What Affects Automated Patch Clamp Data Quality?
Automated patch clamp data quality can be influenced by the platform, cell system, recording conditions and experimental protocol used. Understanding these factors is important when designing an automated patch-clamp study and comparing results generated under different experimental conditions.
Ion-channel capability in early automated systems
IonWorks, an a early developer of automated systems that identified a wide range of ion channels. These included voltage-gated sodium, potassium and calcium channels, transient receptor potential channels, inwardly rectifying and calcium-activated potassium channels, and conductances such as the epithelial sodium channel.
However, early automated systems were not equally suitable for every ion-channel family.
One notable limitation of IonWorks was its ability to investigate fast ligand-gated ion channels. These channels require rapid compound application and recording because their responses can occur and decline quickly.
Holland and colleagues reported recordings from fast ligand-gated GABA ion channels after reducing the time between compound application and recording to approximately eight seconds. This demonstrated how fluidics and application speed could influence which channels were accessible using an automated platform.
Perforated patch recordings and intracellular control
IonWorks recordings used a perforated-patch configuration with amphotericin as the perforating agent.
Amphotericin does not provide complete control over the movement of substances such as calcium and other small molecules between the intracellular solution and the cell. This can be important when studying channels whose activity is influenced by intracellular calcium or other cellular components.
When investigating calcium-activated potassium channels, this limitation could contribute to variability in the data.
Ido and colleagues reported that pre-incubating cells with BAPTA and applying the calcium ionophore ionomycin helped to reduce this variability.
A further development involved using escin as an alternative perforating agent. Escin creates larger pores in the cell membrane, allowing substances including calcium, ATP and GTP to pass through more readily.
This greater level of intracellular access can be useful when studying ion channels affected by intracellular molecules. For example, the presence of ATP may help reduce rundown in assays investigating L-type calcium channels.
These examples demonstrate why the recording configuration and perforating agent must be considered when developing an ion-channel assay.
Limitations of early automated patch-clamp platforms
Although IonWorks provided a substantial increase in throughput, several limitations were recognised.
These included:
- Limited flow-through perfusion
- Lower seal quality compared with conventional patch clamp
- Restricted access to some ion-channel families
- Difficulties studying fast ligand-gated channels
- Variability associated with cells and intracellular conditions
These were limitations of an early generation of automated technology and should not be presented as limitations of every current automated patch-clamp platform.
Developments in platform fluidics and seal quality
Later automated patch-clamp platforms introduced more advanced fluidics. These developments allowed more complete bath exchange and sequential additions of compounds during an experiment.
Seal resistance was also improved by coating areas of the recording plate or chip with silicon dioxide, creating a surface with properties closer to those of the glass pipette used in manual patch clamp.
Some platforms were designed to record electrophysiological activity while dispensing a ligand or compound at the same time. This increased the ability of automated systems to investigate ligand-gated ion channels.
Different platforms were subsequently developed with different recording capacities. Historical examples include:
- Patchliner, with eight recording wells per experiment
- QPatch systems, with 16 to 48 wells
- QUBE and SyncroPatch systems, with 384 or 768 wells
These figures provide historical context for the development of the technology. Current platform capabilities should always be confirmed before they are used to describe a service.
Additional recording capabilities
Automated patch-clamp technology has continued to develop beyond its original voltage-clamp applications.
Later platforms introduced capabilities including:
- Current-clamp recording
- Temperature control
- Dynamic patch clamp
- Improved fluidics
- More flexible compound and ligand application
The relevance of each feature depends on the ion channel, cell system and scientific question being investigated. A greater number of technical features does not automatically mean that a platform is appropriate for every assay.
The importance of the cell system
Recombinant cell lines have traditionally been used extensively in automated patch-clamp assays.
More recently, human induced pluripotent stem cell-derived cells and primary cells have also been investigated because they may provide a more physiologically relevant experimental system for some applications.
However, each cell model has different advantages and limitations. Cell quality, channel expression, biological variability and compatibility with the recording platform may all influence assay performance.
The choice of cell system should therefore be based on the ion channel, the project question and the type of evidence the study needs to produce.
Protocol conditions and data variability
Electrophysiology data contain an inherent degree of variability. Measurements of compound potency can change depending on the experimental conditions used.
Important variables may include:
- Holding potential
- Voltage protocol
- Ligand concentration
- Compound-application timing
- Recording temperature
- Cell system
- Seal quality
- Recording configuration
These factors can influence the measured potency of a compound and should be considered when comparing data from different experiments or laboratories.
This can be particularly important when ion-channel results support downstream analyses. For example, differences in hERG IC 50 measurements may affect inputs used for in silico action-potential prediction.
Kramer and colleagues reported substantial variability in ion-channel data generated by different laboratories, despite efforts to standardise experimental conditions.
This underlines the importance of consistent protocols, suitable controls, clearly reported experimental conditions and expert interpretation.
Designing an appropriate automated patch-clamp study
The suitability of an automated patch-clamp study does not depend on throughput alone.
Platform design, fluidics, cell system, recording configuration, protocol conditions and quality controls should all be considered when selecting an approach.
Careful study design helps ensure that the assay is appropriate for the ion channel and that the results can be interpreted within the context of the project.
Learn more about ApconiX’s Automated Patch Clamp Services and discuss the most appropriate assay approach for your study.
Learn more about automated patch clamp
Learn more about ApconiX’s Automated Patch Clamp Services, including available study options and support for ion-channel screening.