Applications > Drug Discovery > Ion Channels

The MaxCyte STX is based on flow electroporation and is an ideal solution for expressing functional ion channels including multi-subunit or toxic channels. MaxCyte electroporation produces cells with high viability, transfection efficiency, and cell membrane integrity. Whether used immediately or cryopreserved for future use, transfected cells produce superior performance in downstream cellular assays such as calcium flux and automated electrophysiology assays. Results are comparable to stable cell lines, yet assays can be developed and conducted in just a fraction of the time.

MaxCyte Electroporation for Ion Channel Expression:

Express functional multi-subunit, intractable or toxic Ion Channels
Eliminate the reliance on stable cell lines
Proven performance in downstream ion channel assays including FLIPR®, IonWorks® Quattro™ & PatchXpress®
Compatible with primary cells, stems cells or other difficult-to-transfect cell lines
Fully scalable, able to transfect 5x10⁵ cells in seconds up to 1x10¹⁰ cells in < 30 minutes

Multi-subunit Ion Channels
Potassium Channels
STX vs. Stables
STX vs. Lipids
Cryopreservation

Multi-subunit Calcium Channel Expression: Functional Responses Measured using FLIPR

Using the MaxCyte STX Transfection System, multiple plasmids can be co-transfected in defined stoichiometric ratios to express specific functional ion channel complexes. The data in Figure 1 describe the expression of a multi-subunit calcium channel using MaxCyte transient transfection.

Four cDNAs encoding the pore-forming alpha subunit of the voltage-gated calcium channel Cav2.2, the modulatory β subunit, the modulatory α2δ subunit and an inward rectifier potassium channel (Kir2.1) were co-transfected into HEK293 cells using the MaxCyte STX and assayed using the FLIPR® calcium influx assay. Strong calcium flux was evident in cells transfected with all four plasmids, but not in cells transfected without the inward rectifier plasmid or in untransfected control cells. Inclusion of the inward rectifier allowed modulation of resting membrane potential by external potassium and was essential for the FLIPR assay. The Cav2.2 channel was inhibited by ω-conotoxin, a known calcium channel antagonist, illustrating the functionality of the channel and the applicability of transiently transfected cells for compound screening of ion channels.

Figure 1: Calcium Flux Assay Demonstrates the Functionality of the Expressed Multi-subunit Cav2.2 Ion Channel. HEK293 cells were transfected using small-scale electroporation with multiple cDNA constructs encoding 3 calcium channel subunits ± a plasmid expressing the inward rectifier potassium channel, Kir2.1. Calcium influx FLIPR assays were conducted 24 hrs post transfection on untransfected HEK 293 cells, cells transfected with 3 calcium channel subunits ± the inward rectifier. Dye was added in a low potassium, low calcium solution and loaded for 30 minutes. Vehicle control or a calcium channel specific antagonist (600 nM ω-Conotoxin GVIA) was added for an additional 30 minutes. Cells were depolarized with high external K+ (up to 135 mM). Data courtesy of ChanTest Corporation.

Automated Electrophysiology Assays using MaxCyte Transiently Transfected Cells

Potassium channels are a complex class of voltage-gated ion channels both functionally and structurally. Until recently, the ability to perform automated electrophysiology assays have been limited to the use of stably transfected cell lines which are costly and time consuming to create. Using the MaxCyte STX, researchers can express functional ion channels of interest and reproducibly produce cells with proven compatibility with a variety of electrophysiology systems including the IonWorks® Quattro™ and the PatchXpress®. The data summarized in Figure 2 demonstrate the expression of the Kv1.3 channel in HEK cells, the high level of transfection efficiency using the MaxCyte STX and the quality performance of these cells in automated electrophysiology assays.

HEK 293 cells were transfected with three concentrations of the human Kv1.3 cDNA containing a C-terminal GFP tag (OriGene). GFP expression was assessed by FACS as a measure of transfection efficiency. GFP expression was DNA concentration dependent, with approximately 80% of cells GFP+ at the highest concentration of DNA. To assess the function expression of the Kv1.3 channel, transfected cells were tested using the PatchXpress 7000A. 50-65% Gigaohm seal rate was achieved, comparable to results of a stable cell line (data not shown).

Figure 2: Kv1.3 Functional Expression in HEK Cells. A).HEK 293 cells were grown in T175 flasks and split 24 hours prior to transfection. Cells were transfected with 100μg, 200μg or 300μg of the human Kv1.3 cDNA containing a C-terminal GFP tag (OriGene) using the standard MaxCyte HEK electroporation protocol. Channel expression was assessed by FACS. 36 hours post transfection cells were assessed using the PatchXpress 7000A. B).Voltage protocol. C).Sample Kv1.3 currents recorded from a transfected cell. D).Current-voltage relationship of Kv1.3 channel extracted from the same cell as in C. Each data point represents the peak outward current elicited by its respective voltage step. 50-65% Gigaohm seal rate was achieved, comparable to results of a stable cell line (data not shown). Data courtesy of Molecular Devices.

Functional Comparison of MaxCyte Transfected Cells to Stable Cell Lines

Increased attention is being paid to voltage and ligand-gated ion channels as higher throughput, cell-based assays of channel activity have emerged. Current ion channel assays rely heavily on the use of stable cell lines, which are challenging to create due to the multi-subunit nature of many ion channel targets. The use of multiple selection agents during cell line creation can impair cell health and proliferation. Furthermore, ion channels can be toxic when expressed at high levels, creating the need for inducible promoters, which adds an additional layer of complexity. Transient transfection using the MaxCyte STX offers an attractive alternative.

The data summarized in Figure 3 compare the functional responses of the Kv1.5 potassium channel, a therapeutic target in many disease areas, in cells transiently transfected using the MaxCyte STX to a stable cell lines with the same cell background.

CHO K1 cells were transfected with a plasmid encoding the alpha subunit of the Kv1.5 voltage-gated potassium channel and assessed, in both single hole and population patch clamp mode, using the PatchXpress®, an automated electrophysiology assay platform. Transfected cells showed strong, consistent current tracings in response to the application of standard voltage step protocols. To demonstrate the suitability of transiently transfected ion channels for drug screening applications, three independent channel inhibitors, capsaicin, nifedipine and bupivicaine, were tested using transfected cells as well as a Kv1.5 stable cell line. The calculated IC50 were comparable between transiently transfected and stable cells and were in agreement with literature values. Thus, the MaxCyte STX provides a high fidelity alternative to creating stable cells lines which translates into shorter time to screen while using fewer valuable resources.

Figure 3: Automated Electrophysiology Pharmacological Assays with MaxCyte Transiently Transfected Cells. Table: CHO K1 cells transiently transfected with a Kv1.5 expression plasmid or a CHO stable cell line expressing Kv1.5 were incubated with varying concentrations of three potassium channel inhibitors and assayed on the PatchXpress. A). Pharmacological analysis of transiently transfected cells using the PatchXpress instrument. B). Representative current tracings that depict a step-wise loss of Kv1.5 activity in response to increasing concentrations of the inhibitor capsaicin. Table: IC50 values for each compound compared favorably to data obtained using a stable cell line. Data courtesy of BioFocus.

MaxCyte Transfected Cells Perform Better than Lipid Transfected Cells in Ion Channel Screening

Additional factors must be taken into consideration for any assay technology to be successfully integrated within a screening environment. Most importantly, they must have high throughput capacities and reproducible results, while remaining easy to use and affordable. The MaxCyte STX offers complete scalability and high quality performance and is the ideal solution for target screening and profiling.

These data compare the expression levels and performance of CHO cells transfected with a Kv1.5 α-subunit plasmid using the MaxCyte STX or commercial lipid-based transfection reagent. Transfected cells were assayed on the IonWorks® Quattro™ system. Cells transfected using MaxCyte electroporation had extremely higher expression levels as well as high % seals compared with lipid-based transfection.

Table 2: Superior Performance of MaxCyte Transfected Cells in Electrophysiology Assay. CHO K1 cells transiently transfected with a Kv1.5 expression plasmid using a commercial lipid-based transfection reagent or using MaxCyte electroporation. Cells were assayed in the single hole and population patch clamp mode on the IonWorks Quattro system. Data courtesy of BioFocus.

Cells can be Cryopreserved Following MaxCyte Electroporation without Sacrificing Performance in Ion Channel Assays

The MaxCyte STX enables bulk transfection of billions of cells in less than 30 minutes using preprogrammed electroporation protocols. Transfected cells can be used in a wide range of assays immediately following electroporation. If more suitable to assay scheduling, transfected cells can also be aliquoted and cryopreserved for future use. MaxCyte has developed several cryopreservation protocols that enable cell archiving while maximizing cell viability and target expression upon thawing.

To assess the the effects of cell cryopreservation on their performance in downstream ion channel assays, CHO cells were transfected with a Kv1.5 expression plasmid in bulk using MaxCyte flow electroporation and cryopreserved 48 hours post transfection. Transfected cells were thawed approximately one or five months after cryopreservation and assayed using the IonWorks Quattro. The cells demonstrated no significant differences in seal resistance, expression levels or current amplitudes. These levels were also comparable to those cells assayed immediately following cryopreservation (data not shown). These data highlight the ability of the MaxCyte STX to transfect cells in bulk, aliquot and cryopreserve cells without negatively affecting cell performance in screening campaigns.

Table 3: Large-Scale Transfection and Cryopreservation of CHO Cells in Ion Channel Assays. 8x10⁸ CHO K1 cells were transfected with 150μg/ml of Kv1.5 expression plasmid DNA using MaxCyte flow electroporation. Transfected cells were cultured at 37°C for 24 hours, transferred to a 28°C incubator and cultured for an additional 24 hours. Cells were then suspended in freezing medium and cryopreserved in liquid nitrogen using standard methodology. Cells were thawed 1 and 5 months following cryopreservation and assayed in the single hole (SH) and population patch clamp(PPC) mode on the IonWorks Quattro system. Data courtesy of BioFocus.

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