Voltage-gated calcium channels (Cav channels) are involved in neurosecretion and control of muscle contraction and are important targets for the development of therapeutics combating a variety of neurological and cardiovascular diseases. Current drug discovery efforts targeting Cav channels often focus on identifying state- and use-dependent inhibitors, which display maximum inhibitory effects at partially depolarized membrane potentials and thus preserve normal channel function1 . Because characterization of state- and use-dependent inhibitors is traditionally done using manual patch clamp methods, screening for these types of compounds can be difficult. This situation has changed, however, with the IonWorks Barracuda® (IWB) Automated Patch Clamp System. Capable of processing 384-well plates for highthroughput screening and flexible enough to support long assay windows and kinetic studies, the IWB is a robust screening tool for high-throughput characterization and identification of state- and use-dependent Cav channel modulators.
The power of the IWB system for screening Cav channel modulators was recently demonstrated in a peer-reviewed study conducted by Yuri A. Kuryshev, et al., at Charles River Discovery2*. The data highlighted below shows how the IWB system provides clear, actionable information on state dependence and subtype selectivity of a set of wellcharacterized reference inhibitors using recombinant cell lines that stably express either L-, N-, P/Q, or T subtypes of voltagegated calcium channels. These studies highlight how assays performed on the IWB system can be used to screen focused compound libraries for state-dependent Cav channel antagonists, prioritize compounds for potency, or counter-screen for Cav subtype selectivity.
Materials and methods
Full materials and methods can be found in Kuryshev, et al2.
Stable cell lines expressing (under tetracycline induction) human Cav 1.2/ b2/a2d1 (CHO; CACNA1C/CACNB2/ CACNA2D1) (Charles River Discovery PN CT6004), Cav 2.1/b4/a2d1 (CHO; CACNA1A/ CACNB4/CACNA2D1) (Charles River Discovery PN CT6196), Cav 2.2/b3/a2d1 (CHO; CACNA1B/CACNB2/CACNA2D1) (Charles River Discovery PN CT6159), and Cav 3.2 (HEK293; CACNA1H) (Charles River Discovery PN CT6106) voltagegated calcium channel subtypes were constructed3 and grown2 as described. Briefly, the cells were passed in a medium lacking selection antibiotics for 2–4 days. Expression is induced by exposure to tetracycline 16–14 h before recording. Verapamil at 3 µM is included in the induction medium to avoid Ca2+ overload toxicity. Cell density was ~50%–70% confluent at the time of harvest; two 150- mm plates (~1.2 x 107 cells) were used per population patch-clamp (PPC) experiment.
Buffers and reagents
Inhibitor/potentiator compounds were purchased from Sigma-Aldrich (St. Louis, MO) and were of ACS reagent grade purity or higher. Stock solutions of test articles were prepared in dimethyl sulfoxide (DMSO), stored at -20°C, and freshly diluted into extracellular buffer the day of the experiment. Extracellular buffer (EB): 137 mM NaCl, 4 mM KCl, 7 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH adjusted to 7.4 with NaOH. Intracellular buffer (IB): 90 mM CsF, 50 mM CsCl, 2 mM MgCl2, 0.5 mM EGTA, and 10 mM HEPES, pH 7.2 adjusted with CsOH.
Automated electrophysiology methods
A stock solution of amphotericin B was prepared in DMSO (30 mg/mL) and added to the solution at a final concentration of 100 µg/mL. For PPC assays, each well contained 11 µL/well EB, and 9 µL/well cell suspension. After establishment of wholecell configuration, membrane currents are recorded by on-board patch clamp amplifiers in the IWB system with the data acquisition sampling frequency set to 5 kHz, and inward peak current amplitudes measured. Under these conditions, each assay was completed in 45 min, and 5–10 experiments could be conducted each day.
Delivering expected Cav channel subtype-specific voltage-dependent behavior
Using the IWB system, Kuryshev, et al, were able to efficiently measure the expected voltage-dependent behavior of Cav channels in a subtype-specific manner (Figure 1). The high voltageactivated channels Cav 1.2, Cav 2.1, and Cav 2.2 all showed peak responses at positive membrane potentials, whereas low voltage-activated Cav 3.2 channels showed a peak response at negative membrane potential (Figure 1A, 1B). All four Cav channels showed the expected voltage-dependent inactivation curves, with the low-voltage Cav 3.2 inactivation curve shifted almost 40 mV towards more negative potentials (Figure 1C).
Detecting expected subtype-specific pharmacological responses
Cav 1.2 and other L-type channels are sensitive to dihydropyridine activators such as BAY K8644 and inhibitors such as nifedipine4 , whereas other high-voltageactivated and all low-voltage-activated Cav channels show low sensitivity to dihydropyridenes4 . Using the IWB system, Kuryshev, et al, were able to detect these subtype specificities, as illustrated with Cav 1.2 and Cav 2.2 (Figure 2). Cav 1.2 currents (Figure 2Ai) were completely blocked by 1 µM nifedipine and potentiated by 0.3 µM BAY K8644. In contrast, peak Cav 2.2 current amplitudes (Figure 2Aii) were unaffected by 1 µM nifedipine and 0.3 µM BAY K8644.
They found dose-response studies displayed similar subtype specificity (Figure 2B). Nifedipine inhibited Cav 1.2 channels (IC50 = 0.016 mM) and had no effect on Cav 2.2 channels. BAY K8644 produced two-fold potentiation of Cav 1.2 channels (EC50 = 0.006 mM), but inhibited Cav 2.2 channel (IC50 = 14.3 mM).
Capturing expected use- and/or voltage-dependent inhibition by known inhibitors
To successfully evaluate use- or statedependent Cav channel modulators for drug screens, counter-screens, and compound prioritization, an automated electrophysiology system needs to be flexible enough to provide a variety of voltage protocols in order to optimize the balance between sensitivity and success rate. The IWB system fully meets this need, with the ability to deliver precise voltage control and high temporal resolution. Kuryshev, et al, took advantage of the IWB system’s flexibility in order to find an optimal voltage protocol for evaluating the response of Cav channels to a panel of known, well-characterized inhibitors. They first tested several voltage protocols before selecting a multiple-mode voltage protocol (Figure 3A) suitable for assessing Cav 1.2, Cav 2.1, Cav 2.2, and Cav 3.2 channels (data from three different voltage protocols can be found in Kuryshev, et al2.).
Figure 3 and Table 1 describe the protocol voltage wave form and parameters for each ion channel respectively. In brief, a first test pulse (TP1) provides information on the inactivation sensitive component of blockade. TP1 consists of a brief depolarizing stimulus (+30 mV amplitude, 400 ms duration) preceded by a long depolarizing conditioning pulse (-50mV amplitude, 1 s duration) to elicit partial voltage-dependent inactivation. TP1 is applied both pre-baseline and postcompound application.
The second test pulse (TP2) can be used to measure the maximum activation of channels from their resting state when applied at baseline, and the tonic blockade of resting channels when applied after compound addition. TP2 consists of the same depolarizing stimulus as TP1, preceded by a hyperpolarizing conditioning potential (-100 mV amplitude, 2 s duration).
An additional use-dependent blockade arising from the repetitive activation of channels is evaluated by a series of 30 test pulses (TP3 to TP32). TP3-TP32 consists of a train of brief depolarizing pulses (+30mV amplitude, 400 ms duration, from -90 mV holding potential, delivered repetitively at 10 s intervals). TP3, the initial pulse in the train, is preceded by a 30 s conditioning interval at the holding potential (-90 mV) and provides an alternative index of tonic blockade of the resting state. Blockade augmentation (post compound addition) beyond the level attained in the resting state (i.e., use-dependent block) is measured by comparing peak current amplitudes, TP3 versus TP32, normalized to the effects of repetitive stimulation in channels exposed to vehicle alone
Using this multiple-mode voltage protocol and the assay parameters shown in Table 1, Kuryshev, et al, evaluated use- and voltage-dependent blockade of Cav channels by verapamil, mibefradil, and pimozide (Figure 3B), determining IC50 values for these three inhibitors plus nifedipine,as well as the potentiator BAY K8644 (Table 2).
They found that the expected compound specificities for relevant Cav subtypes are mostly conserved, with a few surprises. The two dihydropyridine compounds— agonist nifedipine and potentiator BAY K8644—were only effective in these roles against Cav 1.2 as expected (Table 2). In contrast, while the use-dependent mixed T/L blocker mibefradil was fourfold more potent against Cav 3.2 (T type) than Cav 1.2 (L type), a difference which is in good agreement with published data5, it was found to be equipotent in usedependent experiments against Cav 2.1, Cav 2.2, and Cav 3.2. Pimozide also showed the expected selectivity for T type Cav channels, with strong potency only against Cav 3.2 in the tonic blockade experiment, although this selectivity was broadened to include Cav 1.2 in the use-dependence assessment. Finally, as expected from the literature, they found verapamil to be relatively non-selective6.
|Ion channel||Holdingpotential(mV)||CPpotential(mV)||CPduration (s)||TP potential(TP1-TP32, mV)||TP duration (TP1-TP32, ms)||TP frequency(TP1-TP32, ms)|
|Pre-pulse-50 mV||Pre-pulse-90 mV||0.1 Hz||Pre-pulse-50 mV||Pre-pulse-90mV||0.1 Hz|
|Verapamil||6.5 ± 1.5||25.3 ± 5.2||5.3 ± 1.1||5.0 ± 1.1||22.1 ± 4.7||12.4 ± 3.6|
|Niledipine||0.023 ± 0.007||0.085 ± 0.026||0.010 ± 0.013||>3.0||>3.0||>3.0|
|BAY K8644||(0.006 ± 0.005)2||ND||ND||5.4 ± 0.9||24.8 ± 7.3||10.0 ± 2.6|
|Pimozide||>30.0||27.8 ± 9.3||1.9 ± 0.6||>30.0||>30.0||>30.0|
|Mibefradil||10.8 ± 3.10||16.1 ± 4.6||8.0 ± 1.8||6.5 ± 1.2||4.0 ± 1.1||2.2 ± 0.6|
|Pre-pulse-50 mV||Pre-pulse-90 mV||0.1 Hz||Pre-pulse-50 mV||Pre-pulse-90mV||0.1 Hz|
|Verapamil||13.1 ± 2.7||46.0 ± 8.7 1||17.8 ± 3.1||14.3 ± 3.3||21.2 ± 3.8||12.7 ± 1.8|
|BAY K8644||(13.6, 14.3)||(54.7, 37.2)||(17.3, 16.1)||10.8 ± 1.9||11.5 ± 0.6||7.2 ± 0.7|
|Pimozide||>30.0||>30.0||>30.0||8.9 ± 3.6||7.2 ± 1.2||5.5 ± 1.3|
|Mibefradil||10.8 ± 1.3||8.4 ± 2.1||3.0 ± 0.2||3.30 ± 0.9||3.7 ± 1.0||2.0 ± 0.9|
High-throughput evaluation and characterization of Cav channels, especially in the presence of stateand use-dependent modulators, is of critical importance for drug discovery and development efforts. However, while manual-patch clamp methods provide the high-quality data needed by these studies, these methods cannot support the throughputs necessary for screening, counter-screening, and potency prioritization studies. Kuryshev, et al, have shown how the IWB system delivers not only the throughput needed for this type of work, but also the flexibility for designing the most informative assays while providing the accuracy, sensitivity, and reproducibility required to uncover actionable insights. With the IWB system, high-throughput evaluation of Cav channel state- and use-dependent compounds is now possible.
- Triggle, D. J. Drug targets in the voltage-gated calcium channel family: why some are and some are not. Assay Drug Dev. Technol. 1, 719–733 (2003).
- Kuryshev, Y. A., Brown, A. M., Duzic, E. & Kirsch, G. E. Evaluating state dependence and subtype selectivity of calcium channel modulators in automated electrophysiology assays. Assay Drug Dev. Technol. 12, 110–119 (2014).
- Wible, B. A., Kuryshev, Y. A., Smith, S. S., Liu, Z. & Brown, A. M. An ion channel library for drug discovery and safety screening on automated platforms. Assay Drug Dev. Technol. 6, 765–780 (2008).
- Doering, C. J. & Zamponi, G. W. Molecular pharmacology of high voltage-activated calcium channels. J. Bioenerg. Biomembr. 35, 491–505 (2003).
- Martin, R. L., Lee, J. H., Cribbs, L. L., PerezReyes, E. & Hanck, D. A. Mibefradil block of cloned T-type calcium channels. J.Pharmacol. Exp. Ther. 295, 302–308 (2000).
- Lacinová, L. Voltage-dependent calcium channels. Gen. Physiol. Biophys. 24 Suppl 1, 1–78 (2005).
In October 2014, Chantest was acquired by Charles River Discovery.
Please view the entire article at http://online. liebertpub.com/doi/10.1089/adt.2013.552.
Experimental work and data figures were provided by Charles River Discovery.