Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators

Abstract

T-type voltage-gated Ca²⁺ channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca²⁺ channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca²⁺ channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca²⁺ channels are largely inactivated and unable to open to allow Ca²⁺ influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express K_ir2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca²⁺ sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca²⁺ channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.

Figure 1

Biophysical properties of HEK293 cells stably expressing Ca_V3.1, Ca_V3.2, or Ca_V3.3 T-type channels using high-throughput in vitro electrophysiology. (A) The current–voltage (IV) protocol consists of a sustained depolarization of 1 s from −120 to +20 mV at 10 mV increments (TP1), followed by a voltage step of 300 ms to −20 mV (TP2). The Ca²⁺ currents from the TP1 protocol (activation) were used to construct the “voltage-dependent activation” curve. Ca²⁺ currents from the TP2 protocol (inactivation) were used to establish the “voltage-dependent inactivation” curve. (B) Sample T-type Ca²⁺ currents are shown for Ca_V3.1 (black), Ca_V3.2 (red), and Ca_V3.3 (gray) normalized to the peak current. (C) Voltage-dependent activation curves. V_{1/2-activation} (voltage at which 50% of the channels are activated) = −48.76 mV (n = 47 cells), −47.96 mV (n = 31 cells), and −39.53 mV (n = 38 cells) for Ca_V3.1, Ca_V3.2, and Ca_V3.3, respectively. (D) Voltage-dependent inactivation curves. V_{1/2-inactivation} (voltage at which 50% of the channels are inactivated) = −76.30 mV (n = 47 cells), −76.59 mV (n = 31 cells), and −64.42 mV (n = 38 cells) for Ca_V3.1, Ca_V3.2, and Ca_V3.3, respectively. (E) Averaged peak current density of 67.06 ± 3.84 pA/pF (n = 48 cells), −61.60 ± 3.66 pA/pF (n = 31 cells), and −78.80 ± 3.60 pA/pF (n = 39 cells) for Ca_V3.1, Ca_V3.2, and Ca_V3.3, respectively. Data are expressed as mean ± SEM. (F) KCl-induced changes in membrane potentials were recorded from HEK293 cells expressing K_ir2.3 and Ca_V3.3 measured by whole-cell patch clamp electrophysiology (n = 6–7 cells per treatment). Data are expressed as mean ± SEM.

Figure 2

Development of a novel 384-well-based high-throughput assay to readout T-type Ca²⁺ channel activity using a membrane-tethered calcium sensor GCaMP6s-CAAX. (A) Schematic of GCaMP6s-CAAX in response to T-type Ca²⁺-mediated Ca²⁺ influx. (B) Representative 60× images of HEK293 cells expressing K_ir2.3/Ca_V3.3 with GCaMP6s or GCaMP6s-CAAX. (Left) Differential interference contrast images. (Right) Fluorescence images demonstrating the cytosolic localization of GCaMP6s and the expression of GCaMP6s-CAAX near the plasma membrane. (C) FLIPR signal traces from the HEK293 cells expressing Ca_V3.3/K_ir2.3 in the presence of Fluo-4AM (black) or expressing either GCaMP6s (gray) or GCaMP6s-CAAX (red) when stimulated with 6 mM KCl. (D) KCl CRC of HEK293 cells expressing K_ir2.3 and Ca_V3.3 with GCaMP6s-CAAX (black), GCaMP6m-CAAX (gray), or GCaMP6f-CAAX (white) normalized to baseline fluorescence. (E) KCl CRC of HEK293 cells expressing K_ir2.3 and Ca_V3.3 (black), Ca_V3.2 (red), or Ca_V3.1 (white) normalized to baseline fluorescence. Data are expressed as mean ± SEM (24 replicates and 2 independent experiments). (F) FLIPR signal of HEK293 cells expressing K_ir2.3 and Ca_V3.1 with GCaMP6s-CAAX in response to a different Ca²⁺ concentrations (four replicates and two independent experiments). (G) KCl CRC in HEK293 cells expressing K_ir2.3 and Ca_V3.3 loaded with the Ca²⁺-sensitive dye Fluo-4AM (black) or coexpressing GCaMP6s-CAAX (red). Data are expressed as mean ± SEM (24 replicates and 2 independent experiments).

Figure 3

The novel GCaMP6s-CAAX assay can identify both activators and inhibitors of T-type Ca²⁺ channels. (A) Representative fluorescence sample traces of HEK293 cells expressing Ca_V3.3/K_ir2.3/GCaMP6s-CAAX in the presence of 5.4 mM (low) or 6.7 mM (high) KCl. (B) Sample traces of HEK293 cells expressing Ca_V3.2/K_ir2.3/GCaMP6s-CAAX in the presence of 4.7 mM (low) or 6.0 mM (high) KCl. (C) Sample trace of HEK293 cells expressing Ca_V3.1/K_ir2.3/GCaMP6s-CAAX in the presence of 1.5 mM (low) or 5.0 mM (high) CaCl₂. Cells were treated with DMSO (black) or 1 μM of the pan T-type Ca²⁺ channel inhibitor TTA-A2 (red). Traces were normalized to baseline prestimulation fluorescence. (D) CRC of SAK3 in HEK293 cells expressing K_ir2.3/GCaMP6s-CAAX with Ca_V3.1 (white), Ca_V3.2 (red), or Ca_V3.3 (black). Data are expressed as mean ± SEM of two independent experiments. (E) The IC₅₀ values of 42 different compounds evaluated using GCaMP6s-CAAX are plotted against the IC₅₀ values of the same compounds evaluated using the Ca²⁺-sensitive dye Fluo-4AM. (F) ProTx-I CRC in cells expressing Ca_V3.1 (white), Ca_V3.2 (red), and Ca_V3.3 (black). Data are expressed as mean ± SEM of two independent experiments.

Figure 4

Assays utilizing GCAMP6s-CAAX can be used to effectively evaluate the endogenous activity of GPCRs. (A) Representative sample traces of endogenous LPA₁ receptor activity in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅ in the presence of DMSO (black) or 25 μM AM095 (red) normalized to baseline prestimulation fluorescence. (B) CRC of lysoPA18:1 induced endogenous LPA₁ receptor activity in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅. (C) CRC of the selective LPA₁ receptor antagonist AM095 in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅. (D) Representative sample traces of endogenous C3a receptor activity in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅ in the presence of a buffer (black) or 100 nM BR111 (red) normalized to baseline prestimulation fluorescence. (E) CRC of hC3a induced endogenous C3a receptor activity in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅. (F) CRC of the C3a receptor antagonist BR111 in the presence of 20 nM hC3a in CHOK1 cells expressing GCaMP6s-CAAX and Gα₁₅. Data are expressed as mean ± SEM of four replicates from two independent experiments.