediated excitatory postsynaptic potentials (NMDA-EPSPs) was decreased upon hyperpolarization, owing to the voltage-dependent Mg(2+) blockade of NMDA receptor channels. In contrast, the amplitude of the NMDA-EPSPs in Str neurons of rats undergoing methamphetamine withdrawal (MW) did not show the Mg(2+) blockade and was nearly voltage independent over the membrane potential range of -70 to -110 mV. Application of the specific protein kinase C (PKC) activator, phorbol 12, 13- DL-acetate, blocked the voltage-dependent Mg(2+) blockade of NMDA receptor channels in Str neurons of naive rats. Application of the specific activator of cAMP-dependent protein kinase A (PKA), Sp-cAMPS-triethylamine salt, increased the amplitude of the NMDA receptor-mediated EPSPs at the rest but not at hyperpolarized potentials. Coapplication of the PKC and PKA activators yielded NMDA-EPSPs similar to those seen in Str neurons of MW rats. In Str slices of naive rats, tetanic subcortical white matter stimulation induced long-term depression of field potentials. In Str slices treated with the PKC and/or PKA, the same stimulation induced long-term potentiation of field potentials similar to those observed in slices obtained from MW rats. These results suggest that the enhancement of the NMDA receptor-mediated corticostriatal synaptic transmission plays an important role in behavioral methamphetamine sensitization. This enhancement is probably associated with phosphorylation of NMDA receptors mediated by the simultaneous activation of PKC and PKA.

Basolateral K+ conductance establishes driving force for cation absorption by outer sulcus epithelial cells.

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of -90.4 +/- 0.7 mV (n = 78), close to the Nernst potential for K+ (-95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K+ concentration with a K+/Na+ permeability ratio of 36. The most frequently observed K+ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10-5 to 10-3 m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 microm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K+ conductance, in concert with the Na+,K+-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.

Bidirectional electrogenic transport of peptides by the proton-coupled carrier PEPT1 in Xenopus laevis oocytes: its asymmetry and symmetry.

1. The giant patch clamp technique in the inside-out configuration and the two-electrode voltage clamp technique were used to characterize the bidirectional transport properties of the proton-coupled peptide carrier PEPT1 expressed in Xenopus laevis oocytes. 2. The addition of the neutral dipeptide Gly-L-Gln to the cytoplasmic solution induced a net outward transport current in a membrane potential range between -80 and +60 mV, even in the absence of a pH gradient. 3. The concentration dependency of the outwardly directed transport currents followed Michaelis-Menten-type kinetics, with an apparent K0.5 of 3.28 mM (at pH 7.5 and +60 mV membrane potential). This apparent affinity is around fivefold lower than the apparent affinity measured for the inward transport mode (K0.5 of 0.70 mM (at pH 7.5 and -60 mV) under identical experimental conditions). 4. Apparent K0.5 values were strongly pH and potential dependent only on the external face for inward transport. The transport currents were potential dependent, but essentially pH independent for inward transport and only modestly altered by pH in the reverse direction. In addition to the membrane potential, the transmembrane substrate gradient acts as a driving force and contributes significantly to total transport currents. 5. The differences in apparent substrate affinity under identical experimental conditions suggest major differences in the conformation of the substrate binding pocket of PEPT1 when exposed to the external versus the internal face of the membrane. The lower affinity on the internal face allows the substrate to be released into the cytosolic compartment even in the absence of a proton-motive force. 6. Our study demonstrates for the first time that PEPT1 can transport dipeptides bidirectionally in an electrogenic and proton-coupled symport mode. When substrates are present on both sides of the membrane in sufficiently high concentrations, the direction and rate of transport are solely dependent on the membrane potential, and transport occurs symmetrically.

Rapidly inactivating and non-inactivating calcium-activated potassium currents in frog saccular hair cells.

1. Using a semi-intact epithelial preparation we examined the Ca(2+)-activated K(+) (K(Ca)) currents of frog (Rana pipiens) saccular hair cells. After blocking voltage-dependent K(+) (K(V)) currents with 4-aminopyridine (4-AP) an outward current containing inactivating (I(transient)) and non-inactivating (I(steady)) components remained. 2. The contribution of each varied greatly from cell to cell, with I(transient) contributing from 14 to 90 % of the total outward current. Inactivation of I(transient) was rapid (tau approximately 2-3 ms) and occurred within the physiological range of membrane potentials (V(1/2) = -63 mV). Recovery from inactivation was also rapid (tau approximately 10 ms). 3. Suppression of both I(transient) and I(steady) by depolarizations that approached the Ca(2+) equilibrium potential and by treatments that blocked Ca(2+) influx (application Ca(2+)-free saline or Cd(2+)), suggest both are Ca(2+) dependent. Both were blocked by iberiotoxin, a specific blocker of large-conductance K(Ca) channels (BK), but not by apamin, a specific blocker of small-conductance K(Ca) channels. 4. Ensemble-variance analysis showed that I(transient) and I(steady) flow through two distinct populations of channels, both of which have a large single-channel conductance (~100 pS in non-symmetrical conditions). Together, these data indicate that both I(transient) and I(steady) are carried through BK channels, one of which undergoes rapid inactivation while the other does not. 5. Inactivation of I(transient) could be removed by extracellular papain and could later be restored by intracellular application of the 'ball' domain of the auxiliary subunit (beta2) thought to mediate BK channel inactivation in rat chromaffin cells. We hypothesize that I(transient) results from the association of a similar beta subunit with some of the BK channels and that papain removes inactivation by cleaving extracellular sites required for this association.

Cannabinoid actions on rat superficial medullary dorsal horn neurons in vitro.

1. This study examined the cellular actions of cannabinoids on neurons in the substantia gelatinosa of the spinal trigeminal nucleus pars caudalis, using whole-cell and perforated patch recording in brain slices. 2. The cannabinoid agonist WIN55,212-2 (3 microM) decreased the amplitude of both GABAergic and glycinergic electrically evoked inhibitory postsynaptic currents (IPSCs) by 35 and 41 %, respectively. This inhibition was completely reversed by the CB(1) receptor-selective antagonist N-piperidino-5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) (SR141716A, 3 microM). WIN55,212-2 also produced relative facilitation of the second evoked IPSC to paired stimuli. 3. WIN55,212-2 decreased the rate of both GABAergic and glycinergic miniature IPSCs by 44 and 34 %, respectively, without changing their amplitude distributions or kinetics. 4. WIN55,212-2 did not affect the amplitude of electrically evoked non-NMDA glutamatergic excitatory postsynaptic currents (EPSCs). 5. WIN55,212-2 produced no postsynaptic membrane current and had no significant effect on membrane conductance over a range of membrane potentials (-60 to -130 mV). 6. These results suggest that, within the superficial medullary dorsal horn, cannabinoids presynaptically inhibit GABAergic and glycinergic neurotransmission. At the cellular level, the analgesic action of cannabinoids on these medullary dorsal horn neurons therefore differs from that of mu-opioids, which have both pre- and postsynaptic actions.

In Vivo Effects of Uncoupling Protein-3 Gene Disruption on Mitochondrial Energy Metabolism

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.

Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells.

In guard cells, membrane hyperpolarization in response to a blue light (BL) stimulus is achieved by the activation of a plasma membrane H(+)-ATPase. Using the patch clamp technique on broad bean (Vicia faba) guard cells we demonstrate that both steady-state- and BL-induced pump currents require ATP and are blocked by vanadate perfused into the guard cell during patch clamp recording. Background-pump current and BL-activated currents are voltage independent over a wide range of membrane potentials. During BL-activated responses significant hyperpolarization is achieved that is sufficient to promote K(+) uptake. BL activation of pump current becomes desensitized by three or four pulses of 30 s x 100 micromol m(-2) s(-1) BL. This desensitization is not a result of pump inhibition as maximal responses to fusicoccin are observed after full BL desensitization. BL treatments prior to whole cell recording show that BL desensitization is not due to washout of a secondary messenger by whole cell perfusion, but appears to be an important feature of the BL-stimulated pump response. We found no evidence for an electrogenic BL-stimulated redox chain in the plasma membrane of guard cells as no steady-state- or BL-activated currents are detected with NADH or NADPH added to the cytosol in the absence of ATP. Steady-state- nor BL-activated currents are affected by the inclusion along with ATP of 1 mM NADH in the pipette under saturating red light or by including NADPH in the pipette under darkness or saturating red light. These data suggest that reduced products of photosynthesis do not significantly modulate plasma membrane pump currents and are unlikely to be critical regulators in BL-stimulation of the plasma membrane H(+)-ATPase in guard cells.

Postnatal development of electrophysiological properties of nucleus accumbens neurons.

We have studied the postnatal development of the physiological characteristics of nucleus accumbens (nAcb) neurons in slices from postnatal day 1 (P1) to P49 rats using the whole cell patch-clamp technique. The majority of neurons (102/108) were physiologically identified as medium spiny (MS) projection neurons, and only these were subjected to detailed analysis. The remaining neurons displayed characteristics suggesting that they were not MS neurons. Around the time of birth and during the first postnatal weeks, the membrane and firing characteristics of MS neurons were quite different from those observed later. These characteristics changed rapidly during the first 3 postnatal weeks, at which point they began to resemble those found in adults. Both whole cell membrane resistance and membrane time constant decreased more than fourfold during the period studied. The resting membrane potential (RMP) also changed significantly from an average of -50 mV around birth to less than -80 mV by the end of the third postnatal week. During the first postnatal week, the current-voltage relationship of all encountered MS neurons was linear over a wide range of membrane potentials above and below RMP. Through the second postnatal week, the proportion of neurons displaying inward rectification in the hyperpolarized range increased steadily and after P15, all recorded MS neurons displayed significant inward rectification. At all ages, inward rectification was blocked by extracellular cesium and tetra-ethyl ammonium and was not changed by 4-aminopyridine; this shows that inward rectification was mediated by the same currents in young and mature MS neurons. MS neurons fired single and repetitive Na(+)/K(+) action potentials as early as P1. Spike threshold and amplitude remained constant throughout development in contrast to spike duration, which decreased significantly over the same period. Depolarizing current pulses from rest showed that immature MS neurons fired action potentials more easily than their older counterparts. Taken together, the results from the present study suggest that young and adult nAcb MS neurons integrate excitatory synaptic inputs differently because of differences in their membrane and firing properties. These findings provide important insights into signal processing within nAcb during this critical period of development.

Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60(c-src) kinase activity.

1. Tyrosine kinases have been proposed as regulators of voltage-operated calcium channels. The effects of a range of structurally different inhibitors of protein tyrosine kinases (PTK) were examined on voltage-operated calcium channel currents (I(Ba)) and pp60(c-src) kinase (c-src) activity in vitro. 2. I(Ba) was measured in single myocytes isolated from rabbit ear artery by conventional whole cell voltage-clamp techniques. The activity of purified human c-src was measured in vitro using a non-radioactive assay. 3. Bath application of tyrphostin-23 and genistein (non-selective PTK inhibitors), bistyrphostin (a receptor-PTK-selective inhibitor) and PP1 (a src family-selective inhibitor) inhibited I(Ba) in a concentration-dependent manner over a range of test membrane potentials. Intracellular application of peptide-A, a peptide inhibitor of c-src also inhibited currents. Inhibitor potency series against I(Ba) was PP1 > genistein > tyrphostin 23 > bistyrphostin. 4. Tyrphostin-23, genistein, PP1, and peptide-A shifted the steady-state inactivation curves in a hyperpolarized direction without altering their slope. The inhibitors had no significant effects on I(Ba) activation calculated from current-voltage relationships. 5. The agents inhibited c-src activity in a concentration-dependent manner. The order of potency was PP1 > genistein > peptide-A > tyrphostin-23 > bistyrphostin. The IC(50) for inhibition of c-src activity was similar to the IC(50) for inhibition of I(Ba) in all cases. 6. Western blot analysis with a specific antibody to c-src showed the presence of this cytoplasmic tyrosine kinase in rabbit ear artery cells. 7. A range of structurally dissimilar inhibitors of PTKs inhibit I(Ba) and c-src activity with similar potency. These data provide further evidence implicating endogenous c-src in the modulation of L-type calcium channels in vascular smooth muscle cells.

Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea‐pig gastric pylorus

1. Intracellular recordings made in single bundle strips of a visceral smooth muscle revealed rhythmic spontaneous membrane depolarizations termed slow waves (SWs). These exhibited 'pacemaker' and 'regenerative' components composed of summations of more elementary events termed spontaneous transient depolarizations (STDs). 2. STDs and SWs persisted in the presence of tetrodotoxin, nifedipine and ryanodine, and upon brief exposure to Ca2+-free Cd2+-containing solutions; they were enhanced by ACh and blocked by BAPTA AM, cyclopiazonic acid and caffeine. 3. SWs were also inhibited in heparin-loaded strips. SWs were observed over a wide range of membrane potentials (e.g. -80 to -45 mV) with increased frequencies at more depolarized potentials. 4. Regular spontaneous SW activity in this preparation began after 1-3 h superfusion of the tissue with physiological saline following the dissection procedure. Membrane depolarization applied before the onset of this activity induced bursts of STD-like events (termed the 'initial' response) which, when larger than threshold levels initiated regenerative responses. The combined initial-regenerative waveform was termed the SW-like action potential. 5. Voltage-induced responses exhibited large variable latencies (typical range 0.3-4 s), refractory periods of approximately 11 s and a pharmacology that was indistinguishable from those of STDs and spontaneous SWs. 6. The data indicate that SWs arise through more elementary inositol 1,4,5-trisphosphate (IP3) receptor-induced Ca2+ release events which rhythmically synchronize to trigger regenerative Ca2+ release and induce inward current across the plasmalemma. The finding that action potentials, which were indistinguishable from SWs, could be evoked by depolarization suggests that membrane potential modulates IP3 production. Voltage feedback on intracellular IP3-sensitive Ca2+ release is likely to have a major influence on the generation and propagation of SWs.

Active dendritic membrane properties of Xenopus larval spinal neurons analyzed with a whole cell soma voltage clamp.

Voltage- and current-clamp measurements of inwardly directed currents were made from the somatic regions of Xenopus laevis spinal neurons. Current-voltage (I-V) curves determined under voltage clamp, but not current clamp, were able to indicate a negative slope conductance in neurons that showed strong accommodating action potential responses to a constant current stimulation. Voltage-clamp I-V curves from repetitive firing neurons did not have a net negative slope conductance and had identical I-V plots under current clamp. Frequency domain responses indicate negative slope conductances with different properties with or without tetrodotoxin, suggesting that both sodium and calcium currents are present in these spinal neurons. The currents obtained from a voltage clamp of the somatic region were analyzed in terms of spatially controlled soma membrane currents and additional currents from dendritic potential responses. Linearized frequency domain analysis in combination with both voltage- and current-clamp responses over a range of membrane potentials was essential for an accurate determination of consistent neuronal model behavior. In essence, the data obtained at resting or hyperpolarized membrane potentials in the frequency domain were used to determine the electrotonic structure, while both the frequency and time domain data at depolarized potentials were required to characterize the voltage-dependent channels. Finally, the dendritic and somatic membrane properties were used to reconstruct the action potential behavior and quantitatively predict the dependence of neuronal firing properties on electrotonic structure. The reconstructed action potentials reproduced the behavior of two broad distributions of interneurons characterized by their degree of accommodation. These studies suggest that in addition to the ionic conductances, electrotonic structure is correlated with the action potential behavior of larval neurons.

Subthreshold inward membrane currents in guinea-pig frontal cortex neurons

Current-clamp and single-electrode voltage-clamp recordings were used to study the inward currents activated in the subthreshold membrane potential range of cortical pyramidal neurons. The experiments were done on slices from guinea-pig frontal cortex and all recordings were obtained at a distance of 600–900 μm from the pial surface. In current-clamp recordings and from membrane potentials hyperpolarized to about −70 mV, the depolarization leading to spike firing was partially blocked by 1 μM tetrodotoxin, but not by calcium-free extracellular solution. The calcium-free solution only affected this depolarization when the membrane potential was held at a level more negative than −75 mV. Under voltage-clamp, an inward current was recorded between the resting membrane potential and the level of spike firing. This current was activated at about −60 mV and part of it was blocked by 1 μM tetrodotoxin; the remaining current was blocked by calcium-free extracellular solution. In five neurons both components were recorded and isolated in the same cell. The tetrodotoxin-sensitive component activated at close to −60 mV, was similar to the persistent sodium current (I Na-p). The Ca 2+-sensitive component activated at close to −60 or −65 mV, was less voltage-dependent than I Na-p. This component was similar to the low threshold calcium current (I T). These results suggest that the subthreshold depolarization which led to spike firing was dependent on I Na-p and I T, I Na-p being the most important factor up to resting membrane potentials of −70 or −75 mV. A physiological role of this finding is revealed by the action of dopamine, which (at 10 μM) prevented the firing of action potentials from −60 mV, but not from −80 mV due to the inhibition of I Na-p and the lack of effect on I T.

A patch-clamp study of ion channels in protoplasts prepared from the marine alga Valonia utricularis.

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.