IONFLUX APPLICATIONS

ONE FLEXIBLE SYSTEM - MANY ASSAYS

The IonFlux system, with its advanced fluidic control, plate-integrated recording and compound delivery, is capable of satisfying the needs of the most demanding ion channel screening assays. The speed of recording, managed by the parallel execution of all experimental patterns, married with the system's unique continuous flow system, provides the perfect platform for the study of ligand-gated as well as voltage-gated channels. Below are example of assays performed on the IonFlux System


LIGAND GATED

The microfluidic well plate technology employed by the IonFlux system enables unlimited flexibility and synchronous compound additions across the full plate. This flexibility leads to increased throughput in difficult-to-execute ligand gated assays. Fast compound displacement and continuous perfusion dramatically improve ligand movement and washout, enabling the study of challenging targets such the allosteric modulators (open and closed pore), nACh receptors and NMDA receptors. Below are a few examples:

GABA Receptors

HEK-hGABAA receptor chloride channel recordings. (A) Superimposed plots of ICl amplitude (at Vhold -80mV throughout), nor- malized to the value at 30mM, versus [agonist] for GABA, and isoguvacine. (Inset) IonFlux ensemble ICl traces evoked by applying an ascending series of [GABA] to one ensemble of cells, note the development of increasingly faster desensitization at higher concentrations of GABA. Hill fits to the dose–response curves yielded EC50 values of 4.8 0.8 mM (nH 2.4 0.5, n = 16 ensembles) and 2.2 0.1 mM (nH 1.4 0.1, n = 16 ensembles) for GABA and isoguvacine, respectively. (B) Superimposed dose–response plots for % potentiation of ICl evoked by EC20 [GABA], in the presence of increasing concentrations of diazepam and triazolam. The lines are Hill fits to the data with fitted parameters of EC50 3.6 1.9 nM (nH 0.7 0.3, n = 4 ensembles) for triazolam, and 0.50 0.05 mM (nH 1.6 0.2, n = 5 ensembles) for diazepam. (Inset) Traces showing the increase of ICl evoked by an EC20 GABA concentration after adding 3–30 nM triazolam to the ECS (as indicated). (C) Superimposed raw traces from the cumulative GABA EC50 determination. Comparing the control ensemble current responses (dotted) and responses obtained during simultaneous exposures to GABA and 3mM diazepam shows the potentiation of ICl at submaximal [GABA] (<10mM). (D) Superimposed mean three-point dose–response curves from the control GABA applications (unmodulated) and 3mM diazepam-exposed ensembles. From the Hill plot parameters shown on the graph, it can be seen that this dose of diazepam approximately doubled the apparent sensitivity to GABA. Note the absence of modulation at the highest [GABA] in (C) and (D)

nACh Receptors

hNAChR(a1)ICatrecordings.(A) PlotsofmeanensembleICatamplitude(atVhold -80mVthroughout)versus[ACh]fora1-hNAChRcells (see fitted Hill parameters on the graph) in the IonFlux instrument. (B) Recovery from desensitization in which ICat amplitude evoked by a 10 mM ACh application following closely after an identical initial exposure (Control response) is plotted against the intervening recovery time. The line is an exponential fit to the data (t = 1.9 0.1 s). (C) Superimposed recordings of 10 mM ACh-evoked ICat during agonist exposures of 1 s (dotted trace, gray bar) and 3 s duration (solid trace, black bar). hNAChR, human nicotinic acetylcholine receptor. 


VOLTAGE GATED

Voltage-gated ion channels are responsible for determining the shape, duration, and frequency of action potentials in excitable cells. Given this important physiological role they have been heavily-pursued targets in drug discovery efforts. Voltage-gated channels also control important physiological functions in non-excitable cells, for example secretory epithelial cells. The flexibility in fluidics and the parallel execution of experiments, decreases overall assay time to completion for voltage-gated assays. Continuous flow provides complete washouts and solution exchange, increasing the accuracy of recorded data. Below are a few examples.

hERG (KV 11.1)

CHO-hERG potassium channel recordings. (A) Sample recording of ensemble hERG current (lower trace) determined using the 50/50 voltage command protocol, shown schematically above the trace. All hERG voltage protocols were applied at 6-s intervals. (B) Mean activation voltage dependence for peak hERG tail current (in CHO cells) with the Boltzmann fit parameters displayed on the graph. The inset shows the voltage command protocol. (C) Mean plot for the fully activated IV relationship of the tail current, based on repolarization steps of variable magnitude as shown in the inset. This plot clearly reproduces the known rectification properties of the hERG channel. (D) Dose– response curves for block of peak ensemble outward tail hERG currents, obtained using the 50/50 protocol as in (A). The curves show % block of outward tail current (measured at - 50 mV; large arrows are voltage commands) and IC50s calculated for terfenadine (25 1 nM), cisapride (58 5 nM), and quinidine (0.74 0.13 mM) using Hill fits to the data. hERG, human Ether-a`-go-go–related gene; IV, current versus voltage. 

KV 2.1

hKV2.1 potassium channel recordings. (A) Plot of the longevity of ensemble CHO-hKV2.1 current amplitude, with samples at 5-min intervals, for all ensembles on one IonFlux consumable plate. Vhold - 80 mV, Vtest + 70 mV (30 ms); pulse sequence applied at 0.2 Hz throughout. Actual current sweeps corresponding to the sweeps marking the 5-min intervals are shown above the graph for a repre- sentative ensemble, indicated in the plot by filled symbols. The y-axis reflects the amplitude of ensemble IKV2.1, plotted against elapsed time. (B) Current versus voltage plot for mean ensemble hKV2.1 potassium current recorded (n = 10 ensembles), with the voltage command family (Vtest - 80 to + 100 mV, 30 ms) and data from a single cell in the equivalent conventional patch clamp experiment shown as insets. Note the increase in total current level resulting from summation of the currents from the ensemble cell population. (C) TEA block of hKV2.1 current for a representative ensemble recording channel. The upper panel graphically represents the [TEA] applied cumulatively (with no washout between doses) from successive compound channels, while the lower plot shows peak IKv2.1 (filled symbols) stimulated at a frequency of 0.2 Hz (5 s intervals) versus elapsed time. (D) Dose–response curves for percentage block of hKV2.1 mean ensemble current by TEA+ and quinidine with Hill fits to the data (see text for details). CHO, Chinese hamster ovary; TEA, tetraethylammonium chloride. 

Nav 1.7 and 1.8

HEK-hNaV1.7 and hNaV1.8 sodium channel recordings. (A) Superimposed IV plots for hNaV1.7 (left axis) and 1.8 current (right axis). Vhold - 80 mV, prestep to - 120 mV (50 ms), and Vtest - 70 to + 40 mV (50 ms). Pulse sequence applied at 1 Hz. (B) Plot of INa amplitude versus elapsed time during a cumulative dose–response experiment for NaV1.7. Pulses are as in (A), Vtest -10mV (duration 50ms, frequency 1Hz). Each lidocaine concentration was applied from a different compound channel, pressurized successively. (Inset) The difference current sensitive to 10mM lidocaine for a representative ensemble recording. (C) Plot of INa amplitude versus elapsed time during a dose–response experiment for NaV1.8, with transient washout of lidocaine between successive compound applications. Pulses as in (A), Vtest-10mV (duration 50ms, frequency 1Hz). (Inset) The difference current sensitive to 30mM lidocaine for a representative ensemble. (D) Dose–response plots for % block of hNaV1.7 by lidocaine (filled symbols) and for block of hNaV1.8 by lidocaine (open symbols) and tetracaine (half-filled symbols). The Hill fit parameters were as follows: for NaV1.7 and lidocaine, IC50 2.670.23mM (nH 1.5 0.1, n = 10); for NaV1.8 and lidocaine, IC50 0.32 0.01 mM (nH 0.8 0.02, n = 16); and for NaV1.8 and tetracaine, IC50 0.034 0.006 mM (nH 0.9 0.1, n = 35). HEK, human embryonic kidney.