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Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating. Key words: voltage clamp fluorimetry, Shaker, activation, inactivation, S4 movement, quenching, review. La fluorimetrie en voltage impose (FVI) utilise des sondes fluorescentes qui se lient de maniere covalente aux residus cysteine introduits dans la proteine et qui emettent une lumiere en fonction de leur environnement. La mesure de cette lumiere emise durant la depolarisation de la membrane montre les modifications dans le taux d'emission a mesure que l'environnement du residu marque change. Cette information permet de correler les evenements d'ouverture et de fermeture du canal avec le mouvement de fractions proteiques specifiques, a une resolution temporelle de l'ordre de la nanoseconde. Utilisee d'abord pour examiner les mouvements d'activation des canaux potassiques Shaker, la FVI est devenue une technique tres precieuse pour comprendre le mecanisme d'ouverture et de fermeture des canaux ioniques. Le present article presente de maniere synthetique la theorie et certaines donnees relatives a l'application de la technique FVI. Bien l'emploi de la FVI ne soit plus limite a l'examen des canaux potassiques sensibles au voltage, puisque la technique est utilisee dans de nombreux canaux ioniques sensibles aux ligands et au voltage, nous nous interesserons particulierement aux etudes menees dans les canaux potassiques Shaker et a ce qu'elles nous ont appris sur l'activation et l'inactivation des canaux. Mots-cles : fluorimetrie en voltage impose, Shaker, activation, inactivation, mouvement du segment S4, desactivation, synthese. [Traduit par la Redaction], Introduction The ability of voltage-gated ion channels to sense and modulate cellular membrane potentials, and in doing so influence virtually all essential cellular processes, underscores their importance in all physiological [...]

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium ([Na.sup.+]) channels (ENaC), the rate-limiting protein in transepithelial [Na.sup.+] vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human [alpha]-, [beta]-, and [gamma]-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive [Na.sub.+] currents and [gamma]-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either [alpha]- or [gamma]-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKC[alpha]/[beta]1 and PKC[zeta]. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKC[alpha]/[beta]1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells. Xenopus oocytes; voltage clamp; cell-attached patches; amiloride-sensitive currents; severe acute respiratory syndrome coronavirus; surface epithelial sodium channels; H441 cells

The mechanisms of recovery from inactivation of the L-type calcium current ([I.sub.Ca]) are not well established, and recovery is affected by many experimental conditions. Little is known about developmental changes of recovery from inactivation of [I.sub.Ca]. We studied developmental changes of recovery from inactivation in [I.sub.Ca] using isolated adult and newborn (1-4 days) rabbit ventricular myocytes. We used broken-patch and perforatedpatch techniques with physiological extracellular ionic concentrations of calcium and sodium and interpulse conditioning potentials of -80 or -50 inV. We also maximized [I.sub.Ca] with forskolin. We found that recovery from inactivation did not differ between adult and newborn cells when either EGTA or BAPTA was used to buffer intracellular calcium. Maximizing [I.sub.Ca] with forskolin slowed recovery from inactivation in newborn but not in adult cells. In contrast, when the intracellular buffering of the cell was left nearly intact (perforated patch), recovery from inactivation (half-time of recovery) in the newborn cells was significantly slower than for the adult cells when either a conditioning potential of -80 mV (140 [+ or -] 9 vs. 58 [+ or -] 4 ms, newborn vs. adult; P < 0.05) or -50 mV (641 [+ or -] 106 vs. 168 [+ or -] 15 ms, newborn vs. adult; P < 0.05) was used. Forskolin significantly increased half-time of recovery for both adult and newborn cells. Dialysis with no calcium buffer showed a slower recovery from inactivation in newborn cells. Intracellular dialysis with a calcium buffer masked differences in recovery from inactivation of [I.sub.Ca] between newborn and adult rabbit ventricular cells. voltage clamp; calcium buffering; perforated patch; forskolin

The Na+-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleosidederived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity ([K.sub.o.5]) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current ([I.sub.max]) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased [I.sub.max.]. Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and [I.sub.max.]. Fludarabine and formycin B were also transported with higher [I.sub.max.] than uridine and moderate affinity (102 [+ or -] 10 and 66 [+ or -] 6 [micro]M, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. [K.sub.o.5] for [Na.sup.+] was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two [Na.sup.+] per nucleoside. This discrepancy in the number of [Na.sup.+] molecules that bind rCNT2 may indicate a low degree of cooperativity between the [Na.sup.+] binding sites. two-electrode voltage clamp; concentrative nucleoside transport; presteady-state currents

Epithelial [Na.sup.+] channels (ENaC) are inhibited by extracellular [Na.sup.+], a process referred to as [Na.sup.+] self-inhibition. We previously demonstrated that mutation of key residues within two furin cleavage consensus sites in [alpha], or one site in [gamma] blocked subunit proteolysis and inhibited channel activity when mutant channels were expressed in Xenopus laevis oocytes (Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong Q, Carattino MD, Johnson JP, Stockand JD, and Kleyman TR. J Biol Chem 279: 18111-18114, 2004). Cleavage of subunits was also blocked by these mutations when expressed in Madin-Darby canine kidney cells, and both subunit cleavage and channel activity were blocked when wild-type subunits were expressed in furin-deficient Chinese hamster ovary cells. We now report that channels with mutant [alpha]-subunits lacking either one or both furin cleavage sites exhibited a marked enhancement of the [Na.sup.+] self-inhibition response, while channels with a mutant [gamma]/-subunit showed a modestly enhanced [Na.sup.+] self-inhibition response. Analysis of [Na.sup.+] self-inhibition at varying [Na.sup.+] indicates that channels containing mutant [alpha]-subunits exhibit an increased [Na.sup.+] affinity. At the single-channel level, channels with a mutant [alpha]-subunit had a low open probability ([P.sub.o]) in the presence of a high external [Na.sup.+] in the patch pipette. [P.sub.o] dramatically increased when trypsin was also present, or when a low external [Na.sup.+] was in the patch pipette. Our results suggest that furin cleavage of ENaC subunits activates the channels by relieving [Na.sup.+] self-inhibition and that activation requires that the [alpha]-subunit be cleaved twice. Moreover, we demonstrate for the first time a clear relationship between ENaC [P.sub.o] and extracellular [Na.sup.+], supporting the notion that [Na.sup.+] self-inhibition reflects a [P.sub.o] reduction due to high extracellular [Na.sup.+]. amiloride; open probability; voltage clamp; Xenopus laevis oocyte; mutagenesis

L-type, voltage-dependent calcium ([Ca.sup.2+]) channels, ryanodine-sensitive [Ca.sup.2+] release (RYR) channels, and large-conductance [Ca.sup.2+] activated potassium ([K.sub.Ca]) channels comprise a functional unit that regulates smooth muscle contractility. Here, we investigated whether genetic ablation of caveolin-1 (cav-1), a caveolae protein, alters [Ca.sup.2+] spark to [K.sub.Ca] channel coupling and [Ca.sup.2+] spark regulation by voltage-dependent [Ca.sup.2+] channels in murine cerebral artery smooth muscle cells. Caveolae were abundant in the sarcolemma of control ([cav-1.sup.+/+] cells but were not observed in cav-1-deficient ([cav-1.sup.-/-]) cells. [Ca.sup.2+] spark and transient [K.sub.Ca] current frequency were approximately twofold higher in [cav-1.sup.-/-] than in [cav-1.sup.+/+] cells. Although voltage-dependent [Ca.sup.2+] current density was similar in [cav-1.sup.+/+] and [cav.sup.-1-/-] cells, diltiazem and [Cd.sup.2+], voltage-dependent [Ca.sup.2+] channel blockers, reduced transient [K.sub.Ca] current frequency to ~55% of control in [cav-1.sup.+/+] cells but did not alter transient [K.sub.Ca] current frequency in [cav-1.sup.-/-] cells. Furthermore, although [K.sub.Ca] channel density was elevated in [cav-1.sup.-/-] cells, transient [K.sub.Ca] current amplitude was similar to that in [cav-1.sup.+/+] cells. Higher [Ca.sup.2+] spark frequency in [cav-1.sup.-/-] cells was not due to elevated intracellular [Ca.sup.2+] concentration, sarcoplasmic reticulum [Ca.sup.2+] load, or nitric oxide synthase activity. Similarly, [Ca.sup.2+] spark amplitude and spread, the percentage of [Ca.sup.2+] sparks that activated a transient [K.sub.Ca], current, the amplitude relationship between sparks and transient [K.sub.Ca] currents, and [K.sub.Ca] channel conductance and apparent [Ca.sup.2+] sensitivity were similar in [cav-1.sup.+/+] and [cav-1.sup.-/-] cells. In summary, cav-1 ablation elevates [Ca.sup.2+] spark and transient [K.sub.Ca] current frequency, attenuates the coupling relationship between voltage-dependent [Ca.sup.2+] channels and RyR channels that generate [Ca.sup.2+] sparks, and elevates [K.sub.Ca] channel density but does not alter transient [K.sub.Ca] current activation by [Ca.sup.2+] sparks. These findings indicate that cav-1 is required for physiological [Ca.sup.2+] spark and transient [K.sub.Ca] current regulation in cerebral artery smooth muscle cells. ryanodine-sensitive [Ca.sup.2+] release channel; large-conductance [Ca.sup.2+]-activated potassium channel; caveolae; voltage-dependent [Ca.sup.2+] channel

The relative contributions of C[a.sup.2+]-induced C[a.sup.2+] release (CICR) versus C[a.sup.2+] influx through voltage-dependent C[a.sup.2+] channels (VDCCs) to excitation-contraction coupling has not been defined in most smooth muscle cells (SMCs). The present study was undertaken to address this issue in mouse urinary bladder (UB) smooth muscle cells (UBSMCs). Confocal C[a.sup.2+] images were obtained under voltage- or current-clamp conditions. When UBSMCs were activated by a 30-ms depolarization to 0 mV, intracellular C[a.sup.2+] concentration ([[C[a.sup.2+].sub.i]) increased in several small, discrete areas just beneath the cell membrane. These C[a.sup.2+] 'hot spots' then spread slowly through the myoplasm as C[a.sup.2+] waves, which continued even after repolarization. Shorter depolarizations (5 ms) elicited only a few C[a.sup.2+] sparks, which declined quickly. The number of C[a.sup.2+] sparks, or hot spots, was closely related to the depolarization duration in the range of ~5-20 ms. There was an apparent threshold depolarization duration of ~10 ms within which to induce enough C[a.sup.2+] transients to spread globally and then induce a contraction. Application of 100 [micro]M ryanodine to the pipette solution did not change the resting [[C[a.sup.2+].sub.i] or the VDCC current, but it did abolish C[a.sup.2+] hot spots elicited by depolarization. Application of 3 [micro]M xestospongin C reduced ACh-induced C[a.sup.2+] release but did not affect depolarization-induced C[a.sup.2+] events. The addition of 100 [micro]M ryanodine to tissue segments markedly reduced the amplitude of contractions triggered by direct electrical stimulation. In conclusion, global [[C[a.sup.2+].sub.i] rise triggered by a single action potential is not due mainly to C[a.sup.2+] influx through VDCCs but is attributable to the subsequent two-step CICR. C[a.sup.2+]-induced C[a.sup.2+] release; C[a.sup.2+]-activated [K.sup.+] current; voltage-dependent C[a.sup.2+] channel

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active [Na.sup.+]/[K.sup.+]-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep [[alpha].sub.1]-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ([approximately equal to] 5%) on addition of extracellular [K.sup.+]. When the enzyme was subjected to voltage jumps under [Na.sup.+]/[Na.sup.+]-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the [E.sub.1]P-[E.sub.2]P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of [Na.sup.+] by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating [K.sup.+] concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing [K.sup.+] concentrations. As a result, the stationary distribution between [E.sub.1] and [E.sub.2] states and voltage-dependent relaxation times can be determined at any time and membrane potential under [Na.sup.+]/[Na.sup.+] exchange as well as [Na.sup.+]/[K.sup.+] turnover conditions.

Unintended block of HERG [K.sup.+] channels is a side effect of many common medications and is the most common cause of acquired long QT syndrome associated with increased risk of life-threatening arrhythmias. The molecular mechanism of high-affinity HERG block by structurally diverse compounds has been attributed to [pi]-stacking and cation-[pi] interactions of a drug (e.g., cisapride) with specific aromatic amino acid residues (Tyr-652 and Phe-656) in the S6 [alpha]-helical domain that face the central cavity of the channel. It also has been proposed that strong C-type inactivation of HERG facilitates or is the primary determinant of high-affinity drug binding. The structurally related, but noninactivating eag channel is insensitive to HERG blockers unless inactivation is induced by specific amino acid mutations [Ficker, E., Jarolimek, W. & Brown, A. M. (2001) Mol. Pharmacol. 60, 1343-1348]. Here we examine the relative importance of inactivation vs. positioning of S6 aromatic residues in determining sensitivity of HERG and eag channels to block by cisapride. The repositioning of Tyr-652 or Phe-656 along the S6 [alpha]-helical domain of HERG reduced sensitivity of channels to block by cisapride. Moreover, independent of inactivation, repositioning of the equivalent aromatic residues in Drosophila eag channels induced sensitivity to block by cisapride. These findings suggest that positioning of S6 aromatic residues relative to the central cavity of the channel, not inactivation per se determines drug block of HERG or eag channels. voltage clamp | delayed rectifier | Xenopus oocyte

The hypothesis that there is a highly conserved, positively charged region distal to the second transmembrane domain in [alpha]-ENaC (epithelial sodium channel) that acts as a putative receptor site for the negatively charged COOH-terminal [beta]- and [gamma]-ENaC tails was tested in mutagenesis experiments. After expression in Xenopus oocytes, [alpha]-ENaC constructs in which positively charged arginine residues were converted into negatively charged glutamic acids could not be inhibited by blocking peptides. These observations provide insight into the gating machinery of ENaC. Liddle mutant; amiloride; inside-out patch; voltage clamp; post-M2 region

We present a novel approach to implementing the dynamic-clamp protocol (Sharp et al., 1993), commonly used in neurophysiology and cardiac electrophysiology experiments. Our approach is based on real-time extensions to the Linux operating system. Conventional PC-based approaches have typically utilized single-cycle computational rates of 10 kHz or slower. In this paper, we demonstrate reliable cycle-to-cycle rates as fast as 50 kHz. Our system, which we call model reference current injection (MRCI); pronounced merci is also capable of episodic logging of internal state variables and interactive manipulation of model parameters. The limiting factor in achieving high speeds was not processor speed or model complexity, but cycle jitter inherent in the CPU/motherboard performance. We demonstrate these high speeds and flexibility with two examples: 1) adding action-potential ionic currents to a mammalian neuron under whole-cell patch-clamp and 2) altering a cell's intrinsic dynamics via MRCI while simultaneously coupling it via artificial synapses to an internal computational model cell. These higher rates greatly extend the applicability of this technique to the study of fast electrophysiological currents such fast [Na.sup.+] currents and fast excitory/inhibitory synapses. Index Terms--Computational instrumentation, electrophysiology, neurophysiology.

Results show that a dipeptide derivative Lys[z(NO(sub)2)]-Pro is a competitive inhibitor of the mammalian peptide transporter PEPT1, which binds from extracellular and intracellular sites with a binding affinity of 5-19 micromolar.

Results reveal that amino acid residues on the polar T1 interface influence the voltage-gated potassium channel in the mammalian brain and heart tissues. Data show that an isosteric mutation in the interface leads to T1 tetramer formation, which destabilize the closed channel suggesting a role for the burried polar residues.

Research was conducted to examine the voltage-dependent outward K+ (K(sub V)) current in intermediate cell of stria vascularis of gerbil cochlea using the whole cell patch-clamp method. The objective was to characterize the K(sub V) current and to consider its importance in the physiological function of the intermediate cell. Results suggest that the K(sub V) conductance in the intermediate cell may stabilize the membrane potential, which is apparently closely related to the endocochlear potential, and may offer an additional route for K+ secretion into the intercellular space.

The potassium-channel gene, AKT3, has recently been isolated from an Arabidopsis thaliana cDNA library. By using the whole-mount and in situ hybridization techniques, we found AKT3 predominantly expressed in the phloem. To study the physiological role of this channel type, AKT3 was heterologously expressed in Xenopus oocytes, and the electrical properties were examined with voltage-clamp techniques. Unlike the plant inward-rectifying guard cell [K.sup.+] channels KAT1 and KST1, the AKT3 channels were only weakly regulated by the membrane potential. Furthermore, AKT3 was blocked by physiological concentrations of external [Ca.sup.2+] and showed an inverted pH regulation. Extracellular acidification decreased the macroscopic AKT3 currents by reducing the single-channel conductance. Because assimilate transport in the vascular tissue coincides with both [H.sup.+] and [K.sup.+] fluxes, AKT3 [K.sup.+] channels may be involved in [K.sup.+] transport accompanying phloem loading and unloading processes.

The mammalian sodium-glucose cotransporter (SGLT) can be found in the apical membrane of epithelial cells facing the lumen where it is known to transport glucose using a sodium electrochemical potential difference. SGLT obtained from Xenopus laevis intestines combine properties similar to mammalian SGLT-1 and SGLT-2. To demonstrate the structure-function relationships of SGLT, Xenopus SGLT was cloned and studied using the two-microelectrode voltage-clamp technique. Results showed that SGLT-1-like protein preferred myoinositol to glucose as a substrate when expressed in Xenopus oocytes.

Effects of zinc, an endogenous neuromodulator in the central nervous system, on glycine receptors (GlyRs) in retinal ganglion cells were investigated by using the whole-cell voltage-clamp technique. [Zn.sup.2+] at low concentration (< 2 [[micro]molar]) potentiated the glycine-induced chloride current and at higher concentration (> 10 [[micro]molar]) suppressed it. This biphasic regulatory action of zinc acted selectively on the fast component of the glycine-induced current mediated by the strychnine-sensitive GlyRs, but not on the slow component mediated by the 5,7-dichlorokynurenic acid-sensitive GlyRs. Dose-response studies showed that 1 [[micro]molar] [Zn.sup.2+] increased the maximum glycine response ([I.sub.[varies]]) and shifted the E[C.sub.50] to the left, suggesting that [Zn.sup.2+] at low concentrations acts as an allosteric activator of the strychnine-sensitive GlyRs. [Zn.sup.2+] at a concentration of 100 [[micro]molar] did not alter [I.sub.[varies]] and shifted the E[C.sub.50] to the right, indicating that [Zn.sup.2+] at high concentrations acts as a competitive inhibitor of the GlyRs. Physiological functions of zinc modulation of GlyRs in retinal ganglion cells are discussed.

Human wild-type M1592V mutant alpha-subunits were expressed in the beta1-subunit of Xenopus laevis oocyte to investigate whether the clinical hyperkalemic periodic paralysis is associated with similar biophysical defects in the human skeletal muscle Na+ channel function. Results showed that the activation curve shifted to less depolarized potentials in M1592 channels to allow expression of a noninactivating component of the Na+ current. The mutated residues were found in domains far from the putative voltage sensor domains of the Na+ channel.

The effects of temperature on pH dependent gating and H+ permeation in voltage-activated H+ channels are explored. Whole-cell and excised-patch tight-seal voltage clamp techniques were used. The observed temperature sensitivity was greater than for most other channels and for H+ conduction in aqueous solution, although was in the range reported for H+ transport mechanisms.

Little is known about the nature of the conformational rearrangements of the voltage sensor and gates, or how the voltage sensor is coupled to the gates. Voltage clamp fluorometry was used to study the voltage sensor (S4) and pore region (P-region) protein motions underlying the slow inactivation of the Shaker K+ channel. Two sequential rearrangements were seen with channels entering first the P-type and then the C-type inactivated state.

Voltage-dependent channels rearrange their voltage sensors in response to changes in the transmembrane electric field. Studies of ion channel gating initially relied on the ability of channels to conduct ions at rates of 10(super6)-10(super7) ions/s. Spectroscopic approaches allow investigations of most of the conformational space of a macromolecule. The challenges of voltage-clamp fluorimetry (VCF) include identification of changes with specific steps along the channel activation/inactivation path.

A two-electrode voltage clamp was applied and Na+/Pi cotransporter (NaPi-2) in Xenopus laevis oocytes were expressed to examine the voltage dependence of the rat renal type II NaPi-2. With inorganic phosphate as the variable substrate, the apparent affinity constant was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. The findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind and the observed voltage dependence arose from the unloaded carrier and the first Na+ binding step.

Experiments were performed to explore the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human or rat isoforms respectively. Saturation at physiological external Cl- concentrations were observed when sodium chloride was substituted with either glucose or glutamate in the external solution. Although an increased rate of deactivation of inward currents were also observed after the substitution of HCO3- and BrO3- as in the case of glucose, a possible interaction between Cl- and the anions are postulated.

By voltage clamp technique and intracellular calcium measurements, we recorded in prawn oocytes simultaneous [[[Ca.sup.2+]].sub.i] and ionic current changes stimulated by external [Mg.sup.2+]. The [[[Ca.sup.2+]].sub.i] response consists of an oscillation period followed by a second state of sustained [[[Ca.sup.2+]].sub.i] level. The oscillation period successively comprises a first [[[Ca.sup.2+]].sub.i] peak, a series of [[[Ca.sup.2+]].sub.i] transients, and a [[[Ca.sup.2+]].sub.i] oscillatory plateau respectively concurrent with an initial transient outward [Mathematical Expression Omitted] current, an inward [Mathematical Expression Omitted] current, and a final [Mathematical Expression Omitted] outward current. By using inhibitor (heparin) or sensitizers (thimerosal or caffeine) of calcium release ER channels, and caged Ins[P.sub.3], we established that Ins[P.sub.3] is the sole second messenger releasing [Ca.sub.2+] from intracellular stores. By sequential substitutions and reapplications of external [Ca.sup.2+], and using econazole (50 [[micro]molar]), a [Ca.sup.2+] influx inhibitor, we documented [Ca.sup.2+] influx during the [[[Ca.sup.2+]].sub.i] oscillatory plateau. The intracellular [Ca.sup.2+] store was depleted with thapsigargin (75-350 nM) in [Ca.sup.2+]-free ASW. Reapplication of external [Ca.sup.2+] evoked a rise in [[[Ca.sup.2+]].sub.i], indicating a store-dependent capacitative [Ca.sup.2+] influx, correlated with a [Mathematical Expression Omitted] outward current increase. No measurable [Ca.sup.2+] release-activated [Ca.sup.2+] current ([I.sub.crac]) could be detected, but was indirectly demonstrated using the sensitivity of the [Mathematical Expression Omitted] channels to [[[Ca.sup.2+]].sub.i]. We propose that the involvement of external [Ca.sup.2+], in the physiological [[[Ca.sup.2+]].sub.i] response of prawn oocytes to external [Mg.sup.2+], consists of a store-dependent capacitative [Ca.sup.2+] influx.

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-D-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal [poly(A).sup.+] mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal [poly(A).sup.+] mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal [poly(A).sup.+] mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.

Electrophysiological measurements were performed on RNA-injected Xenopus oocytes to study the interaction between metabotropic glutamate receptors (mGluRs) and the inwardly rectifying G protein-activated K+ channel (GIRK). The results confirmed that G(sub i)-coupled mGluRs activate the GIRK channel when expressed in Xenopus egg cells in a pertussis toxin-sensitive manner. It was also observed that PLC-coupled mGluRs and GIRK are negatively coupled which is thought to be mediated by the stimulation of the G(sub q)-phospholipase C pathway as well as that of a protein kinase C subtype.

The inactivation of delayed rectified K conductance (g(sub K)) in giant fiber lobe neurons and in squid giant axons was investigated. The results confirmed that a fast phase of inactivation is largely dependent on temperature and on the presence of internal ATP. There also exists a slower component of inactivation which was found to be relatively insesitive to such agents. It was thus indicated that complex inactivation kinetics that are distinct from the known C- and N-type of inactivation and which are susceptible to modification, characterizes the squid delayed rectifier g(sub K).

Whole-cell and excised-patch voltage-clamp technique were used to investigate the effects of substituting heavy water (deuterium oxide, D2O) for water (protium oxide, H2O) on permeation and gating of proton channels in rat alveolar epithelium. The results revealed that D+ permeates proton channels and that D+ interacts specifically with the channel during permeation. It was also found that the time constant of H+ current activation is three times slower in D2O than in H2O. The results further indicated that H+ channel activation requires deprotonation of the channel.

RT4-B8 cells belong to the cell line classified as neuronal type because they do not express glial marker proteins. Standard electrophysiological techniques reveal that this cell line has functional sodium (Na) channels that are of the cardiac phenoype. Different kinds of assays show that RT4-B8 cells do not contain other ion channels and that no other Na isoform exists in these cells.

We have used capacitance measurements with a 1-[mu]s voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500[s.sup.-1] at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-[s.sup.-1], pump transition (the [E.sub.1]-[E.sub.2] conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GATT ([gamma]-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GATT transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

A study of the voltage dependence of charge movement, calcium current and calcium transients, and their inter-relation in normal skeletal muscle cells and in pCAC6 or pCARD1-injected dysgenic myotubes, revealed a sigmoidal dependence of calcium transient. This dot disappear even after the addition of 0.5 millimoles of Cd ions and 0.1 millimoles of La ions in dysgenic myotubes expressing pCAC6. Calcium transient in dysgenic myotubes with pCARD1 expressed a bell shaped voltage dependence and disappeared completely with addition of Cd and La ions.

Intracellular current-clamp and individual voltage-clamp techniques facilitated the analysis of the role of vasoactive intestinal contractor (VIC) during receptor-activated ionic conduction in neurons located in feline colonic parasympathetic ganglia. Experimental results demonstrated the potential of VIC to induce receptor-mediated channel and regulate the calcium channels stimulated by the omega-conotoxin-dependent voltages. These calcium channels are propagated through the endothelin(B) receptor subgroups of neurons.

Examination of the response of colonic myocytes to acetylcholine (Ach) revealed that cationic channel-inhibitors reduced ca ion currents that varied with voltage. Ach also caused an inward current when the depolarization voltage was clamped at -70mv in the presence of nifedipine or in dialyzed cells. Selective blockers can be developed on the basis of these results and used to control the contractions in the gastro-intestinal tract. They can also be used to evaluate the role of Ca ion currents in physiological responses during the use of Ach.

The mechanism and location of operation at the molecular level of 4-aminopyridine (4AP), a preferential blocker of voltage-activated K+ currents in excitable membranes, were studied in Kv3.1 and Kv3.1, mammalian representatives of the Drosophila Show and Shab subfamilies of voltage-gated K+ channels. The gating mechanism protects the region to which 4AP binds in both channels.

Voltage-clamp methods were used to analyze the membrane currents obtained from follicle stimulating hormone (FSH) and acetylcholine (ACh) in follicle enclosed oocytes of Xenopus laevis. A gradual uniform inward component related to a rise in membrane conductance, mostly to Cl- (Sin) was observed for FSH and ACh responses. Osmolarity did not affect the rapid and temporary Cl- current (Fin), particularly evident for ACh. The follicular cells contain FSH and ACh receptors and the Cl- channels mediate the Fin and Sin currents.

Salamander rods need to exhibit an increased concentration of 8-aminoethylamino-cGMP (AEA-cGMP) rather than 8-Br-cGMP to ensure the opening of the channels in excised membrane patches. The cGMP moiety is linked to an affinity resin, and the replacement of the two cGMP phases serve as an attachment between the two. Amine replacement at C8 causes the lack of influence of AEA-cGMP. A positive charge at physiological pH is noted at C8, which is the basic amine tail. Patch-clamp examinations are conducted to study the analogues of products derived from modifying the locations of the cGMPases.

The whole-cell patch clamp method and a perfused pipette were used to estimate calcium currents (Ica) in frog venticular myocytes. Ica, stimulated by isoproterenol or forskolin, was studied for the influence of internal perfusion with hydrolysis resistant guanosine 5'-thiotriphosphate (GTP-gamma-S), guanylyl 5'-imidodiphosphate (GppNHp) or guanosine 5'-triophosphate analogues, to understand the function of G proteins in the control of Ica in intact cells. The GTP analogue with which the G protein is activated could influence the effectiveness of G protein effector molecule interactions.

Discrete AT2 receptor-mediated effects of angiotensin II (ANG II) were proved in IA and IK in neonate neurons that were specially cultured. Data obtained from the study provided an electrophysiological basis for behavioral and physiological effects induced by the ANG II receptor subtype within the brain. Results of previous studies suggested that ANG II induces an augmentation in the net outward ionic current within neurons that are cocultured from neonate rat hypothalamus and brain stem. In the present study, whole cell voltage clamp methods were employed to determine the currents regulated by AT2 receptors. It was concluded that transient K+ current and delayed-rectifier K+ current are induced in cultured neurons.

The patch-clamp method was used to directly study the densities of Ca2+ currents in single circular smooth muscle cells isolated from the rat colon. The correlation between the age-related alterations in Ca2+ channels and the motility function of the colon was analyzed. Evidence to prove that the average Ica density of circular smooth muscle cells of rat colon is altered during growth and aging was obtained.

Rapidly inactivating A-type K+ current was seen in all the adrenal zona fasciculata (AZF) cells which had been dissociated by enzymes. A Boltzmann function was used to study the voltage-dependent activation and inactivation. Transitions dependent on and independent of voltage in pathways between closed, open and inactivated states were included in the gating kinetics of IA. The sigmoidal kinetics which were dependent on voltage activated with the IA to fit the n (to the 4th power) h formalism. Many minutes of whole cell recording showed slowing of the IA inactivation kinetics. Temporary K+ currents in neurons and muscle resemble the IA in AZF cells biophysically and pharmacologically. The inactive and crime cells, which produce steroid hormones, and their reaction to the K+ currents is unclear.

Voltage-gates Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells were examined by the patch clamp technique. Low voltage-activated, rapidly inactivating Ca2+ current was observed in most of the cells while the other cells had features of T-type Ca2+ current. Boltzmann function was adopted to fit the activation of the current. AZF cells expressed a single Ca2+ channel subtype and the voltage-dependent gating and kinetic properties of T current were studied. The T channel activation, inactivation, deactivation and reactivation show voltage independent transition that controls the rate at high voltages. T-type Ca2+ channels, which are found in secretory cells, contain bovine AZF cells. The Ca2+ current enters through these channels, and cortisol secretion is mediated in these cells.

Pretreatment of K+-deprived rat diaphragm with insulin caused a 38% increase in input resistance and a decline in K+ current in the skeletal muscle, resulting in membrane depolarization and deactivation. Depolarization in K+-depleted muscle is induced by insulin-associated blockage of K+ channels and is maintained by triggering noninactivating Na+ channels. Conductance in K+-depleted fibers is opposed by a large leakage current in the absence of insulin. The diminished outward current is distinct from the inward current carried by Ca2+, as demonstrated by the decline in insulin-induced current in nitrendipine.

Sodium current (I(Na)) gating and its voltage dependence are investigated by making use of the loose-patch voltage-clamp technique on 19 fast-twitch human intercoastal skeletal muscle fibers I(Na), inactivated by the accumulation of potassium in the extracellular space surrounding a muscle fiber, resulting in its depolarization. Gating properties and the amplitude of INa are different for the two types of fibers, fast-twitch oxidative-glycolytic type IIa and the fast-twitch glycolytic type IIb. Type IIb fibers exhibit greater INa amplitude and are more susceptible to inactivation of INa at greater negative potentials than type IIa fibers.

Guanosine 3',5'-cyclic monophosphate (cGMP) and adenylate cyclase-adenosine 3'5'-cyclic monophosphate (cAMP) systems are responsible for inducing calcium currents ICa in frog skeletal muscle. cGMP and cAMP play opposite roles in the regulation of slow ICa in the mammalian cardiac muscle, with cAMP inducing the current and cGMP retarding it. cAMP and cGMP, when applied to the cut end of fibers, cause a decline in the time of peak current in the membrane potentials and shift the current-voltage characteristics to incline toward negative potentials.

Permeation properties of the cystic fibrosis transmembrane conductance regulator (CTFR) were studied with the aid of diphenylamine-2-carboxylate (DPC) and flufenamic acid (FFA) blocking agents. The blockade is dependent on voltage, affecting currents at positive potentials alone, which implies an open-channel block. Depolarizing membrane voltages cause continuous bursts for up to ten seconds, hyperpolarizing voltages cause interrupted bursts. Vm flickers attested to the permeation of drugs through the membrane. The binding site for both drugs was the same FFA and DPC voltage-dependent blockage aid in the study of CTFR structure-functions.

Subpopulations of cells of lingual epithelium from Necturus maculosus were distinguished by the presence of voltage-gated currents. Of the two groups identified, one contained Na+, K+ and Ca2+ currents and the other K+ currents alone. This is also observed in basal taste cells. In electrically coupled taste cells, cell coupling was stronger in dye-coupled receptor cells. Cells possessing voltage-gated currents were electrically coupled. Variation in chemosensitivity is the basis of electrophysiological differentiation in taste receptor cells.