Sodium channels have been challenging to study on automated platforms due to the precise voltage control and high temporal resolution needed to control and measure the rapid conformational changes of these ion channels. In this application note, a high-throughput electrophysiology assay was optimized and validated for hit identification and confirmation of NaV1.5 inhibitors. The IonWorks Barracuda® Automated Patch Clamp System provides a continuous voltage clamp, sophisticated voltage protocols, flexible recording parameters and a long assay window to identify and characterize NaV1.5 blockers.
Sodium channels are integral membrane proteins that form ion channels within the plasma membrane of living cells. They play crucial roles in excitable cells since they serve as the pathway for rapid, excitatory currents, which cause rapid depolarization of the transmembrane potential. Over the years a number of sodium channel blockers have been developed into drugs, with the two most important mechanisms of action as “usedependent” (e.g. local anesthetics) and “state-dependent” (e.g. anticonvulsants). The “use-dependent” block allows these drugs to preferentially act on cells or tissues exhibiting higher excitability, which provides therapeutic selectivity much desired in new drug development. To study such properties, the patch-clamp method remains the “gold standard” because it provides precise voltage control and high temporal resolution, which are necessary to accurately measure these fast ionic currents. Here, we designed and validated a cell-based assay for the NaV1.5 channel, using the IonWorks Barracuda system. We set up a single concentration mock screen of known sodium channel blockers to identify the location of the hits in a 384-compound plate. Follow-up experiments on the hits were then performed to identify use- and state-dependent blockade using a train of depolarizing pulses. The potency of these compounds was assessed using elevenpoint concentration curves performed in duplicate on 384-well compound plates. The system features 384 simultaneous recording sites capable of generating an unsurpassed throughput of > 1,100 data points per hour.
Material and methods
Chinese hamster lung (CHL) cells stably expressing the human SCNA5 (NaV1.5) gene were used. All test compounds were obtained from Sigma-Aldrich and freshly prepared each day following a double-blind procedure. The final test plate contained 0.1% DMSO. All experiments were performed in the Population Patch Clamp™ (PPC) mode.1,2 Internal buffer contained (in mM): 100 K+ gluconate, 30 KCl, 3.2 MgCl2, 5.0 EGTA, 5.0 HEPES, pH 7.25 with KOH.1,2 External buffer was Phosphate Buffered Saline (PBS, Gibco Cat #14040). Two data filtering criteria were implemented: 1) seal resistance > 50 MΩ, and 2) seal resistance change < 50% (pre vs. post). The data were analyzed and plotted using Prism 5 software.
Validation of NaV1.5 screening protocol
Voltage protocols with a train of repetitive depolarizations are typically used to analyze the use-dependent block of sodium channels, as drug affinity varies with gating state of the channel. To determine the appropriate voltage protocol for this assay, we first characterized the biophysical parameters of NaV1.5 channels on the IonWorks Barracuda system (Figure 1).
Based on the biophysical profiles of NaV1.5 channel, we designed and validated a 30-pulse, 10 Hz train protocol for this study (Figure 2). In wells with buffer only added, the currents remain stable under all conditions.
Screening for use-dependent NaV1.5 blockers
High-throughput screening of a compound library typically starts with a single-point screening assay, which is followed by a multiple concentration confirmation of ontarget activity. We chose a number of wellcharacterized blockers of NaV1.5 channels such as lidocaine and tetracaine for usedependent block as well as tetrodotoxin for state-dependent block.
For single-point screening, the final concentration of all compounds was 10 μM in 0.1% DMSO. These compounds were randomly added to a single well of a 384-well compound plate. In order to simulate a true screening of a compound library, a double-blind procedure was used to prevent bias in the results.
As shown in Figure 3, tetracaine blocked the NaV1.5 currents in a use-dependent manner, in which the 30th pulse of the train protocol exhibited more pronounced current reduction than the first pulse. This characteristic “use-dependent block” is not observed with tetrodotoxin.
We further analyzed the hits from a single point screening assay by assessing hit potency in duplicate using eleven point concentration curves mapped out on 384-well plates for lidocaine, tetracaine and TTX (Figure 4).
Potency estimates at 1st pulse and 30th pulse were calculated using a fourparameter logistic fit of the concentration response curves. As shown in Table 1, IC50 values for lidocaine and tetracaine were significantly higher at first pulse when compared with those at 30th pulse, thereby validating the use-dependent nature of the block (Figure 5).
|Exp||Pulse 30 IC50 (μM)||Pulse 1 IC50 (μM)|
Table 1. Summary of IC50 values. Data scatter from three repeats of the same experiment indicating highly consistent results.
We designed and validated a highthroughput electrophysiology assay for screening and characterizing inhibitors of the NaV1.5 channel. By using known blockers of Na channels, we were able to separate the compounds based on their use-dependent characteristics. Tetracaine showed the most use-dependence in this study with a shift in potency of ~30 fold from pulse 1 to pulse 30. Lidocaine showed moderate use-dependence and TTX showed very little use-dependence. This study validates the ability of the IonWorks Barracuda system to separate compounds based on their use-dependence of blockade. The system enables continuous voltage clamp, sophisticated voltage protocols, flexible recording parameters, and a prolonged assay window, all important features for identifying usedependent compounds in a drug screening setting.
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