bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the low threshold Ca current in single frog atrial cardiomyocytes. A comparison with the high threshold Ca current.
Authors: Alvarez J L , Vassort G
Published: May 2004

The properties of the low threshold Ca current (ICaT) in bullfrog (Rana catesbeiana) isolated atrial cardiomyocytes were studied using the whole-cell recording patch-clamp technique and compared with those of the high threshold Ca current (ICaL). In 91% of atrial cells we observed both ICaT and ICaL when collagenase and trypsin were used to dissociate the cells. But when pronase was used, only 30% of the cells exhibited ICaT. ICaT was never found in ventricular cells. ICaT could be investigated more easily when ICaL was inhibited by Cd ions (50 microM). Its kinetics were unchanged by substituting Ba for Ca, or in the presence of high concentrations of Ba. Both ICaT and ICaL exhibited reduced inactivation after high depolarizing prepulses. ICaT was found to be sensitive to dihydropyridines: 1 microM nifedipine decreased this current while 1 microM BAY K 8644 increased it; this occurred without significant variations in the steady-state inactivation curve. ICaT was more sensitive than ICaL to alpha 1-adrenergic and P2-purinergic stimulations, while ICaL was more sensitive to beta-adrenergic stimulation. Isoproterenol was still able to increase ICaT in the presence of high intracellular cAMP. Both currents were increased by 1 microM ouabain (although ICaL only transiently) and decreased by 10 microM ouabain. It is concluded that the two types of Ca channels can be observed in bullfrog atrial cells and that they are specifically altered by pharmacological agents and neuromediators. This may have implications for cardiac behavior.
Actions of chiriquitoxin on frog skeletal muscle fibers and implications for the tetrodotoxin/saxitoxin receptor.
Authors: Yang L , Kao C Y
Published: May 2004

Chiriquitoxin (CqTX) from the Costa Rican frog Atelopus chiriquensis differs from tetrodoxin (TTX) only in that a glycine residue replaces a methylene hydrogen of the C-11 hydroxymethyl function. On the voltage-clamped frog skeletal muscle fiber, in addition to blocking the sodium channel and unrelated to such an action, CqTX also slows the activation of the fast potassium current in approximately 40% of the muscle fiber population. At pH 7.25, CqTX is as potent as TTX in blocking the sodium channel, with an ED50 of 3.8 nM. Its ED50's at pH 6.50 and 8.25 are 6.8 and 2.3 nM, contrasted with 3.8 and 4.3 nM for TTX. These differences are attributable to changes in the chemical states in the glycine residue. The equipotency of CqTX with TTX at pH 7.25 is explainable by an intramolecular salt bridge between the amino and carboxyl groups of the glycine function, all other surface groups in TTX and CqTX being the same. From available information on these groups and those in saxitoxin (STX), the TTX/STX binding site is deduced to be in a pocket 9.5 A wide, 6 A high, and 5 A deep. The glycine residue of CqTX probably projects out of the entrance to this pocket. Such a view of the binding site could also account for the actions of STX analogues, including the C-11 sulfated gonyautoxins and the 21-sulfocarbamoyl analogues. In the gonyautoxins the sulfate groups are equivalently placed as the glycine in CqTX, whereas in the sulfocarbamoyl toxins the sulfate groups extend the carbamoyl side-chain, leading to steric hinderance to productive binding.
Voltage dependence of acetylcholine receptor channel gating in rat myoballs.
Authors: Chabala L D
Published: May 2004

Whole-cell currents from nicotinic acetylcholine receptor (AChR) channels were studied in rat myoballs using a light-activated agonist to determine the voltage dependence of the macroscopic opening and closing rate constants. Myoballs were bathed in a solution containing a low concentration of the inactive isomer of the photoisomerizable azobenzene derivative, cis-Bis-Q. A light flash was then presented to produce a known concentration jump of agonist, trans-Bis-Q, across a wide range of membrane potentials in symmetrical solutions (NaCl or CsCl on both sides) or asymmetrical solutions (NaCl in the bath and CsCl in the pipette). At the low agonist concentration used in this study, the reciprocal of the macroscopic time constants gives an unambiguous measure of the effective closing rate. It showed an exponential decrease with membrane hyperpolarization between +20 and -100 mV, but tended to level off at more depolarized and at more hyperpolarized membrane potentials. The relative effective opening rate was derived from the steady-state conductance, the single-channel conductance, and the apparent closing rate; it decreased sharply in the depolarizing region and tended to level off and then turn up in the hyperpolarizing region. The two effective rate constants were shown to depend on the first, second, and third power of membrane potential.
Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.
Authors: Wollmuth L P , Hille B
Published: May 2004

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.
Adaptation-promoting effect of IP3, Ca2+, and phorbol ester on the sugar taste receptor cell of the blowfly, Phormia regina.
Authors: Ozaki M , Amakawa T
Published: May 2004

The fly has a receptor cell highly specialized for the taste of sugars. We introduced inositol 1,4,5-trisphosphate (IP3), Ca2+, or a phorbol ester, 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA), into the cell and investigated their effects on the response to sucrose. The sugar receptor cell generates impulses during constant stimulation with sucrose, but the impulse frequency gradually declines as the cell adapts to the stimulus. Thus, this gradual reduction of the impulse frequency is a direct manifestation of adaptation of the cell. These reagents accelerated the gradual reduction of the impulse frequency, although the initial impulse frequency was little affected. In contrast to these reagents, glycoletherdiamine-tetraacetate (EGTA) retarded the gradual reduction of the impulse frequency. Moreover, when IP3 and DPBA were applied together, the gradual reduction of the impulse frequency was more accelerated than when either IP3 or DPBA was applied. When IP3 and EGTA were applied together, however, the accelerating effect of IP3 tended to be canceled. Based on these results, we hypothesized that an intracellular cascade involving inositol phospholipid hydrolysis, intracellular Ca2+ mobilization, and protein kinase C-mediated phosphorylation promotes adaptation of the sugar receptor cell.
Nature of increase in quantal release by the thallous ion at frog end plates with and without nerve stimulation.
Authors: Talbot P A
Published: May 2004

The monovalent thallous ion (Tl) was evaluated at the frog end plate in vitro with intracellular microelectrodes. Recordings included end plate potentials (EPPs), and miniature end plate potentials (MEPPs). Replacement of extracellular potassium (K) by 2.5 mM Tl (a) caused increases in MEPP and EPP amplitudes, MEPP frequency, and quantal content, and (b) caused complete recovery of the EPP facilitation index at BAPTA-loaded nerve terminals. Tl's effects were reversible and concentration dependent, and persisted for > 3 h. The increase in MEPP frequency and its rate of decline due to Tl washout were more pronounced at 0 calcium (Ca)-2 mM EGTA than at 0.3 mM EGTA, suggesting that Tl's effects were not due to elevation of internal Ca. Unlike heavy metal ions reportedly capable of substituting for Ca, 0.2 mM Tl did not block, but further enhanced, elevated MEPP frequencies, occurring after nerve stimulation or in high K, to greater levels with barium (Ba) than with Ca. 200 nM omega-conotoxin (omega-CTX) blocked Tl's effect, indicating that Tl primarily entered the nerve terminal via Ca channels. A 50% reduction in sodium (Na) did not modify Tl's effect, although removal of K in the presence of 20 microM ouabain and 2.5 mM Tl caused an exaggerated increase in MEPP frequency, which decreased with a 50% reduction in Na. Based on the analysis, Tl neither substituted for Ca nor elevated internal Ca and Na, nor were its effects antagonized by ouabain; Tl increased quantal secretion, possibly by a fusogenic mechanism, after its entry into the nerve terminal.
Altered stereoselectivity of cocaine and bupivacaine isomers in normal and batrachotoxin-modified Na+ channels.
Authors: Wang G K , Wang S Y
Published: May 2004

The inhibitory effects of local anesthetics (LAs) of cocaine and bupivacaine optical isomers on Na+ currents were studied in clonal GH3 cells under whole-cell patch clamp conditions. At holding potential of -100 mV, all four isomers inhibited peak Na+ currents when the cell was stimulated infrequently. The dose-response curves of this tonic block of peak Na+ currents by (-)/(+) cocaine and (-)/(+) bupivacaine were well fitted by the Langmuir isotherm, suggesting that one LA isomer blocked one Na+ channel. Each pair of isomers showed no greater than a twofold difference in stereoselectivity toward Na+ channels. Additional block of Na+ currents occurred when the cell was stimulated at 2 Hz. This use-dependent block was also observed in all four isomers, which again displayed little stereoselectivity. The voltage dependence of the use-dependent block produced by cocaine isomers did not overlap with the activation of Na+ channels but did overlap with the steady-state inactivation (h infinity), indicating that cocaine can bind directly to the inactivated state of Na+ channels before channel opening. In comparison, the peak batrachotoxin (BTX)-modified Na+ currents were little inhibited by cocaine and bupivacaine isomers. However, the maintained BTX-modified Na+ currents were highly sensitive toward the (-) form of cocaine and bupivacaine isomers during a prolonged depolarization. As a result, a profound time-dependent block of BTX-modified Na+ currents was evident in the presence of these LA isomers. The estimated values of the equilibrium dissociation constant (KD in micromolar) at +50 mV were 35.8, 661, 7.0, and 222 for (-)/(+) cocaine and (-)/(+) bupivacaine, respectively. Although chloramine-T (CT) also modified the fast inactivation of Na+ channels and gave rise to a maintained Na+ current during a prolonged depolarization, LA isomers showed no greater stereoselectivity in blocking this maintained current than in blocking the normal transient Na+ current. We conclude that (a) cocaine and bupivacaine isomers exhibit only weak stereoselectivity toward the LA receptor in normal and CT-treated Na+ channels, (b) BTX drastically modifies the configuration of the LA binding site so that the LA stereoselectivity of the open Na+ channels is altered by an order of magnitude, and (c) the (-) forms of cocaine and bupivacaine interact strongly with the open state of BTX-modified Na+ channels but only weakly, if at all, with the closed state. The last finding may explain why most LA drugs were reported to be less effective toward BTX-modified Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Two functionally distinct 4-aminopyridine-sensitive outward K+ currents in rat atrial myocytes.
Authors: Boyle W A , Nerbonne J M
Published: May 2004

In the experiments here, the detailed kinetic properties of the Ca(2+)-independent, depolarization-activated outward currents (Iout) in enzymatically dispersed adult rat atrial myocytes were studied. Although there is only slight attenuation of peak Iout during brief (100 ms) voltage steps, substantial decay is evident during long (10 s) depolarizations. The analyses here reveal that current inactivation is best described by the sum of two exponential components, which we have termed IKf and IKs to denote the fast and slow components, respectively, of Iout decay. At all test potentials, IKf inactivates approximately 20-fold more rapidly than IKs. Neither the decay time constants nor the fraction of Iout remaining at the end of 10-s depolarizations varies over the potential range of 0 to +50 mV, indicating that the rates of inactivation and recovery from inactivation are voltage independent. IKf recovers from inactivation completely, independent of the recovery of IKs, and IKf recovers approximately 20 times faster than IKs. The pharmacological properties of IKf and IKs are similar: both components are sensitive to 4-aminopyridine (1-5 mM) and both are relatively resistant to externally applied tetraethylammonium (50 mM). Taken together, these findings suggest that IKf and IKs correspond to two functionally distinct K+ currents with similar voltage-dependent properties and pharmacologic sensitivities, but with markedly different rates of inactivation and recovery from inactivation. From the experimental data, several gating models were developed in which voltage-independent inactivation is coupled either to channel opening or to the activation of the individual channel subunits. Experimental testing of predictions of these models suggests that voltage-independent inactivation is coupled to activation, and that inactivation of only a single subunit is required to result in functional inactivation of the channels. This model closely approximates the properties of IKf and IKs, as well as the composite outward currents, measured in adult rat atrial myocytes.
Interplay between passive tension and strong and weak binding cross-bridges in insect indirect flight muscle. A functional dissection by gelsolin-mediated thin filament removal.
Authors: Granzier H L , Wang K
Published: May 2004

The interplay between passive and active mechanical properties of indirect flight muscle of the waterbug (Lethocerus) was investigated. A functional dissection of the relative contribution of cross-bridges, actin filaments, and C filaments to tension and stiffness of passive, activated, and rigor fibers was carried out by comparing mechanical properties at different ionic strengths of sarcomeres with and without thin filaments. Selective thin filament removal was accomplished by treatment with the actin-severving protein gelsolin. Thin filament, removal had no effect on passive tension, indicating that the C filament and the actin filament are mechanically independent and that passive tension is developed by the C filament in response to sarcomere stretch. Passive tension increased steeply with sarcomere length until an elastic limit was reached at only 6-7% sarcomere extension, which corresponds to an extension of 350% of the C filament. The passive tension-length relation of insect flight muscle was analyzed using a segmental extension model of passive tension development (Wang, K, R. McCarter, J. Wright, B. Jennate, and R Ramirez-Mitchell. 1991. Proc. Natl. Acad. Sci. USA. 88:7101-7109). Thin filament removal greatly depressed high frequency passive stiffness (2.2 kHz) and eliminated the ionic strength sensitivity of passive stiffness. It is likely that the passive stiffness component that is removed by gelsolin is derived from weak-binding cross-bridges, while the component that remains is derived from the C filament. Our results indicate that a significant number of weak-binding cross-bridges exist in passive insect muscle at room temperature and at an ionic strength of 195 mM. Analysis of rigor muscle indicated that while rigor tension is entirely actin based, rigor stiffness contains a component that resists gelsolin treatment and is therefore likely to be C filament based. Active tension and active stiffness of unextracted fibers were directly proportional to passive tension before activation. Similarly, passive stiffness due to weak bridges also increased linearly with passive tension, up to a limit. These correlations lead us to propose a stress-activation model for insect flight muscle in which passive tension is a prerequisite for the formation of both weak-binding and strong-binding cross-bridges.
Actions of cholinergic drugs in the nematode Ascaris suum. Complex pharmacology of muscle and motorneurons.
Authors: Segerberg M A , Stretton A O
Published: May 2004

The cholinergic agonists acetylcholine (ACh), nicotine, and pilocarpine produced depolarizations and contractions of muscle of the nematode Ascaris suum. Dose-dependent depolarization and contraction by ACh were suppressed by about two orders of magnitude by 100 microM d-tubocurarine (dTC), a nicotinic antagonist, but only about fivefold by 100 microM N-methyl-scopolamine (NMS), a muscarinic antagonist. NMS itself depolarized both normal and synaptically isolated muscle cells. The muscle depolarizing action of pilocarpine was not consistently antagonized by either NMS or dTC. ACh receptors were detected on motorneuron classes DE1, DE2, DI, and VI as ACh-induced reductions in input resistance. These input resistance changes were reversed by washing in drug-free saline or by application of dTC. NMS applied alone lowered input resistance in DE1, but not in DE2, DI, or VI motorneurons. In contrast to the effect of ACh, the action of NMS in DE1 was not reversed by dTC, suggesting that NMS-sensitive sites may not respond to ACh. Excitatory synaptic responses in muscle evoked by depolarizing current injections into DE1 and DE2 motorneurons were antagonized by dTC; however, NMS antagonized the synaptic output of only the DE1 and DE3 classes of motorneurons, an effect that was more likely to have been produced by motorneuron conduction failure than by pharmacological blockade of receptor. The concentration of NMS required to produce these changes in muscle polarization and contraction, ACh antagonism, input resistance reduction, and synaptic antagonism was 100 microM, or more than five orders of magnitude higher than the binding affinity for [3H]NMS in larval Ascaris homogenates and adult Caenorhabditis elegans (Segerberg, M. A. 1989. Ph.D. thesis. University of Wisconsin-Madison, Madison, WI). These results describe a nicotinic-like pharmacology, but muscle and motorneurons also have unusual responses to muscarinic agents.
Modulation of calcium channels in arterial smooth muscle cells by dihydropyridine enantiomers.
Authors: Hering S , Hughes A D , Timin E N , Bolton T B
Published: May 2004

The actions of the optical enantiomers of BAY K 8644 and Sandoz 202,791 were studied on barium inward currents recorded using the whole-cell configuration of the patch clamp technique from enzymatically isolated smooth muscle cells from the rabbit ear artery. The enantiomers were applied by bath perfusion or rapidly by a concentration jump technique, which enabled the study of drug action under equilibrium and nonequilibrium conditions. A larger effect of agonists was seen on peak inward current in 110 mM Ba when small rather than large depolarizations were applied. The midpoint voltage of the steady-state inactivation curve of IBa was -12.8 +/- 1.9 mV (n = 4) in the absence of drug, -16.4 +/- 2.5 mV (n = 4) in 1 microM (+)202,791, and -31.4 +/- 0.4 mV (n = 4) in 1 microM (-)202,791. The rate of onset of action of the agonist and antagonist enantiomers of BAY K 8644 and Sandoz 202,791 was studied by rapid application during 20-ms depolarizing steps from different holding potentials to +30 mV at 1 or 0.2 Hz. The drugs were applied as concentration jumps between two single pulses of a pulse train. The rates of onset of drug action on peak IBa during a 1-Hz pulse train were concentration dependent over the range of 100 nM-3 microM for both (+) and (-)202,791. The rate of onset of inhibition of peak current by antagonist enantiomers was not significantly influenced by the test pulse frequency. At a holding potential of -60 mV, the onset rate of the increase in peak IBa on application of 1 microM of agonist enantiomers (+)202,791 or (-)BAY K 8644 during a train of pulses occurred with mean time constants of 2.1 +/- 0.7 s (n = 7) and 2.3 +/- 0.2 s (n = 4), respectively. The onset of current increase on application of 1 microM (+)202,791 during a single voltage clamp step to 20 mV was faster, with a mean time constant of 380 +/- 80 ms (n = 3).
Anion modulation of taste responses in sodium-sensitive neurons of the hamster chorda tympani nerve.
Authors: Rehnberg B G , MacKinnon B I , Hettinger T P , Frank M E
Published: May 2004

Beidler's work in the 1950s showed that anions can strongly influence gustatory responses to sodium salts. We have demonstrated "anion inhibition" in the hamster by showing that the chorda tympani nerve responds more strongly to NaCl than to Na acetate over a wide range of concentrations. Iontophoretic presentation of Cl- and acetate to the anterior tongue elicited no response in the chorda tympani, suggesting that these anions are not directly stimulatory. Drugs (0.01, 1.0, and 100 microM anthracene-9-carboxylate, diphenylamine-2-carboxylate, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate, and furosemide) that interfere with movements of Cl- across epithelial cells were ineffective in altering chorda tympani responses to 0.03 M of either NaCl or Na acetate. Anion inhibition related to movements of anions across epithelial membranes therefore seems unlikely. The chorda tympani contains a population of nerve fibers highly selective for Na+ (N fibers) and another population sensitive to Na+ as well as other salts and acids (H fibers). We found that N fibers respond similarly to NaCl and Na acetate, with spiking activity increasing with increasing stimulus concentration (0.01-1.0 M). H fibers, however, respond more strongly to NaCl than to Na acetate. Furthermore, H fibers increase spiking with increases in NaCl concentration, but generally decrease their responses to increasing concentrations of Na acetate. It appears that anion inhibition applies to taste cells innervated by H fibers but not by N fibers. Taste cells innervated by N fibers use an apical Na+ channel, whereas those innervated by H fibers may use a paracellularly mediated, basolateral site of excitation.
Modulation of Cl-, K+, and nonselective cation conductances by taurine in olfactory receptor neurons of the mudpuppy Necturus maculosus.
Authors: Dubin A E , Dionne V E
Published: May 2004

Odors are transduced by processes that modulate the membrane conductance of olfactory receptor neurons. Olfactory neurons from the aquatic salamander, Necturus maculosus, were acutely isolated without enzymes and studied with a resistive whole-cell method to minimize loss of soluble intracellular constituents. 55 of 224 neurons responded to the test compound taurine at concentrations between 10 nM and 100 microM. Four different conductance changes were elicited by taurine: an increased Cl- conductance (33%), an increased nonselective cation conductance (15%), a decreased Cl- conductance (15%), and a decreased K+ conductance (15%); in addition, responses too small to be characterized were elicited in some neurons. In most cases, taurine appeared to modulate only a single conductance in any particular cell. Modulation of each conductance was dose dependent, and each response ran down quickly in the normal whole-cell mode, presumably due to washout of a diffusible component in the transduction pathway. Modulation of taurine-sensitive conductances caused either inhibitory or excitatory responses. A similar diversity of responses in vivo would produce a complex pattern of electrical activity that could encode the identity and characteristics of an odor.
The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis.
Authors: Campbell D L , Rasmusson R L , Qu Y , Strauss H C
Published: May 2004

Enzymatically isolated myocytes from ferret right ventricles (12-16 wk, male) were studied using the whole cell patch clamp technique. The macroscopic properties of a transient outward K+ current I(to) were quantified. I(to) is selective for K+, with a PNa/PK of 0.082. Activation of I(to) is a voltage-dependent process, with both activation and inactivation being independent of Na+ or Ca2+ influx. Steady-state inactivation is well described by a single Boltzmann relationship (V1/2 = -13.5 mV; k = 5.6 mV). Substantial inactivation can occur during a subthreshold depolarization without any measurable macroscopic current. Both development of and recovery from inactivation are well described by single exponential processes. Ensemble averages of single I(to) channel currents recorded in cell-attached patches reproduce macroscopic I(to) and indicate that inactivation is complete at depolarized potentials. The overall inactivation/recovery time constant curve has a bell-shaped potential dependence that peaks between -10 and -20 mV, with time constants (22 degrees C) ranging from 23 ms (-90 mV) to 304 ms (-10 mV). Steady-state activation displays a sigmoidal dependence on membrane potential, with a net aggregate half-activation potential of +22.5 mV. Activation kinetics (0 to +70 mV, 22 degrees C) are rapid, with I(to) peaking in approximately 5-15 ms at +50 mV. Experiments conducted at reduced temperatures (12 degrees C) demonstrate that activation occurs with a time delay. A nonlinear least-squares analysis indicates that three closed kinetic states are necessary and sufficient to model activation. Derived time constants of activation (22 degrees C) ranged from 10 ms (+10 mV) to 2 ms (+70 mV). Within the framework of Hodgkin-Huxley formalism, Ito gating can be described using an a3i formulation.
The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine.
Authors: Campbell D L , Qu Y , Rasmusson R L , Strauss H C
Published: May 2004

Block of the calcium-independent transient outward K+ current, I(to), by 4-aminopyridine (4-AP) was studied in ferret right ventricular myocytes using the whole cell patch clamp technique. 4-AP reduces I(to) through a closed state blocking mechanism displaying "reverse use-dependent" behavior that was inferred from: (a) development of tonic block at hyperpolarized potentials; (b) inhibition of development of tonic block at depolarized potentials; (c) appearance of "crossover phenomena" in which the peak current is delayed in the presence of 4-AP at depolarized potentials; (d) relief of block at depolarized potentials which is concentration dependent and parallels steady-state inactivation for low 4-AP concentrations (V1/2 approximately -10 mV in 0.1 mM 4-AP) and steady-state activation at higher concentrations (V1/2 = +7 mV in 1 mM 4-AP, +15 mV in 10 mM 4-AP); and (e) reassociation of 4-AP at hyperpolarized potentials. No evidence for interaction of 4-AP with either the open or inactivated state of the I(to) channel was obtained from measurements of kinetics of recovery and deactivation in the presence of 0.5-1.0 mM 4-AP. At hyperpolarized potentials (-30 to -90 mV) 10 mM 4-AP associates slowly (time constants ranging from approximately 800 to 1,300 ms) with the closed states of the channel (apparent Kd approximately 0.2 mM). From -90 to -20 mV the affinity of the I(to) channel for 4-AP appears to be voltage insensitive; however, at depolarized potentials (+20 to +100 mV) 4-AP dissociates with time constants ranging from approximately 350 to 150 ms. Consequently, the properties of 4-AP binding to the I(to) channel undergo a transition in the range of potentials over which channel activation and inactivation occurs (-30 to +20 mV). We propose a closed state model of I(to) channel gating and 4-AP binding kinetics, in which 4-AP binds to three closed states. In this model 4-AP has a progressively lower affinity as the channel approaches the open state, but has no intrinsic voltage dependence of binding.
Functionally distinct phospho-forms underlie incremental activation of protein kinase-regulated Cl- conductance in mammalian heart.
Authors: Hwang T C , Horie M , Gadsby D C
Published: May 2004

The regulation of cardiac Cl- conductance by cAMP-dependent protein kinase (PKA) and cellular phosphatases was studied in isolated guinea pig ventricular myocytes by using wide-tipped, perfused pipettes to record whole-cell currents. Exposure to forskolin (Fsk) or isoproterenol (Iso) elicits a Cl- conductance that results exclusively from PKA-dependent phosphorylation because it can be completely abolished, or its activation fully prevented, by switching to pipette solution containing PKI, a synthetic peptide inhibitor of PKA. The Cl- conductance activated by micromolar concentrations of either agonist reached its steady-state amplitude in 1-2 min and was deactivated promptly and entirely, usually within 2 min, upon washing out the agonist, implying a continuous high level of activity of endogenous protein phosphatases. Accordingly, intracellular application of okadaic acid or microcystin, both potent inhibitors of protein phosphatases 1 and 2A, during exposure to Fsk enhanced the steady-state Cl- conductance and slowed its deactivation after washing out the Fsk. Maximal potentiation of the conductance, by approximately 60%, was obtained with pipette concentrations of approximately 10 microM okadaic acid (or approximately 5 microM microcystin) and did not result from an increase in the apparent affinity for Fsk. In the presence of maximally effective concentrations of okadaic acid and/or microcystin, deactivation of the enhanced Cl- conductance upon washout of agonist was incomplete, with about half of the conductance persisting indefinitely. That residual conductance did not reflect continued action of PKA because it was insensitive to PKI, but was identified as a fraction of the activated Cl- conductance by its biophysical characteristics. The results suggest that complete deactivation of the PKA-regulated cardiac Cl- conductance requires dephosphorylation by a type 1 and/or 2A phosphatase, but that partial deactivation can be accomplished by activity of some other phosphatase(s). These findings are consistent with sequential phosphorylation of a protein, probably the Cl- channel itself, at two different kinds of sites. The resulting phosphoproteins can be distinguished on the basis of their different contributions to whole-cell Cl- conductance.
Mechanism of apical K+ channel modulation in principal renal tubule cells. Effect of inhibition of basolateral Na(+)-K(+)-ATPase.
Authors: Wang W H , Geibel J , Giebisch G
Published: May 2004

The effects of inhibition of the basolateral Na(+)-K(+)-ATPase (pump) on the apical low-conductance K+ channel of principal cells in rat cortical collecting duct (CCD) were studied with patch-clamp techniques. Inhibition of pump activity by removal of K+ from the bath solution or addition of strophanthidin reversibly reduced K+ channel activity in cell-attached patches to 36% of the control value. The effect of pump inhibition on K+ channel activity was dependent on the presence of extracellular Ca2+, since removal of Ca2+ in the bath solution abolished the inhibitory effect of 0 mM K+ bath. The intracellular [Ca2+] (measured with fura-2) was significantly increased, from 125 nM (control) to 335 nM (0 mM K+ bath) or 408 nM (0.2 mM strophanthidin), during inhibition of pump activity. In contrast, cell pH decreased only moderately, from 7.45 to 7.35. Raising intracellular Ca2+ by addition of 2 microM ionomycin mimicked the effect of pump inhibition on K+ channel activity. 0.1 mM amiloride also significantly reduced the inhibitory effect of the K+ removal. Because the apical low-conductance K channel in inside-out patches is not sensitive to Ca2+ (Wang, W., A. Schwab, and G. Giebisch, 1990. American Journal of Physiology. 259:F494-F502), it is suggested that the inhibitory effect of Ca2+ is mediated by a Ca(2+)-dependent signal transduction pathway. This view was supported in experiments in which application of 200 nM staurosporine, a potent inhibitor of Ca(2+)-dependent protein kinase C (PKC), markedly diminished the effect of the pump inhibition on channel activity. We conclude that a Ca(2+)-dependent protein kinase such as PKC plays a key role in the downregulation of apical low-conductance K+ channel activity during inhibition of the basolateral Na(+)-K(+)-ATPase.
Kinetics of residual chloride transport in human red blood cells after maximum covalent 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid binding.
Authors: Gasbjerg P K , Funder J , Brahm J
Published: May 2004

Irreversible inhibition, 99.8% of control values for chloride transport in human red blood cells, was obtained by well-established methods of maximum covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The kinetics of the residual chloride transport (0.2%, 106 pmol.cm-2 x s-1) at 38 degrees C, pH 7.2) was studied by means of 36Cl- efflux. The outside apparent affinity, expressed by Ko1/2,c, was 34 mM, as determined by substituting external KCl by sucrose. The residual flux was reversibly inhibited by a reexposure to DIDS, and by 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), phloretin, salicylate, and alpha-bromo-4-hydroxy-3,5-dinitroacetophenone (Killer III) (Borders, C. L., Jr., D. M. Perez, M. W. Lafferty, A. J. Kondow, J. Brahm, M. B. Fenderson, G. L. Breisford, and V. B. Pett. 1989. Bioorganic Chemistry. 17:96-107), to approximately 0.001% of control cells, which is a flux as low as in lipid bilayers. The reversible DIDS inhibition of the residual chloride flux depended on the extracellular chloride concentration, but was not purely competitive. The half-inhibition concentrations at [Cl(o)] = 150 mM in control cells (Ki,o) and covalently DIDS-treated cells (Ki,c) were: DIDS, Ki,c = 73 nM; DNDS, Ki,o = 6.3 microM, Ki,c = 22 microM; phloretin, Ki,o = 19 microM, Ki,c = 17 microM; salicylate, Ki,o = 4 mM, Ki,c = 8 mM; Killer III, Ki,o = 10 microM, Ki,c = 10 microM.
Chloride channels activated by osmotic stress in T lymphocytes.
Authors: Lewis R S , Ross P E , Cahalan M D
Published: May 2004

We have used whole-cell and perforated-patch recording techniques to characterize volume-sensitive Cl- channels in T and B lymphocytes. Positive transmembrane osmotic pressure (intracellular osmolality > extracellular osmolality) triggers the slow induction of a Cl- conductance. Membrane stretch caused by cellular swelling may underlie the activation mechanism, as moderate suction applied to the pipette interior can reversibly oppose the induction of Cl- current by an osmotic stimulus. Intracellular ATP is required for sustaining the Cl- current. With ATP-free internal solutions, the inducibility of Cl- current declines within minutes of whole-cell recording, while in whole-cell recordings with ATP or in perforated-patch experiments, the current can be activated for at least 30 min. The channels are anion selective with a permeability sequence of I- > SCN- > NO3-, Br- > Cl- > MeSO3- > acetate, propionate > ascorbate > aspartate and gluconate. GCl does not show voltage- and time-dependent gating behavior at potentials between -100 and +100 mV, but exhibits moderate outward rectification in symmetrical Cl- solutions. Fluctuation analysis indicates a unitary chord conductance of approximately 2 pS at -80 mV in the presence of symmetrical 160 mM Cl-. The relationship of mean current to current variance during the osmotic activation of Cl- current implies that each cell contains on the order of 10(4) activatable Cl- channels, making it the most abundant ion channel in lymphocytes yet described. The current is blocked in a voltage-dependent manner by DIDS and SITS (Ki = 17 and 89 microM, respectively, at +40 mV), the degree of blockade increasing with membrane depolarization. The biophysical and pharmacological properties of this Cl- channel are consistent with a role in triggering volume regulation in lymphocytes exposed to hyposmotic conditions.
Expression of multiple water channel activities in Xenopus oocytes injected with mRNA from rat kidney.
Authors: Echevarria M , Frindt G , Preston G M , Milovanovic S , Agre P , Fischbarg J , Windhager E E
Published: May 2004

To test the hypothesis that renal tissue contains multiple distinct water channels, mRNA prepared from either cortex, medulla, or papilla of rat kidney was injected into Xenopus oocytes. The osmotic water permeability (Pf) of oocytes injected with either 50 nl of water or 50 nl of renal mRNA (1 microgram/microliter) was measured 4 d after the injection. Pf was calculated from the rate of volume increase on exposure to hyposmotic medium. Injection of each renal mRNA preparation increased the oocyte Pf. This expressed water permeability was inhibited by p-chloromercuriphenylsulfonate and had a low energy of activation, consistent with the expression of water channels. The coinjection of an antisense oligonucleotide for CHIP28 protein, at an assumed > 100-fold molar excess, with either cortex, medulla, or papilla mRNA reduced the expression of the water permeability by approximately 70, 100, and 30%, respectively. Exposure of the oocyte to cAMP for 1 h resulted in a further increase in Pf only in oocytes injected with medulla mRNA. This cAMP activation was not altered by the CHIP28 antisense oligonucleotide. These results suggest that multiple distinct water channels were expressed in oocytes injected with mRNA obtained from sections of rat kidney: (a) CHIP28 water channels in cortex and medulla, (b) cAMP-activated water channels in medulla, and (c) cAMP-insensitive water channels in papilla.
Selectivity of lingual nerve fibers to chemical stimuli.
Authors: Wang Y , Erickson R P , Simon S A
Published: May 2004

The cell bodies of the lingual branch of the trigeminal nerve were localized in the trigeminal ganglion using extracellular recordings together with horseradish peroxidase labeling from the tongue. Individual lingual nerve fibers were characterized with regard to their conduction velocities, receptive fields, and response to thermal, mechanical, and chemical stimuli. Fibers were classified as C, A delta, A beta, cold, and warm. The chemical stimuli included NaCl, KCl, NH4Cl, CaCl2, menthol, nicotine, hexanol, and capsaicin. With increasing salt concentration the latency of the response decreased and the activity increased. The responses elicited by salts (to 2.5 M), but not nonpolar stimuli such as menthol, were reversibly inhibited by 3.5 mM of the tight junction blocker, LaCl3. These data suggest that salts diffuse into stratified squamous epithelia through tight junctions in the stratum corneum and stratum granulosum, whereupon they enter the extracellular space. 11 C fibers were identified and 5 were characterized as polymodal nociceptors. All of the C fibers were activated by one or more of the salts NaCl, KCl, or NH4Cl. Three C fibers were activated by nicotine (1 mM), but none were affected by CaCl2 (1 M), menthol (1 mM), or hexanol (50 mM). However, not all C fibers or even the subpopulation of polymodals were activated by the same salts or by nicotine. Thus, it appears that C fibers display differential responsiveness to chemical stimuli. A delta fibers also showed differential sensitivity to chemicals. Of the 35 characterized A delta mechanoreceptors, 8 responded to NaCl, 9 to KCl, 9 to NH4Cl, 0 to CaCl2, menthol, or hexanol, and 2 to nicotine. 8 of 9 of the cold fibers (characterized as A delta's) responded to menthol, none responded to nicotine, 8 of 16 were inhibited by hexanol, 9 of 19 responded to 2.5 M NH4Cl, 5 of 19 responded to 2.5 M KCl, and 1 of 19 responded to 2.5 M NaCl. In summary, lingual nerve fibers exhibit responsiveness to chemicals introduced onto the tongue. The differential responses of these fibers are potentially capable of transmitting information regarding the quality and quantity of chemical stimuli from the tongue to the central nervous system.
Intracellular injection of heparin and polyamines. Effects on phototransduction in limulus ventral photoreceptors.
Authors: Faddis M N , Brown J E
Published: May 2004

Heparin is thought to inhibit InsP3 binding to receptors involved in the intracellular release of Ca2+. Injection of heparin into Limulus ventral photoreceptors to high intracellular concentrations reduces the amplitude and slows the rate of rise of voltage-clamp currents induced by brief flashes, tends to make the responses to long flashes more "square," and tends to block the light-induced rise in [Ca2+]i detected by arsenazo III. In these ways, intracellular heparin mimics the effects of high concentrations of intracellular BAPTA or EGTA. In addition, the effects of heparin are attenuated by prior injection of BAPTA to high intracellular concentrations. Neomycin and spermine are thought to inhibit phospholipase C activity. Injections of spermine or neomycin to low intracellular concentrations largely mimic the effects of intracellular heparin. These findings suggest that the predominant effect of polyamines is to inhibit light-induced production of InsP3 by phospholipase C activity and thereby reduce the light-induced increase in [Ca2+]i. Our findings suggest that excitation can proceed in the absence of InsP3-induced increases in [Ca2+]i, but (a) the gain and speed of transduction are reduced and (b) adaptation is largely blocked.
In retinal cones, membrane depolarization in darkness activates the cGMP-dependent conductance. A model of Ca homeostasis and the regulation of guanylate cyclase.
Authors: Miller J L , Korenbrot J I
Published: May 2004

We measured outer segment currents under voltage clamp in solitary, single cone photoreceptors isolated from the retina of striped bass. In darkness, changes in membrane voltage to values more positive than 10 mV activate a time- and voltage-dependent outward current in the outer segment. This dark, voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course up to a steady-state value, reached in 0.75-1.5 s. DVAC is entirely suppressed by light, and its current-voltage characteristics and reversal potential are the same as those of the light-sensitive currents. DVAC, therefore, arises from the activation by voltage in the dark of the light-sensitive, cGMP-gated channels of the cone outer segment. Since these channels are not directly gated by voltage, we explain DVAC as arising from a voltage-dependent decrease in cytoplasmic Ca concentration that, in turn, activates only guanylate cyclase and results in net synthesis of cGMP. This explanation is supported by the finding that the Ca buffer BAPTA, loaded into the cytoplasm of the cone outer segment, blocks DVAC. To link a decrease in cytoplasmic Ca concentration to the synthesis of cGMP and the characteristics of DVAC, we develop a quantitative model that assumes cytoplasmic Ca concentration can be continuously calculated from the balance between passive Ca influx via the cGMP-gated channel and its active efflux via a Na/Ca,K exchanger, and that further assumes that guanylate cyclase is activated by decreasing cytoplasmic Ca concentration with characteristics identical to those described for the enzyme in rods. The model successfully simulates experimental data by adjusting the Ca conductance of the cGMP-gated channels as a function of voltage and the Ca buffering power of the cytoplasm. This success suggests that the activity of guanylate cyclase in cone outer segments is indistinguishable from that in rods.
Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.
Authors: McCarty N A , McDonough S , Cohen B N , Riordan J R , Davidson N , Lester H A
Published: May 2004

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.
Regulation of the Na-K pump of the rat cortical collecting tubule by aldosterone.
Authors: Palmer L G , Antonian L , Frindt G
Published: May 2004

Activities of Na channels and Na pumps were studied in the rat cortical collecting tubule (CCT) during manipulation of the animals' mineralocorticoid status in vivo using a low-Na diet, diuretics, or administration of exogenous aldosterone. Tubules were isolated and split open to expose the luminal membrane surface. Using the whole-cell patch-clamp technique, activities of the apical Na channels and the basolateral Na pumps were measured in principal cells as the currents inhibited by amiloride (10 microM) and ouabain (1 mM), respectively. Na channel current (INa) was not measurable in CCTs from control animals on a normal diet. INa was approximately 200 pA/cell in CCTs from animals on a low-Na diet or infused with aldosterone using osmotic minipumps. Currents attributable to the Na pump (Ipump) were similar in control animals and animals on a low-Na diet. Maximal currents were approximately 35 pA/cell in both groups, and decreased with hyperpolarization of the cell membrane. In contrast, administration of exogenous aldosterone increased Ipump fourfold. Coinfusion of aldosterone and amiloride in vivo through the minipumps did not affect the induction of INa but reduced the induction of Ipump by 80%. We conclude that the induction of channel activity in this tissue is a direct action of aldosterone, whereas the induction of pump activity may be a consequence of the increased Na traffic through the epithelial cells.