Ion channel screening for non-experts

 

Ion channels consist of proteins and are embedded in all cell membranes of all living organisms. They are responsible for electrical signaling within cells and between cells. The signal is carried by ions (i.e., ‘salt’) that normally cannot pass the cell membrane but can diffuse passively (no energy expenditure) through ion channels, when they open in response to certain conditions. This electrical signaling is crucial for all physiological processes.

Examples of such processes are:

  • Generation of electrical activity in nerves
  • Control of contractile activity in the heart and muscles
  • Nutrient uptake
  • Hormone secretion

There are many ion channels

A typical cell has 100-1000 ion channels of different types. Most ion channels allow only one specific ion to permeate through them. This is reflected in the common names of the ion channels, e.g., K+, Na+, Ca2+ and Clion channels. The sequencing of the human genome has recently led to the identification of more than 400 putative ion channels. Only about 100 of these have been cloned and functionally tested so far.

Ion channels play a role in many diseases

A number of human diseases, including pathological pain conditions, epilepsy, cystic fibrosis and a variety of neural and muscular disorders, are caused by defects in the function of ion channels.

Many drugs affect ion channels

The large number of physiological processes regulated by ion channels and their role in many diseases make ion channels highly interesting as targets for new drugs. Today, about 20% of all registered drugs target ion channels. The drugs modulate specific ion channels, resulting in altered cellular behavior.

Ion channels are difficult to explore

Ion channels are important targets in drug discovery, but they are also difficult to study. Despite significant research efforts, there is still limited knowledge about ion channel function and how ion channels are related to specific diseases. This is mainly a result of the limitations and complexity in the technologies that are available to ion channel research. Ion channels are therefore considered to be largely unexploited compared to other target classes.

Methods to explore ion channels

There are two main methods to investigate ion channels: direct and indirect methods. The only direct method is called ‘patch clamp’. In brief, traditional patch clamp is very accurate, but also very time consuming (i.e., has low throughput), while the indirect methods are less accurate, but typically much faster.

Direct methods – Patch clamping

The patch clamp technique is considered the gold standard in ion channel research. The technique was developed in the 1970’s by Erwin Neher and Bert Sakmann, who received the Nobel Prize in Physiology and Medicine (1991) for their work. In a traditional patch clamp experiment, a tiny glass pipette containing an electrode is attached or ‘sealed’ tightly to the cell membrane. The cell membrane is ruptured manually by applying suction through the glass pipette. The tiny currents (10-12-10-9 amperes) through the ion channels can be measured via the electrode, which is connected to an amplifier. The typical throughput is three to ten successful patch clamp experiments per day and requires patience and high-level insight in electrophysiology.

Indirect methods to study ion channels

There are several indirect methods for studying ion channels. The most important indirect methods are based on fluorescent dyes that are loaded into cells and then analyzed using specialized plate readers. The readers detect a change in the concentration of certain ions that is the result of ion channel activity or a change in the voltage across the cell membrane, also caused by the flow of ion through the channel. The main benefits of these methods are their high throughput and low cost per data point. The main limitations of the methods are low accuracy and sensitivity, and lack of control of the voltage across the cell membrane, making it possible to study only certain types of ion channels and rather simple ways of drugs interacting with the ion channels.

Automated patch clamp technologies

QPatch is Sophion’s product family for medium-throughput automated patch clamp, and Qube is the newest high-throughput automated patch clamp system. With the QPatch and Qube, patch clamp experiments are done on a planar ‘glass’ surface that creates a connection between the cell and the amplifiers that are very similar to the manual ‘gold standard’ patch clamping. QPatch and Qube operate automatically and these systems increase patch clamp throughput due to the high degree of parallelism.

QPatch and Qube therefore combine the accuracy of traditional patch clamp with the high throughput of indirect methods.

The drug discovery process in short

The drug discovery process in pharmaceutical companies can begin either with a certain target, e.g. a specific ion channel, or a certain disease and a search for relevant therapeutic targets to modulate.  One important class of targets comprises ion channels. After a target validation phase, a test system (an assay) is developed to measure interactions with the target. Pharmaceutical companies have huge libraries of chemical compounds, the largest of which contain up to a couple of million compounds. One or more of these compounds may be identified as so-called hits with a desired effect on a specific target. From this hit, chemical modifications  can make the identified molecule suitable as a drug. This process is iterative and called lead optimization. For every modification, new patch clamp tests are needed to check that the desired effect is preserved or improved. Further tests in disease relevant animal-models and safety test are carried out before the development in the clinical trials (test in humans) can lead to an approval by health authorities to market a new drug.

Finding the hits is like finding a needle in a haystack! For the last 20 years it has been routine to test either the entire compound library, or part of it, with indirect methods in a so-called High Throughput Screening (HTS) department in the pharmaceutical company that identifies the initial hits.