Whole blood leukocytes isolation with microfabricated filter for cell analysis.

The flow cytometric analysis of leukocytes in whole blood usually requires isolation of leukocytes from other components of whole blood. Density gradient centrifugation and red blood cell lysis are the most commonly used methods to separate leukocytes but come with significant limitations. We report the results of the evaluation of a microfabricated filtration device for blood preparation that separates erythrocytes from leukocytes based on their size and mechanical properties. The microfabricated filter evaluated here requires a rapid and simple procedure and results in high leukocytes recovery without introducing bias among the leukocyte subpopulations. The filter removes erythrocytes, platelets, plasma proteins, and unbound staining reagent. This gentle filtration process produces very clean stained leukocytes for cytometric analysis without any apparent damage to leukocytes.

Ca(2+)-dependent components of inactivation of unitary cardiac L-type Ca(2+) channels.

A Ca(2+) ion-dependent inactivation (CDI) of L-type Ca(2+) channels (LCC) is vital in limiting and shaping local Ca(2+) ion signalling in a variety of excitable cell types. However, under physiological conditions the unitary LCC properties that underlie macroscopic inactivation are unclear. Towards this end, we have probed the gating kinetics of individual cardiac LCCs recorded with a physiological Ca(2+) ion concentration (2 mM) permeating the channel, and in the absence of channel agonists. Upon depolarization the ensemble-averaged LCC current decayed with a fast and a slow exponential component. We analysed the unitary behaviour responsible for this biphasic decay by means of a novel kinetic dissection of LCC gating parameters. We found that inactivation was caused by a rapid decrease in the frequency of LCC reopening, and a slower decline in mean open time of the LCC. In contrast, with barium ions permeating the channel ensemble-averaged currents displayed only a single, slow exponential decay and little time dependence of the LCC open time. Our results demonstrate that the fast and slow phases of macroscopic inactivation reflect the distinct time courses for the decline in the frequency of LCC reopening and the open dwell time, both of which are modulated by Ca(2+) influx. Analysis of the evolution of CDI in individual LCC episodes was employed to examine the stochastic nature of the underlying molecular switch, and revealed that influx on the order of a thousand Ca(2+) ions may be sufficient to trigger CDI. This is the first study to characterize both the unitary kinetics and the stoichiometry of CDI of LCCs with a physiological Ca(2+) concentration. These novel findings may provide a basis for understanding the mechanisms regulating unitary LCC gating, which is a pivotal element in the local control of Ca(2+)-dependent signalling processes.

Physiologic gating properties of unitary cardiac L-type Ca2+ channels.

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca(2+) ions through sarcolemmal L-type Ca(2+) channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca(2+) ions permeating the channel. Unitary LCC currents were recorded with 2mM external Ca(2+) ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of -12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53+/-0.02 ms at -30 mV to 1.08+/-0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca(2+) ion entry. Thus, the average amount of Ca(2+) ions entering through an individual LCC opening ( approximately 300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca(2+) ion concentration. Moreover, they provide insight into local Ca(2+)-induced Ca(2+) release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.

Automated voltage-clamp technique.

The voltage-clamp electrophysiology method is the gold standard for measuring the function of ion channels. In the past, this technique has had limited applicability in pharmaceutical drug discovery because of its low throughput, steep learning curve, and challenges in standardization of the experiments. Recently, new electrophysiology platforms have been developed, which are based on the use of planar electrodes. One key advantage of the new electrode geometry is that it makes the process of cell-to-electrode sealing amenable to automation, thus increasing the throughput and significantly reducing the skill-set needed to run the experiments. The further addition of computer-controlled fluidics, voltage-clamping electronics, and automated data handling makes it possible to perform multiple electrophysiology experiments in parallel with a high degree of consistency and in completely automated mode. Among the new offerings for automated voltage clamp, one of the systems, PatchXpress/Sealchip, is quickly becoming the new gold standard for the quantification of ion channel function. In this chapter, we provide an overview of the new planar patch-clamping platforms and describe how electrophysiology experiments are performed on the PatchXpress/Sealchip automated system.

Efficient characterization of use-dependent ion channel blockers by real-time monitoring of channel state.

Ion channels are important therapeutic targets for the treatment of a variety of conditions. Among ion channel blocking agents, use-dependent inhibitors can be especially effective therapeutic agents. Use dependence allows the selective inhibition of hyperactive neurons or tachycardiac myocytes, while minimizing effects on cells with normal activity. For voltage-gated channels, the use-dependent compounds typically bind to and inhibit a particular kinetic state that is induced by specific voltage changes. Drug discovery programs that focus on this class of drugs need to rank the use dependence of the compounds. A meaningful comparison among different molecules requires voltage clamp-based assays with continuous voltage control and compensation for or elimination of electrode drift-related effects. A method was developed based on automated electrophysiology in which voltage and frequency dependence of voltage-gated ion channel blockers can be compared using a protocol in which voltage error is compensated for in real time.

Ca(2+) signaling in cardiac myocytes overexpressing the alpha(1) subunit of L-type Ca(2+) channel.

Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.

Identifying modulators of hERG channel activity using the PatchXpress planar patch clamp.

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.

A benchmark study with sealchip planar patch-clamp technology.

Although conventional patch-clamp methods provide high information content, they are labor-intensive and suffer from low throughput and high overall cost. Several approaches for achieving high throughput electrophysiology are under development, among which microchip-based patch-clamp systems uniquely achieve a higher degree of miniaturization, faster perfusion and mixing, and lower reagent cost without losing information content. The goal of this study was to establish a benchmark for our biochip technology with 52 chips tested sequentially. We demonstrate that our microfabrication and processing technology is sufficiently mature to produce a consistent hole size. We further demonstrate high-quality planar whole-cell patch clamping with >75% overall success rate at achieving gigaohm seals, followed by stable whole-cell access lasting at least 15 min with access resistance (Ra) below 15 MOmega and membrane resistance (Rm) above 200 MOmega. These biochips are ideally suited for high throughput compound screening for ion channel targets.

Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds.

Unintended inhibition of the cardiac potassium channel human ether-a-go-go-related gene (hERG) is considered the main culprit in drug-induced arrhythmias known as torsades de pointes. Electrophysiology is the most reliable in vitro screening method for identifying potential cardiac hERG liabilities, but only the recent advent of planar electrode-based voltage clamp electrophysiology promises sufficient throughput to support the drug testing needs of most drug discovery programs. We have assessed the reliability of this new format of the voltage clamp technology in measuring the activity of small molecules on the hERG channel. Based on the results herein of a screening against a panel of well-characterized hERG-active and -inactive molecules, we demonstrate that planar electrode electrophysiology, utilizing the Sealchip and PatchXpress technology platform (AVIVA Biosciences Corp., San Diego, CA), is comparable to traditional electrophysiology based on glass micropipettes in its reliability and data content. The new technology will allow significantly higher throughput and more thorough testing of pharmaceutical compounds.

Roles of PKC Isoforms in PMA-Induced Modulation of the hERG Channel (Kv11.1).

Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCß, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R(2)= 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.

Modulation of the gating of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions.

Although a considerable number of studies have characterized inactivation and facilitation of macroscopic L-type Ca(2+) channel currents, the single channel properties underlying these important regulatory processes have only rarely been examined using Ca(2+) ions. We have compared unitary L-type Ca(2+) channel currents recorded with a low concentration of Ca(2+) ions with those recorded with Ba(2+) ions to elucidate the ionic dependence of the mechanisms responsible for the prepulse-dependent modulation of Ca(2+) channel gating kinetics. Conditioning prepulses were applied across a wide range of voltages to examine their effects on the subsequent Ca(2+) channel activity, recorded at a constant test potential. All recordings were made in the absence of any Ca(2+) channel agonists. Moderate-depolarizing prepulses resulted in a decrease in the probability of opening of the Ca(2+) channels during subsequent test voltage steps (inactivation), the extent of which was more dramatic with Ca(2+) ions than Ba(2+) ions. Facilitation, or increase of the average probability of opening with strong predepolarization, was due to long-duration mode 2 openings with Ca(2+) ions and Ba(2+) ions, despite a decrease in Ca(2+) channel availability (inactivation) under these conditions. The degree of both prepulse-induced inactivation and facilitation decreased with increasing Ba(2+) ion concentration. The time constants (and their proportions) describing the distributions of Ca(2+) channel open times (which reflect mode switching) were also prepulse-, and ion-dependent. These results support the hypothesis that both prior depolarization and the nature and concentration of permeant ions modulate the gating properties of cardiac L-type Ca(2+) channels.

Modulation of the conductance of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions.

The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca(2+) channel currents using Ca(2+) and Ba(2+) ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca(2+) channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca(2+) channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba(2+), 18 pS for 5 mM Ba(2+), and 6 pS for 5 mM Ca(2+) ions; these values were 70% greater than the conductance of Ca(2+) channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca(2+) channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca(2+) channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.

Ion-channel assay technologies: quo vadis?

Although many existing methods are used to study the functions of ion channels and to screen lead compounds for important ion-channel targets, new technologies are being developed for improved performance. It is important to identify the advantages and disadvantages of each technology. In this review, we segment the ion-channel assay market according to distinguishable applications, and compare the needs of each market segment with the capabilities of different technologies in terms of multiple assay attributes. We further discuss the future directions in the development of ion-channel assays.

Alterations in properties of L-type Ca channels in aging rat heart.

Previous studies of whole-cell L-type Ca currents in aging heart have demonstrated an increase in the peak Ca current magnitude in proportion to the increase in membrane area, and a slowing of the time course for inactivation. However, the single-channel mechanisms underlying this upregulation, and for the slowed inactivation are not known. We have therefore compared the properties of single L-type Ca channel currents recorded from ventricular myocytes obtained from young adult (3 month), adult (6-8 month) and aging (24 month) Wistar rats, using 5 m m Ba ions as the permeant ion. We report that the peak ensemble-averaged single Ca channel currents from aging heart (-280+/-57 fA) were enhanced compared to those from young adult (-137+/-16 fA), or adult hearts (-144+/-38 fA). This surprising result was related, in part, to an apparent increase in the number of active Ca channels per patch in aging (1.90+/-0.23) v young adult (1.33+/-0.19) or adult heart (1.50+/-0.2). Moreover, there was an increase in the time constant for inactivation of the ensemble-averaged Ca currents of aging (471+/-169 ms), compared with young adult (198+/-43 ms), or adult heart (196+/-32 ms). The aging-related changes were also traced to alterations in single Ca channel gating, including an increase in the average probability of being open, and an increase in the availability of single Ca currents in aging heart. In contrast, the unitary Ca current amplitude was unchanged with aging. These novel findings suggest that the compensatory increase in the L-type Ca currents during aging is a consequence of an apparent increase in both the number, and the activity of individual L-type Ca channels.

The ryanodine receptor modulates the spontaneous beating rate of cardiomyocytes during development.

In adult myocardium, the heartbeat originates from the sequential activation of ionic currents in pacemaker cells of the sinoatrial node. Ca(2+) release via the ryanodine receptor (RyR) modulates the rate at which these cells beat. In contrast, the mechanisms that regulate heart rate during early cardiac development are poorly understood. Embryonic stem (ES) cells can differentiate into spontaneously contracting myocytes whose beating rate increases with differentiation time. These cells thus offer an opportunity to determine the mechanisms that regulate heart rate during development. Here we show that the increase in heart rate with differentiation is markedly depressed in ES cell-derived cardiomyocytes with a functional knockout (KO) of the cardiac ryanodine receptor (RyR2). KO myocytes show a slowing of the rate of spontaneous diastolic depolarization and an absence of calcium sparks. The depressed rate of pacemaker potential can be mimicked in wild-type myocytes by ryanodine, and rescued in KO myocytes with herpes simplex virus (HSV)-1 amplicons containing full-length RyR2. We conclude that a functional RyR2 is crucial to the progressive increase in heart rate during differentiation of ES cell-derived cardiomyocytes, consistent with a mechanism that couples Ca(2+) release via RyR before an action potential with activation of an inward current that accelerates membrane depolarization.

Manifestaciones clínicas bucales en pacientes con giardiasis y amebiasis / Oral clinical manifestations in patients with giardiasis and amebiasis

El presente trabajo se realizó con el propósito de evidenciar las posibles manifestaciones clínicas bucales encontradas en pacientes conn giardiasis y amebiasis. Para la elaboración del estudio se estableció una población conformada por 120 pacientes que acudieron al Departamento de Pediatría del Hospital Hipólito Unánue en un lapso de 3 meses, a quienes se les elaboró un registro clínico que evaluaba el estado de severidad de la enfermedad, edad y sexo. Los resultados muestran lo siguiente: los datos obtenidos sugieren que se presenta mayor númnero de manifestaciones bucales en pacientes con giardiasis que en los casos de amebiasis