bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
Authors: Sheppard D N , Welsh M J
Published: May 2004

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.
A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production.
Authors: Scamps F , Rybin V , Puceat M , Tkachuk V , Vassort G
Published: May 2004

P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.
Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists.
Authors: Cohen C J , Spires S , Van Skiver D
Published: May 2004

Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.
Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.
Authors: Haynes L W
Published: May 2004

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.
A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.
Authors: Restrepo D , Cronise B L , Snyder R B , Knauf P A
Published: May 2004

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.
A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid.
Authors: Kim D
Published: May 2004

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.
Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.
Authors: Doyle D D , Guo Y , Lustig S L , Satin J , Rogart R B , Fozzard H A
Published: May 2004

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel.
Authors: Xu L , Jones R , Meissner G
Published: May 2004

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)-conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.
Microinjection of strong calcium buffers suppresses the peak of calcium release during depolarization in frog skeletal muscle fibers.
Authors: Csernoch L , Jacquemond V , Schneider M F
Published: May 2004

The effects of high intracellular concentrations of various calcium buffers on the myoplasmic calcium transient and on the rate of release of calcium (Rrel) from the sarcoplasmic reticulum (SR) were studied in voltage-clamped frog skeletal muscle fibers. The changes in intracellular calcium concentration (delta[Ca2+]) for 200-ms pulses to 0-20 mV were recorded before and after the injection of the calcium buffer and the underlying Rrel was calculated. If the buffer concentration after the injection was high, the initial rate of rise of the calcium transient was slower after injection than before and was followed by a slow increase of [Ca2+] that resembled a ramp. The increase in myoplasmic [Mg2+] that accompanies the calcium transient in control was suppressed after the injection and a slight decrease was observed instead. After the injection the buffer concentration in the voltage-clamped segment of the fiber decreased as the buffer diffused away toward the open ends. The calculated apparent diffusion coefficient for fura-2 (Dapp = 0.40 +/- 0.03 x 10(-6) cm2/s, mean +/- SEM, n = 6) suggests that approximately 65-70% of the indicator was bound to relatively immobile intracellular constituents. As the concentration of the injected buffer decreased, the above effects were reversed. The changes in delta[Ca2+] were underlined by characteristic modification of Rrel. The early peak component was suppressed or completely eliminated; thus, Rrel rose monotonically to a maintained steady level if corrected for depletion. If Rrel was expressed as percentage of SR calcium content, the steady level after injection did not differ significantly from that before. Control injections of anisidine, to the concentration that eliminated the peak of Rrel when high affinity buffers were used, had only a minor effect on Rrel, the peak was suppressed by 26 +/- 5% (mean +/- SE, n = 6), and the steady level remained unchanged. Thus, the peak component of Rrel is dependent on a rise in myoplasmic [Ca2+], consistent with calcium-induced calcium release, whereas the steady component of Rrel is independent of myoplasmic [Ca2+].
Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles in cardiac ventricular myocytes. Effects via modification of surface potential.
Authors: Ji S , Weiss J N , Langer G A
Published: May 2004

Modulation of voltage-dependent sodium and potassium currents by charged amphiphiles was investigated in cardiac ventricular myocytes using the patch-clamp technique. Negatively charged sodium dodecylsulfate (SDS) increased amplitude of INa, whereas positively charged dodecyltrimethylammonium (DDTMA) decreased INa. Furthermore, SDS shifted the steady-state activation and inactivation of INa in the negative direction, whereas DDTMA shifted the curves in the opposite direction. These shifts provided an explanation for the changes in current amplitude. Activation and inactivation kinetics of INa were accelerated by SDS but slowed by DDTMA. These changes in both steady-state gating and kinetics of INa are consistent with a decrease of the intramembrane field by SDS and an increase of the field by DDTMA due to an alteration of surface potential after their insertion into the outer monolayer of the sarcolemma. The effect of SDS on the steady-state inactivation of INa was concentration dependent and partially reversed by screening surface charges with increased extracellular [Ca2+]. These amphiphiles also altered the activation of the delayed rectifier K+ current (IK,del), producing a shift in the negative direction by SDS but in the positive direction by DDTMA. These results suggest that the insertion of charged amphiphiles into the cell membrane alters the behavior of voltage-dependent INa and IK,del by changing the surface charge density, and consequently the surface potential and implies, although indirectly, that the lipid surface charges are important to the voltage-dependent gating of these channels.
Block by calcium of ATP-activated channels in pheochromocytoma cells.
Authors: Nakazawa K , Hess P
Published: May 2004

We have investigated the effects of Ca2+ on Na+ influx through ATP-activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1-21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.
High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state.
Authors: Seow C Y , Ford L E
Published: May 2004

The effects of varying pH and ionic strength on the force-velocity relations and tension transients of skinned rabbit skeletal muscle were studied at 1-2 degrees C. Both decreasing pH from 7.35 to 6.35 and raising ionic strength from 125 to 360 mM reduced isometric force by about half and decreased sarcomere stiffness by about one-fourth, so that the stiffness/force ratio was increased by half. Lowering pH also decreased maximum shortening velocity by approximately 29%, while increasing ionic strength had little effect on velocity. These effects on velocity were correlated with asymmetrical effects on stiffness. The increase in the stiffness/force ratio with both interventions was manifest as a greater relative force change associated with a sarcomere length step. This force difference persisted for a variable time after the step. At the high ionic strength the force difference was long-lasting after stretches but relaxed quickly after releases, suggesting that the structures responsible would not impose much resistance to steady-state shortening. The opposite was found in the low pH experiments. The force difference relaxed quickly after stretches but persisted for a long time after releases. Furthermore, this force difference reached a constant value of approximately 8% of isometric force with intermediate sizes of release, and was not increased with larger releases. This value was almost identical to the value of an internal load that would be sufficient to account for the reduction in maximum velocity seen at the low pH. The results are interpreted as showing that both low pH and high ionic strength inhibit the movement of crossbridges into the force-generating parts of their cycle after they have attached to the actin filaments, with very few other effects on the cycle. The two interventions are different, however, in that detained bridges can be detached readily by shortening when the detention is caused by high ionic strength but not when it is caused by low pH.
A rapidly activating and slowly inactivating potassium channel cloned from human heart. Functional analysis after stable mammalian cell culture expression.
Authors: Snyders D J , Tamkun M M , Bennett P B
Published: May 2004

The electrophysiological properties of HK2 (Kv1.5), a K+ channel cloned from human ventricle, were investigated after stable expression in a mouse Ltk- cell line. Cell lines that expressed HK2 mRNA displayed a current with delayed rectifier properties at 23 degrees C, while sham transfected cell lines showed neither specific HK2 mRNA hybridization nor voltage-activated currents under whole cell conditions. The expression of the HK2 current has been stable for over two years. The dependence of the reversal potential of this current on the external K+ concentration (55 mV/decade) confirmed K+ selectivity, and the tail envelope test was satisfied, indicating expression of a single population of K+ channels. The activation time course was fast and sigmoidal (time constants declined from 10 ms to < 2 ms between 0 and +60 mV). The midpoint and slope factor of the activation curve were Eh = -14 +/- 5 mV and k = 5.9 +/- 0.9 (n = 31), respectively. Slow partial inactivation was observed especially at large depolarizations (20 +/- 2% after 250 ms at +60 mV, n = 32), and was incomplete in 5 s (69 +/- 3%, n = 14). This slow inactivation appeared to be a genuine gating process and not due to K+ accumulation, because it was present regardless of the size of the current and was observed even with 140 mM external K+ concentration. Slow inactivation had a biexponential time course with largely voltage-independent time constants of approximately 240 and 2,700 ms between -10 and +60 mV. The voltage dependence of slow inactivation overlapped with that of activation: Eh = -25 +/- 4 mV and k = 3.7 +/- 0.7 (n = 14). The fully activated current-voltage relationship displayed outward rectification in 4 mM external K+ concentration, but was more linear at higher external K+ concentrations, changes that could be explained in part on the basis of constant field (Goldman-Hodgkin-Katz) rectification. Activation and inactivation kinetics displayed a marked temperature dependence, resulting in faster activation and enhanced inactivation at higher temperature. The current was sensitive to low concentrations of 4-aminopyridine, but relatively insensitive to external TEA and to high concentrations of dendrotoxin. The expressed current did not resemble either the rapid or the slow components of delayed rectification described in guinea pig myocytes. However, this channel has many similarities to the rapidly activating delayed rectifying currents described in adult rat atrial and neonatal canine epicardial myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites.
Authors: Venglarik C J , Singh A K , Wang R , Bridges R J
Published: May 2004

Outwardly rectifying 30-50-pS Cl- channels mediate cell volume regulation and transepithelial transport. Several recent reports indicate that rectifying Cl- channels are blocked after addition of ATP to the extracellular bath (Alton, E. W. F. W., S. D. Manning, P. J. Schlatter, D. M. Geddes, and A. J. Williams. 1991. Journal of Physiology. 443:137-159; Paulmichl, M., Y. Li, K. Wickman, M. Ackerman, E. Peralta, and D. Clapham. 1992. Nature. 356:238-241). Therefore, we decided to conduct a more detailed study of the ATP binding site using a higher affinity probe. We tested the ATP derivative, 2',3',O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5'-triphosphate (TNP-ATP), which has a high affinity for certain nucleotide binding sites. Here we report that TNP-ATP blocked colonic Cl- channels when added to either bath and that blockade was consistent with the closed-open-blocked kinetic model. The TNP-ATP concentration required for a 50% decrease in open probability was 0.27 microM from the extracellular (cis) side and 20 microM from the cytoplasmic (trans) side. Comparison of the off rate constants revealed that TNP-ATP remained bound 28 times longer when added to the extracellular side compared with the cytoplasmic side. We performed competition studies to determine if TNP-ATP binds to the same sites as ATP. Addition of ATP to the same bath containing TNP-ATP reduced channel amplitude and increased the time the channel spent in the open and fast-blocked states (i.e., burst duration). This is the result expected if TNP-ATP and ATP compete for block, presumably by binding to common sites. In contrast, addition of ATP to the bath opposite to the side containing TNP-ATP reduced amplitude but did not alter burst duration. This is the result expected if opposite-sided TNP-ATP and ATP bind to different sites. In summary, we have identified an ATP derivative that has a nearly 10-fold higher affinity for reconstituted rectifying colonic Cl- channels than any previously reported blocker (Singh, A. K., G. B. Afink, C. J. Venglarik, R. Wang, and R. J. Bridges. 1991. American Journal of Physiology. 260 [Cell Physiology. 30]:C51-C63). Thus, TNP-ATP should be useful in future studies of ion channel nucleotide binding sites and possibly in preliminary steps of ion channel protein purification. In addition, we have obtained good evidence that there are at least two nucleotide binding sites located on opposite sides of the colonic Cl- channel and that occupancy of either site produces a blocked state.
Modification of cardiac sodium channels by carboxyl reagents. Trimethyloxonium and water-soluble carbodiimide.
Authors: Dudley S C , Baumgarten C M
Published: May 2004

In TTX-sensitive nerve and skeletal muscle Na+ channels, selective modification of external carboxyl groups with trimethyloxonium (TMO) or water-soluble carbodiimide (WSC) prevents voltage-dependent Ca2+ block, reduces unitary conductance, and decreases guanidinium toxin affinity. In the case of TMO, it has been suggested that all three effects result from modification of a single carboxyl group, which causes a positive shift in the channel's surface potential. We studied the effect of these reagents on Ca2+ block of adult rabbit ventricular Na+ channels in cell-attached patches. In unmodified channels, unitary conductance (gamma Na) was 18.6 +/- 0.9 pS with 280 mM Na+ and 2 mM Ca2+ in the pipette and was reduced to 5.2 +/- 0.8 pS by 10 mM Ca2+. In contrast to TTX-sensitive Na+ channels, Ca2+ block of cardiac Na+ channels was not prevented by TMO; after TMO pretreatment, gamma Na was 6.1 +/- 1.0 pS in 10 mM Ca2+. Nevertheless, TMO altered cardiac Na+ channel properties. In 2 mM Ca2+, TMO-treated patches exhibited up to three discrete gamma Na levels: 15.3 +/- 1.7, 11.3 +/- 1.5, and 9.8 +/- 1.8 pS. Patch-to-patch variation in which levels were present and the absence of transitions between levels suggests that at least two sites were modified by TMO. An abbreviation of mean open time (MOT) accompanied each decrease in gamma Na. The effects on channel gating of elevating external Ca2+ differed from those of TMO pretreatment. Increasing pipette Ca2+ from 2 to 10 mM prolonged the MOT at potentials positive to approximately -35 mV by decreasing the open to inactivated (O-->I) transition rate constant. On the other hand, even in 10 mM Ca2+ TMO accelerated the O-->I transition rate constant without a change in its voltage dependence. Ensemble averages after TMO showed a shortening of the time to peak current and an acceleration of the rate of current decay. Channel modification with WSC resulted in analogous effects to those of TMO in failing to show relief from block by 10 mM Ca2+. Further, WSC caused a decrease in gamma Na and an abbreviation of MOT at all potentials tested. We conclude that a change in surface potential caused by a single carboxyl modification is inadequate to explain the effects of TMO and WSC in heart. Failure of TMO and WSC to prevent Ca2+ block of the cardiac Na+ channel is a new distinction among isoforms in the Na+ channel multigene family.
Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina.
Authors: Takahashi K , Dixon D B , Copenhagen D R
Published: May 2004

A sustained high voltage-activated (HVA), nifedipine- and cadmium-sensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina. pH indicator dyes showed that superfusion with NH4Cl alkalinized these cells and that washout of NH4Cl or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4Cl but was suppressed upon NH4Cl washout or application of Na-acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4Cl and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extracellular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4Cl and reduced by washout of NH4Cl or superfusion with Na-acetate. The Na-acetate and NH4Cl washout-dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium-dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.
Swelling activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process.
Authors: Dunham P B , Klimczak J , Logue P J
Published: May 2004

K-Cl cotransport in LK sheep erythrocytes is activated by osmotic swelling and inhibited by shrinkage. The mechanism by which changes in cell volume are transduced into changes in transport was investigated by measuring time courses of changes in transport after osmotic challenges in cells with normal and reduced Mg concentrations. When cells of normal volume and normal Mg are swollen, there is a delay of 10 min or more before the final steady-state flux is achieved, as there is for swelling activation of K-Cl cotransport in erythrocytes of other species. The delay was shown to be independent of the extent of swelling. There was also a delay after shrinkage inactivation of cotransport. Reducing cellular Mg concentration activates cotransport. Swelling of low-Mg cells activates cotransport further, but with no measurable delay. In contrast, there is a delay in shrinkage inactivation of cotransport in low-Mg cells. The results are interpreted in terms of a three-state model: [formula see text] in which A state, B state, and C state transporters have relatively slow, intermediate, and fast transport rates, respectively. Most transporters in shrunken cells with normal Mg are in the A state. Swelling converts transporters to the B state in the rate-limiting process, followed by rapid conversion to the C state. Reducing cell Mg also promotes the A-->B conversion. Swelling of low-Mg cells activates transport rapidly because of the initial predominance of B state transporters. The results support the following conclusions about the rate constants of the three-state model: k21 is the rate constant for a Mg-promoted process that is inhibited by swelling; k12 is not volume sensitive. Both k23 and k32 are increased by swelling and reduced by shrinkage; they are rate constants for a single process, whereas k12 and k21 are rate constants for separate processes. Finally, the A-->B conversion entails an increase in Jmax of the transporters, and the B-->C conversion entails an increase in the affinity of the transporters for K.
Tension transients initiated by photogeneration of MgADP in skinned skeletal muscle fibers.
Authors: Lu Z , Moss R L , Walker J W
Published: May 2004

Addition of MgADP to skinned skeletal muscle fibers causes a rise in Ca(2+)-activated isometric tension. Mechanisms underlying this tension increase have been investigated by rapid photogeneration of ADP within skinned single fibers of rabbit psoas muscle. Photolysis of caged ADP (P2-1(2-nitrophenyl)ethyladenosine 5'-diphosphate) resulted in an exponential increase in isometric tension with an apparent rate constant, kADP, of 9.6 +/- 0.3 s-1 (mean +/- SE, n = 28) and an amplitude, PADP, of 4.9 +/- 0.3% Po under standard conditions (0.5 mM photoreleased MgADP, 4 mM MgATP, pH 7.0, pCa 4.5, 0.18 M ionic strength, 15 degrees C). PADP depended upon the concentration of photoreleased MgADP as well as the concentration of MgATP. A plot of 1/PADP vs. 1/[MgADP] at three MgATP concentrations was consistent with competition between MgADP and MgATP for the same site on the crossbridge. The rate of the transient, kADP, also depended upon the concentration of MgADP and MgATP. At both 4 and 1 mM MgATP, kADP was not significantly different after photorelease of 0.1-0.5 mM MgADP, but was reduced by 28-40% when 3.5 mM MgADP was added before photorelease of 0.5 mM MgADP. kADP was accelerated by about twofold when MgATP was varied from 0.5 to 8 mM MgATP. These effects of MgATP and MgADP were not readily accounted for by population of high force-producing states resulting from reversal of the ADP dissociation process. Rather, the results suggest that competition between MgADP and MgATP for crossbridges at the end of the cycle slows detachment leading to accumulation of force-generating crossbridges. Elevation of steady-state Pi concentration from 0.5 to 30 mM caused acceleration of kADP from 10.2 +/- 0.5 to 27.8 +/- 1.8 s-1, indicating that the tension rise involved crossbridge flux through the Pi dissociation step of the cycle.
Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter.
Authors: Whisenant N , Khademazad M , Muallem S
Published: May 2004

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR-106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3-h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.
Authors: Copello J , Heming T A , Segal Y , Reuss L
Published: May 2004

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- > SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.
Two types of sugar-binding protein in the labellum of the fly. Putative taste receptor molecules for sweetness.
Authors: Ozaki M , Amakawa T , Ozaki K , Tokunaga F
Published: May 2004

Flies have taste cells specifically sensitive to sweetness. It has been suggested that the cells possess two types of receptor sites covering the receptive field of sweetness. By affinity electrophoresis with the site-specific inhibitory polysaccharides, two types of sugar-binding protein were isolated from the labellar extract of the blowfly. These proteins showed consistent sugar-binding specificities and affinities with the two types of receptor sites for sweetness, respectively. The dissociation constant of the protein-sugar complex varies 100-400 mM and the molecular weight of one type of the protein is 27,000, while that of the other is 31,000 or 32,000. Both proteins were water insoluble and were also detected in the isolated chemosensilla. Thus they are probably located on the taste receptor membrane, and the proteins are likely to act as the taste receptor molecules for sweetness in the fly.