bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of tetraethylammonium ion-resistant K+ channels in the photoreceptor membrane of the giant barnacle.
Authors: Edgington D R , Stuart A E
Published: May 2004

After the offset of illumination, barnacle photoreceptors undergo a large hyperpolarization that lasts seconds or minutes. We studied the mechanisms that generate this afterpotential by recording afterpotentials intracellularly from the medial photoreceptors of the giant barnacle Balanus nubilus. The afterpotential has two components with different time-courses: (a) an earlier component due to an increase in conductance to K+ that is not blocked by extracellular tetraethylammonium ion (TEA+) or 3-aminopyridine (3-AP) and (b) a later component that is sensitive to cardiac glycosides and that requires extracellular K+, suggesting that it is due to an electrogenic Na+ pump. The K+ conductance component increases in amplitude with increasing CA++ concentration and is inhibited by extracellular Co++; the Co++ inhibition can be overcome by increasing the Ca++ concentration. Thus, the K+ conductance component is Ca++ dependent. An afterpotential similar to that evoked by a brief flash of light is generated by depolarization with current in the dark and by eliciting Ca++ action potentials in the presence of TEA+ in the soma, axon, or terminal regions of the photoreceptor. The action potential undershoot is generated by an increase in conductance to K+ that is resistant to TEA+ and 3-AP and inhibited by Co++. The similarity in time-course and pharmacology of the hyperpolarization afterpotentials elicited by (a) a brief flash of light, (b) depolarization with current, and (c) an action potential indicates that Ca++-dependent K+ channels throughout the photoreceptor membrane are responsible for all three hyperpolarizing events.
Analyses of bipolar cell responses elicited by polarization of horizontal cells.
Authors: Toyoda J , Kujiraoka T
Published: May 2004

Simultaneous intracellular recordings were made from a bipolar cell and a horizontal cell in the carp retina. The properties of the bipolar cell were studied while injecting current into the horizontal cell. Hyperpolarization of horizontal cells, irrespective of their type, elicited a hyperpolarizing response in on-center bipolar cells and a depolarizing response in off-center bipolar cells. Analyses of the ionic mechanisms of bipolar cell responses revealed that depolarization of horizontal cells simulated and hyperpolarization opposed the effect of central illumination. The effect of polarization was exerted in such a manner that each type of horizontal cells modified the transmission from those photoreceptors from which they receive main inputs. In on-center bipolar cells, for example, the L-type horizontal cells receiving inputs mainly from red cones modified the cone-bipolar transmission accompanied by a conductance change of K+ and/or Cl- channels, and the intermediate horizontal cells receiving inputs from rods modified the rod-bipolar transmission accompanied by a conductance change of Na+ channels. In off-center bipolar cells, the effect of polarization of any type of horizontal cells was mediated mainly by conductance changes of Na+ channels. Feedback mechanisms from horizontal cells to photoreceptors could explain these results reasonably well.
On the ionic mechanism underlying adrenergic-cholinergic antagonism in ventricular muscle.
Authors: Josephson I , Sperelakis N
Published: May 2004

In atrial muscle, acetylcholine (ACh) decreases the slow inward current (Isi) and increases the time-independent outward K+ current. However, in ventricular muscle, ACh produces a marked negative inotropic effect only in the presence of positive inotropic agents that elevate cyclic adenosine monophosphate (AMP). A two-microelectrode voltage-clamp method was used on cultured reaggregates of cells from 16--20-d-old embryonic chick ventricles to determine the effects of ACh on Isi and outward current during beta-adrenergic stimulation. Only double penetrations displaying low-resistance coupling were voltage-clamped. Cultured reaggregates are advantageous because their small size (50--250 microns) permits better control of membrane potential and adequate space clamp. Tetrodotoxin (10(-6) M) and a holding potential of --50 to --40 mV were used to eliminate the fast Na+ current. Depolarizing voltage steps above --40 mV caused a slow inward current to flow that was sensitive to changes in [Ca]o and was depressed by verapamil (10(-6) M). Maximal Isi was obtained at --10 mV and the reversal potential was about +25 mV. Isoproterenol (10(-6) M) increased Isi at all clamp potentials. Subsequent addition of ACh (10(-6) M) rapidly reduced Isi to control values (before isoproterenol) without a significant effect on the net outward current measured at 300 ms. The effects of ACh were reversed by muscarinic blockade with atropine (5 X 10(-6) M). We conclude that the anti-adrenergic effects of ACh in ventricular muscle are mediated by a reduction in Ca2+ influx during excitation.
Volume regulation by human lymphocytes. Role of calcium.
Authors: Grinstein S , Dupre A , Rothstein A
Published: May 2004

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++-containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).
Functional stoichiometry at the nicotinic receptor. The photon cross section for phase 1 corresponds to two bis-Q molecules per channel.
Authors: Sheridan R E , Lester H A
Published: May 2004

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q. A 1-microsecond laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans leads to cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes. At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule.
The periaxonal space of crayfish giant axons.
Authors: Shrager P , Starkus J C , Lo M V , Peracchia C
Published: May 2004

The influence of the glial cell layer on effective external ion concentrations has been studied in crayfish giant axons. Excess K ions accumulate in the periaxonal space during outward K+ current flow, but at a rate far below that expected from the total ionic flux and the measured thickness of the space. At the conclusion of outward current flow, the external K+ concentration returns to normal in an exponential fashion, with a time constant of approximately 2 ms. This process is about 25 times faster than is the case in squid axons. K+ repolarization (tail) currents are generally biphasic at potentials below about -40 mV and pass through a maximum before approaching a final asymptotic level. The initial rapid phase may in part reflect depletion of excess K+. After block of inactivation and reversal of the Na+ concentration gradient, we could demonstrate accumulation and washout of excess Na ions in the periaxonal space. Characteristics of these processes appeared similar to those of K+. Crayfish glial cell ultrastructure has been examined both in thin sections and after freeze fracture. Layers of connective tissue and extracellular fluid alternate with thin layers of glial cytoplasm. A membranous tubular lattice, spanning the innermost glial layers, may provide a pathway allowing rapid diffusion of excess ions from the axon surface.
An optical determination of the series resistance in Loligo.
Authors: Salzberg B M , Bezanilla F
Published: May 2004

The resistance in series with the membrane capacitance in the giant axon of the squid Loligo pealei was measured using potentiometric probes that exhibit absorbance changes proportional to the voltage across the plasma membrane proper. The method relies upon the fact that a voltage drop across the series resistance produces a deviation in the true transmembrane voltage from that imposed by a voltage clamp. Optical measurement of the true transmembrane potential, together with electrical measurement of the ionic current, permits the immediate determination of the series resistance by Ohm's law. An alternative method monitored the amount of electronic series resistance compensation required to force the optical signal to match the shape of the reference potential. The value of the series resistance measured in artificial seawater was 3.78 +/- 0.95 omega X cm2. The estimated value of the contribution of the Schwann cell layer to the series resistance was 2.57 +/- 0.89 omega X cm2.
Theoretical effects of transmembrane electroneutral exchange on membrane potential.
Authors: Jacob R , Piwnica-Worms D , Horres C R , Lieberman M
Published: May 2004

Transmembrane electroneutral transport mechanisms [e.g., Na/H exchange, Cl/HCO3 exchange, (K + Cl) cotransport] have recently been identified in a wide variety of cell types. If these exchanges sum to give a net electroneutral Na/K exchange, they may hyperpolarize the membrane potential beyond the value calculated from the Mullins-Noda equation, provided the cell maintains steady state intracellular ionic concentrations. In extreme circumstances, the membrane potential could hyperpolarize beyond the potassium reversal potential. This effect is mediated by the electrogenic Na/K pump. If either Na or K exchanges electroneutrally against a third ion (e.g., Na/Ca exchange), then the exchange may depolarize the membrane potential.
The active ion transport properties of canine lingual epithelia in vitro. Implications for gustatory transduction.
Authors: Desimone J A , Heck G L , Mierson S , Desimone S K
Published: May 2004

The electrophysiological properties of the dorsal and ventral canine lingual epithelium are studied in vitro. The dorsal epithelium contains a special ion transport system activated by mucosal solutions hyperosmotic in NaCl or LiCl. Hyperosmotic KCl is significantly less effective as an activator of this system. The lingual frenulum does not contain the transport system. In the dorsal surface it is characterized by a rapid increase in inward current and can be quantitated as a second component in the time course of either the open-circuit potential or short-circuit current when the mucosal solution is hyperosmotic in NaCl or LiCl. The increased inward current (hyperosmotic response) can be eliminated by amiloride (10(-4) M). The specific location of this transport system in the dorsal surface and the fact that it operates over the concentration range characteristic of mammalian salt taste suggests a possible link to gustatory transduction. This possibility is tested by recording neural responses in the rat to NaCl and KCl over a concentration range including the hyperosmotic. We demonstrate that amiloride specifically blocks the response to NaCl over the hyperosmotic range while affecting the KCl response significantly less. The results suggest that gustatory transduction for NaCl is mediated by Na entry into the taste cells via the same amiloride-sensitive pathway responsible for the hyperosmotic response in vitro. Further studies of the in vitro system give evidence for paracellular as well as transcellular current paths. The transmural current-voltage relations are linear under both symmetrical and asymmetrical conditions. After ouabain treatment under symmetrical conditions, the short-circuit current decays to zero. The increase in resistance, though significant, is small, which suggests a sizeable shunt pathway for current. Flux measurements show that sodium is absorbed under symmetrical conditions. Mucosal solutions hyperosmotic in various sugars also induce an amiloride-sensitive inward current. In summary, this work provides evidence that the sodium taste receptor is most probably a sodium transport system, specifically adapted to the dorsal surface of the tongue. The transport paradigm of gustation also suggests a simple model for electric taste and possible mechanisms for sweet taste.
Frog rod outer segments with attached inner segment ellipsoids as an in vitro model for photoreceptors on the retina.
Authors: Biernbaum M S , Bownds M D
Published: May 2004

Purified suspensions of frog rod outer segments still attached to the mitochondria-rich inner segment portion of the receptor cell (OS-IS) can be obtained in quantities (0.1 mg/retina) sufficient for chemical analysis. In oxygenated glucose-bicarbonate Ringer's medium with added Percoll, they display normal dark currents, light sensitivity, and photocurrent kinetics for several hours. Two millimolar cytoplasmic levels of ATP and GTP are maintained, fivefold higher than in isolated OS. The levels are not altered by abolition of the dark current with ouabain. Nucleoside triphosphates are more effectively buffered than in isolated OS, and their levels remain constant during changes in external calcium levels. 32Pi is incorporated into endogenous ATP and GTP pools twice as efficiently as in isolated OS, and is used in the phosphorylation of rhodopsin. OS-IS take up and release 45Ca++ by Na+-, Ca++-, and IBMX-sensitive mechanisms. Illumination causes release of 45Ca++, which confirms retinal studies by other groups using Ca++-sensitive electrodes. Thus, OS-IS suspensions model the behavior of photoreceptors still attached to the living retina. Their availability permits the simultaneous assay and correlation of electrophysiological and chemical changes occurring during excitation and adaptation.
Permeation and interaction of divalent cations in calcium channels of snail neurons.
Authors: Byerly L , Chase P B , Stimers J R
Published: May 2004

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.
Circadian regulation of retinomotor movements. I. Interaction of melatonin and dopamine in the control of cone length.
Authors: Pierce M E , Besharse J C
Published: May 2004

In lower vertebrates, cone retinomotor movements occur in response to changes in lighting conditions and to an endogenous circadian clock. In the light, cone myoids contract, while in the dark, they elongate. In order to test the hypothesis that melatonin and dopamine may be involved in the regulation of cone movement, we have used an in vitro eyecup preparation from Xenopus laevis that sustains light- and dark-adaptive cone retinomotor movement. Melatonin mimics darkness by causing cone elongation. Dark- and melatonin-induced cone elongation are blocked by dopamine. Dopamine also stimulates cone contraction in dark-adapted eyecups. The effect of dopamine appears to be mediated specifically by a dopamine receptor, possibly of the D2 type. The dopamine agonist apomorphine and the putative D2 agonist LY171555 induced cone contraction. In contrast, the putative D1 agonist SKF38393-A and specific alpha 1-, alpha 2-, and beta-adrenergic receptor agonists were without effect. Furthermore, the dopamine antagonist spiroperidol not only blocked light-induced cone contraction, but also stimulated cone elongation in the light. These results suggest that dopamine is part of the light signal for cone contraction, and that its suppression is part of the dark signal for cone elongation. Melatonin may affect cone movement indirectly through its influence on the dopaminergic system.
Dynamics of cockroach ocellar neurons.
Authors: Mizunami M , Tateda H , Naka K
Published: May 2004

The incremental responses from the second-order neurons of the ocellus of the cockroach, Periplaneta americana, have been measured. The stimulus was a white-noise-modulated light with various mean illuminances. The kernels, obtained by cross-correlating the white-noise input against the resulting response, provided a measure of incremental sensitivity as well as of response dynamics. We found that the incremental sensitivity of the second-order neurons was an exact Weber-Fechner function; white-noise-evoked responses from second-order neurons were linear; the dynamics of second-order neurons remain unchanged over a mean illuminance range of 4 log units; the small nonlinearity in the response of the second-order neuron was a simple amplitude compression; and the correlation between the white-noise input and spike discharges of the second-order neurons produced a first-order kernel similar to that of the cell's slow potential. We conclude that signal processing in the cockroach ocellus is simple but different from that in other visual systems, including vertebrate retinas and insect compound eyes, in which the system's dynamics depend on the mean illuminance.
Inhibition of the Na,K pump by vanadate in high-Na solutions. Modification of the reaction mechanism by external Na acting at a high-affinity site.
Authors: Sachs J R
Published: May 2004

We have examined vanadate inhibition of the Na,K pump in the presence of external Na (Nao). Nao protects against inhibition of the Na,K pump by vanadate, but not against inhibition by phosphate or arsenate. Protection by Nao is reversed by external K (Ko). Although the site at which Na exerts its protective effect has properties similar to the two transport sites for K at the outside of the pump, it is not one of the transport sites. The data can be qualitatively accounted for if it is postulated that there is a protective site, separate from the transport sites, at which Nao and Ko compete. When the site is empty or bound to K, vanadate combines with high affinity with pumps that have two K ions bound to the transport sites, but not with pumps that have Na bound to the protective site, even if K is bound to the transport sites. The protective site has a high affinity for both Na and K; the apparent K 1/2 for external Na is less than 2 mM, which is similar to that of a previously described site at which Nao inhibits a number of the partial reactions of the pump. Nao protects against vanadate inhibition of the K-K exchange in the absence of cell Na, and against vanadate inhibition of p-nitrophenylphosphatase activity of the pump in the absence of ATP. The protective site is a manifestation of an E2 conformation of the pump. The protective effect of Nao is not changed by altering the intracellular Mg2+ concentration.
"Imposed" and "inherent" mucosal activity patterns. Their composite representation of olfactory stimuli.
Authors: Mozell M M , Sheehe P R , Hornung D E , Kent P F , Youngentob S L , Murphy S J
Published: May 2004

Both regional differences in mucosal sensitivity and a gas chromatography-like process along the mucosal sheet have been separately proposed in two sets of earlier studies to produce different odorant-dependent activity patterns across the olfactory mucosa. This investigation evaluated, in one study, whether and to what degree these two mechanisms contribute to the generation of these activity patterns. Summated multiunit discharges were simultaneously recorded from lateral (LN) and medial (MN) sites on the bullfrog's olfactory nerve to sample the mucosal activity occurring near the internal and external nares, respectively. Precisely controlled sniffs of four odorants (benzaldehyde, butanol, geraniol, and octane) were drawn through the frog's olfactory sac in both the forward (H1) and reverse (H2) hale directions. By combining the four resulting measurements, LNH1, LNH2, MNH1, and MNH2, in different mathematical expressions, indexes reflecting the relative effects of the chromatographic process, regional sensitivity, and hale direction could be calculated. Most importantly, the chromatographic process and the regional sensitivity differences both contributed significantly to the mucosal activity patterns. However, their relative roles varied markedly among the four odorants, ranging from complete dominance by either one to substantial contributions from each. In general, the more strongly an odorant was sorbed by the mucosa, the greater was the relative effect of the chromatographic process; the weaker the sorption, the greater the relative effect of regional sensitivity. Similarly, the greater an odorant's sorption, the greater was the effect of hale direction. Other stimulus variables (sniff volume, sniff duration, and the number of molecules within the sniff) had marked effects upon the overall size of the response. For strongly sorbed odorants, the effect of increasing volume was positive; for a weakly sorbed odorant, it was negative. The reverse may be true for duration. In contrast, the effect of increasing the number of molecules was uniformly positive for all four odorants. However, there was little evidence that these other stimulus variables had a major influence upon the effects of the chromatographic process and regional sensitivity differences in their generation of mucosal activity patterns.
Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength.
Authors: Anderson C S , MacKinnon R , Smith C , Miller C
Published: May 2004

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.
Differential effects of tetracaine on charge movements and Ca2+ signals in frog skeletal muscle.
Authors: Csernoch L , Huang C L , Szucs G , Kovacs L
Published: May 2004

The effects of tetracaine on charge movements and on antipyrylazo III signals monitoring intracellular delta [Ca2+] were compared in cut frog semitendinosus muscle fibers in a single vaseline gap-voltage clamp. Low tetracaine concentrations (25-40 microM) markedly reduced delta [Ca2+] signals and shifted the rheobase. However, they neither influenced charge movement nor that peak delta [Ca2+] value associated with the contractile threshold. Higher tetracaine concentrations (100-200 microM) partly inhibited charge movements in cut fibers. They separated a steeply voltage-sensitive charge, some of whose features resembled 'q gamma' reported in intact fibers, and whose movement preceded delta [Ca2+] signals at threshold. These findings: (a) directly confirm an earlier suggestion that tetracaine acts on steps in excitation-contraction coupling rather than myofilament activation; (b) show that tetracaine at low concentrations can directly interfere with sarcoplasmic reticular calcium release without modifying charge movement; (c) show that the tetracaine-sensitive charge, first found in intact fibers, also exists in cut fibers; and (d) make it unlikely that tetracaine-sensitive charge transfer is a consequence of Ca2+ release as suggested on earlier occasions.
Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents.
Authors: Papke R L , Oswald R E
Published: May 2004

The functional mechanisms of noncompetitive blockade of the nicotinic acetylcholine receptor from the BC3H-1 cell line were examined using single-channel currents recorded from cell-attached patches. Channel open times were distributed as sums of two exponentials and the closed times as sums of at least four exponentials. The single-channel currents of the receptor were analyzed in terms of activation schemes in which the receptor exists in two open states and a number of closed or blocked states. The existence of two distinct open states for the acetylcholine receptor allows for predictions to be made that will distinguish between different mechanisms of blockade. Notably, predictions could be made based on the model for the sequential block of open channels, that would allow us to discriminate such a mechanism, even for ligands that appear to dissociate so slowly that sequential openings of the same channel do not appear as distinct bursts. Four noncompetitive blockers of the acetylcholine receptor were studied: tetracaine, phencyclidine, and the (+) and (-) isomers of N-allylnormetazocine (SKF-10047). All four of these ligands decreased the duration of single-channel currents without increasing the number of fast closures per burst. The data suggest that the ligands block the channel in at least two distinct ways, one of which involves a specific interaction with open channels and the other is most consistent with the blockade of channels that may be either open or closed. In addition, the duration of the open state may be allosterically lengthened by the interaction of certain blockers with another class of sites.
Ionic channels of the inner segment of tiger salamander cone photoreceptors.
Authors: Barnes S , Hille B
Published: May 2004

Cone photoreceptors were isolated enzymatically and their ionic currents studied by the whole-cell, gigaseal voltage-clamp technique. Five nonsynaptic currents were identified. A prominent, poorly selective cation current, Ih, activated after a delay during hyperpolarizations and then deactivated with a delay on return to potentials greater than -50 mV. An empirical model for Ih gating kinetics is developed with three open and two closed states. Depolarization elicits a small, voltage-gated calcium current (ICa). Block by nitrendipine, nickel, cadmium, and cobalt, increase of current with barium, lack of rapid inactivation, and relatively high threshold suggest an L-type Ca channel. No evidence was found for low-threshold Ca channels. An anion current ICl(Ca) was present after pulses that led to a significant inward ICa (but not IBa) and was not elicited when cobalt was present. Tails of ICl(Ca) were short (100 ms) after short depolarizations and were longer after longer depolarizations. Two TEA-sensitive K currents were also elicited by depolarizations. One, IK(Ca), was calcium sensitive. We looked for modulation of Ih, ICa, and ICl(Ca) by a number of neurotransmitters. No changes of Ih were seen, but ICa and ICl(Ca) were depressed in a few cones when GABA or adenosine were applied. We discuss how this modulation might contribute to the feedback effects of horizontal cells on cones when surrounding cones are illuminated.
Inactivation of A currents and A channels on rat nodose neurons in culture.
Authors: Cooper E , Shrier A
Published: May 2004

Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10-40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation.
Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog.
Authors: Cota G , Stefani E
Published: May 2004

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.
Cytosolic pH regulation in osteoblasts. Regulation of anion exchange by intracellular pH and Ca2+ ions.
Authors: Green J , Yamaguchi D T , Kleeman C R , Muallem S
Published: May 2004

Measurements of cytosolic pH (pHi) 36Cl fluxes and free cytosolic Ca2+ concentration ([Ca2+]i) were performed in the clonal osteosarcoma cell line UMR-106 to characterize the kinetic properties of Cl-/HCO3- (OH-) exchange and its regulation by pHi and [Ca2+]i. Suspending cells in Cl(-)-free medium resulted in rapid cytosolic alkalinization from pHi 7.05 to approximately 7.42. Subsequently, the cytosol acidified to pHi 7.31. Extracellular HCO3- increased the rate and extent of cytosolic alkalinization and prevented the secondary acidification. Suspending alkalinized and Cl(-)-depleted cells in Cl(-)-containing solutions resulted in cytosolic acidification. All these pHi changes were inhibited by 4',4',-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) and H2DIDS, and were not affected by manipulation of the membrane potential. The pattern of extracellular Cl- dependency of the exchange process suggests that Cl- ions interact with a single saturable external site and HCO3- (OH-) complete with Cl- for binding to this site. The dependencies of both net anion exchange and Cl- self-exchange fluxes on pHi did not follow simple saturation kinetics. These findings suggest that the anion exchanger is regulated by intracellular HCO3- (OH-). A rise in [Ca2+]i, whether induced by stimulation of protein kinase C-activated Ca2+ channels, Ca2+ ionophore, or depolarization of the plasma membrane, resulted in cytosolic acidification with subsequent recovery from acidification. The Ca2+-activated acidification required the presence of Cl- in the medium, could be blocked by DIDS, and H2DIDS and was independent of the membrane potential. The subsequent recovery from acidification was absolutely dependent on the initial acidification, required the presence of Na+ in the medium, and was blocked by amiloride. Activation of protein kinase C without a change in [Ca2+]i did not alter pHi. Likewise, in H2DIDS-treated cells and in the absence of Cl-, an increase in [Ca2+]i did not activate the Na+/H+ exchanger in UMR-106 cells. These findings indicate that an increase in [Ca2+]i was sufficient to activate the Cl-/HCO3- exchanger, which results in the acidification of the cytosol. The accumulated H+ in the cytosol activated the Na+/H+ exchanger. Kinetic analysis of the anion exchange showed that at saturating intracellular OH-, a [Ca2+]i increase did not modify the properties of the extracellular site. A rise in [Ca2+]i increased the apparent affinity for intracellular OH- (or HCO3-) of both net anion and Cl- self exchange. These results indicate that [Ca2+]i modifies the interaction of intracellular OH- (or HCO3-) with the proposed regulatory site of the anion exchanger in UMR-106 cells.
ATP-induced pore formation in the plasma membrane of rat peritoneal mast cells.
Authors: Tatham P E , Lindau M
Published: May 2004

We have investigated the ATP-induced permeabilization of rat peritoneal mast cells using three different techniques: (a) by measuring uptake of fluorescent membrane and DNA marker dyes, (b) by voltage-clamp measurements using the patch-clamp technique, and (c) by measurements of exocytosis in response to entry of Ca2+ and GTP gamma S into permeabilized cells. In the absence of divalent cations cells become highly permeable at ATP concentrations as low as 3 microM. In normal saline containing 1 mM MgCl2 and 2 mM CaCl2, dye uptake and electric conductance are detectable at 100 microM ATP corresponding to 4 microM ATP4-. The permeabilization is half-maximal at an ATP4- concentration of 5-20 microM with a Hill coefficient near 2. The ATP-induced whole-cell conductance at saturating ATP concentrations was 35-70 nS, exhibiting only weak cation selectivity. The activation is very fast with a time constant less than or equal to 65 ms. Pores which are large enough to allow for permeation of substances of 300-900 D are expected to have a unit conductance of approximately 200-400 pS. However, in whole cells as well as outside-out patches, discrete openings and closings of channels could not be observed at a resolution of approximately 40 pS and the single-channel conductance obtained from noise analysis is approximately 2-10 pS. Entry of Ca2+ into cells permeabilized with ATP stimulates exocytosis at low but not at high ATP concentrations indicating loss of an essential intracellular component or components at a high degree of permeabilization. This inactivation is removed when GTP gamma S is provided in the medium and this leads to enhanced exocytosis. The enhancement only occurs at high ATP concentrations. These results strongly suggest that the ATP-induced pores are of variable size and can increase or decrease by very small units.
Signal transmission through the dark-adapted retina of the toad (Bufo marinus). Gain, convergence, and signal/noise.
Authors: Copenhagen D R , Hemilä S , Reuter T
Published: May 2004

Responses to light were recorded from rods, horizontal cells, and ganglion cells in dark-adapted toad eyecups. Sensitivity was defined as response amplitude per isomerization per rod for dim flashes covering the excitatory receptive field centers. Both sensitivity and spatial summation were found to increase by one order of magnitude between rods and horizontal cells, and by two orders of magnitude between rods and ganglion cells. Recordings from two hyperpolarizing bipolar cells showed a 20 times response increase between rods and bipolars. At absolute threshold for ganglion cells (Copenhagen, D.R., K. Donner, and T. Reuter. 1987. J. Physiol. 393:667-680) the dim flashes produce 10-50-microV responses in the rods. The cumulative gain exhibited at each subsequent synaptic transfer from the rods to the ganglion cells serves to boost these small amplitude signals to the level required for initiation of action potentials in the ganglion cells. The convergence of rod signals through increasing spatial summation serves to decrease the variation of responses to dim flashes, thereby increasing the signal-to-noise ratio. Thus, at absolute threshold for ganglion cells, the convergence typically increases the maximal signal-to-noise ratio from 0.6 in rods to 4.6 in ganglion cells.
Membrane capacitance in frog cut twitch fibers mounted in a double vaseline-gap chamber.
Authors: Chandler W K , Hui C S
Published: May 2004

In experiments on cut muscle fibers mounted in a double Vaseline-gap chamber, electrical measurements are usually made by measuring the voltage V1(t) in one end pool and by passing current I2(t) from the other end pool to the central pool, which is usually clamped to earth potential. The voltage in the current-passing end pool is denoted by V2(t). This article describes how the value of the holding current, Ih, and the values of delta V2(infinity)/delta V1(infinity) and delta I2(infinity)/delta V1(infinity) that are associated with a small change in V1(t) can be used to estimate the linear cable parameters rm, ri, and re in a cut fiber that has been equilibrated with a Cs-containing internal solution. rm, ri, and re represent, respectively, the resistance of the plasma membranes, the internal longitudinal resistance, and the external longitudinal resistance under the Vaseline seals, all for a unit length of fiber. The apparent capacitance, Capp, of the preparation is defined to equal integral of infinity 0 delta I2,tr(t) dt/delta V1(infinity), in which delta I2,tr(t) represents the transient component of current that is associated with a change in V1(t) of amplitude delta V1(infinity). A method is described to estimate cm, the capacitance of the plasma membranes per unit length of fiber, from Capp and the values of rm, ri, and re. In experiments carried out with a tetraethylammonium chloride (TEA.Cl) solution at 13-14 degrees C in the central pool, cm remained stable for as long as 3-4 h. The values of cm, 0.19 microF/cm on average, and their variation with fiber diameter are similar to published results from intact fibers. This article also describes the different pathways that are taken by the current that flows from the current-passing end pool to the central pool. Approximately two-thirds of delta I2,tr(t) flows across the capacitance of the plasma membranes in the central-pool region. The rest flows either across plasma membranes that are under the two Vaseline seals or directly from the current-passing end pool to the central pool, across the external longitudinal resistance under the Vaseline seal. [There is also a current that flows directly from the voltage-measuring end pool to the central pool but this does not contribute to delta I2,tr(t).]
The mechanism of rectification of iK1 in canine Purkinje myocytes.
Authors: Oliva C , Cohen I S , Pennefather P
Published: May 2004

We have characterized the inward rectifying background potassium current, iK1, of canine cardiac Purkinje myocytes in terms of its reversal potential, voltage activation curve, and "steady-state" current-voltage relation. The latter parameter was defined from the difference current between holding currents in the presence and absence of 20 mM cesium. Our data suggest that iK1 rectification does not arise exclusively from voltage-dependent gating or exclusively from voltage-dependent blockade by internal magnesium ions. The voltage activation curve constructed from tail currents fit to a Boltzmann two-state model predicts less outward current than is actually observed. The magnesium-dependent rectification due to channel blockade is too fast to account for the time-dependent gating of iK1 that gives rise to the tail currents. We propose a new model of rectification that assumes that magnesium blockade of the channel occurs simultaneously with voltage-dependent gating. The new model incorporates the kinetic schema elaborated by Matsuda, H. (1988. J. Physiol. 397:237-258) to explain the appearance of subconducting states of the iK1 channel in the presence of blocking ions. That schema suggested that iK1 channels were composed of three parallel pores, each of which could be blocked independently. In our model we considered the consequences of partial blockade of the channel. If the channels are partially blocked at potentials where normally they are mostly gated closed, and if the partially blocked channels cannot close, then blockade will have the paradoxical result of enhancing the current carried by iK1.
Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts. Single channel current kinetics reveal distinct agonist binding affinities.
Authors: Sine S M , Claudio T , Sigworth F J
Published: May 2004

The experiments described examine single channel currents recorded through Torpedo acetylcholine receptor channels stably expressed by a mouse fibroblast cell line. Closed-duration histograms were constructed from currents elicited by 0.5-300 microM acetylcholine (ACh). The concentration dependence of closed durations is well described by a four-state linear scheme with the addition of open-channel block by ACh. Analysis of closed durations measured at low concentrations gives estimates of the rate of opening of doubly liganded receptors, beta, the rate of dissociation of ACh from doubly liganded receptors, k-2, and the rate of channel closing, alpha. The rate of ACh dissociation from singly liganded receptors, k-1, is then deduced from closed-duration histograms obtained at intermediate ACh concentrations. With k-1, k-2 and beta determined, the rates of ACh association, k+1 and k+2, are estimated from fitting closed-duration histograms obtained over a range of high ACh concentrations. A complete set of rate constants is presented for three experimental conditions: (a) Ca2(+)-free extracellular solution containing 1 mM free Mg2+ at 22 degrees C, (b) Ca2(+)-free solution at 12 degrees C, and (c) extracellular Ca2+ and Mg2+, both at 0.5 mM, at 22 degrees C. For all three conditions the dissociation constant for the first agonist binding site is approximately 100-fold lower than that for the second site. The different affinities are due primarily to different dissociation rates. Both the association and dissociation rates depend strongly on temperature. At 22 degrees C ACh associates at diffusion-limited rates, whereas at 12 degrees C association is 30- to 60-fold slower. Also slowed at 12 degrees C are beta (4-fold), k-2 (3-fold), k-1 (25-fold), and alpha (15-fold). In contrast to the activation rate constants, those for ACh-induced block decrease only twofold between 22 and 12 degrees C. Changing from a Ca2(+)-free to a Ca2(+)-containing extracellular solution does not affect k+1 and k+2, but increases beta (twofold) and decreases k-2, k-1, and alpha (all twofold). Spectral analysis of single channel currents supports the parameter estimates obtained from fitting the open- and closed-duration histograms, and improves resolution of brief channel blockages produced by ACh.