High Throughput Electrophysiology
Ion Channels as a Therapeutic Target Class
[U.S. Patent 6,488,829]Ion channels are an important target class in drug discovery, accounting for over 6 billion dollars in sales per annum. Ion channels underlie many physiological processes, including neuronal signaling, cardiovascular function, immune cell activation and muscle contraction. Currently, there are active pharmaceutical research and development programs involving ion channels in a number of areas, including asthma, inflammation, arrhythmia and epilepsy.
In spite of their tremendous physiological importance, ion channels have remained an untapped therapeutic target class, certainly in comparison to other target areas, such as G-protein coupled receptors or kinases. This is due, in part, to the complex nature of ion channel signaling and to the absence of high-throughput instrumentation to analyze them. The gold-standard measurement technique for studying ion channels is called the patch clamp. Although very powerful, this technique is extremely labor-intensive and thereby limited to a throughput of 10-20 individual cell measurements per day. The low measurement throughput has mandated the use of other less specific and less sensitive technologies for high-throughput screening of ion channel targets. Electrophysiology instrumentation developed at Essen Instruments will alleviate this trade off, allowing for high measurement throughput and fidelity, and thereby enabling the exploitation of ion channels as drug targets.
Characteristics of Ion Channels
Ion channels are proteins that span the lipid bilayer of the cell membrane. Their basic function is to regulate the electrochemical gradient of physiological ions across the cell membrane by variable conductance of specific ions. It is the flow of these ions and the subsequent separation of electrical charges across the non-conducting cell membrane that account for the cell’s membrane potential.
Some of the common characteristics of ion channels are denoted above in a depiction of a voltage-gated ion channel. As shown, the channel spans the cell membrane and separates the extracellular domain (high Ca++, high Na+) from the intracellular domain (high K+). Different ion channels exhibit specific permeability differences to the various physiological ions, (e.g., Na channels preferentially conduct Na ions) as specified by the selectivity filter of the pore. Another characteristic of ion channels is a gate mechanism, simplistically shown in this model on the intracellular side of the membrane. The gate regulates the flow of ions through the channel pore and can be modulated by various physiological mechanisms, including the membrane potential, a chemical ligand or a pharmacological agent. One of the challenging aspects of monitoring ion channel activity in the laboratory is the fact that ion channel gating can be extremely fast, often occurring on the time scale of a few milliseconds.
One exploitable characteristic of ion channels as drug targets is state dependence. Ion channels undergo conformational state changes in response to changes in voltage and/or chemical ligands. The state changes are complex, often multi-staged and ion channel-type specific. In order to exploit this feature, it is useful and often necessary to be able to control the cell’s membrane potential, thereby controlling the activation/inactivation state of the channel. Ideally, this control can be used to place the ion channel in a state consistent with the believed mechanism of action of the pharmacological compound. Indeed, many pharmacological ion channel modulators bind preferentially to specific conformational states of a given channel. By specifically targeting the abnormal or diseased state of the ion channel, this control can also be a tremendous advantage to the pharmaceutical researcher.
The Patch Clamp Technique
As mentioned previously, the gold-standard measurement technique for characterizing and studying ion channels is the patch clamp. Invented over twenty years ago by Sakmann and Neher [Pflugers Arch 375: 219-228, 1978], this technique allows for the direct electrical measurement of ion channel currents while simultaneously controlling the cell’s membrane potential. The figure below is a conceptual diagram of the patch-clamp setup.
As shown, the technique relies on using a fine-tipped glass capillary to make contact with a biological cell or membrane in order to form a high resistance seal (denoted in red). Conventionally, this requires the use of a technician, a microscope, and a micropositioning device connected to the capillary. A sensing electrode inside the capillary is connected to a high-gain, trans-impedance amplifier circuit that converts the current flowing through the electrode to an output voltage. The purpose of the high-resistance seal is to insure that the flow of current from the sensing electrode inside the capillary to the ground electrode in the bath solution is predominantly due to ions flowing through the cell membrane.
There are many potential measurement configurations of the patch-clamp technique. One that is typically used in the pharmaceutical industry is termed a "whole-cell clamp". In this configuration, the part of the membrane inside the capillary is electrically permeabilized, thereby creating a low-resistance pathway between the sensing electrode and the cytoplasm. From an electrical viewpoint, this puts the sensing electrode in the glass capillary inside the cell. This allows for the control of the cell membrane via the non-inverting input of the trans-impedance amplifier (Vcmd as shown). The current measurement between the sensing electrode and the bath electrode is then the resultant sum of all of the ion currents in the remainder of the cell, which, due to the small geometry of the capillary, comprises the majority of the ion channel currents in the "whole cell".
The power of the patch-clamp technique lies in the ability to directly measure the fast electrical currents of the ion channel activity while simultaneously controlling the cell’s membrane potential. The ability to control membrane potential and the subsequent advantage of exploiting the state dependence of certain ion channels targets have already been described. As the patch clamp is a direct electrical measurement, it offers the additional advantage of stimulating and recording signals with a high degree of temporal resolution, often as fast 10 kHz. This makes it possible to isolate specific ion channel temporal signatures and, hence, verify that the measured signal is due to the ion channel of interest. Other ion channel measurement platforms, e.g., those that measure membrane potential, do not kinetically resolve the ion channel event and are subject to other non-specific changes. These factors have all combined to make the patch-clamp technique the most powerful method to ascertain the function and pharmacology of ion channels.
One disadvantage of current implementations of the patch-clamp technique is the high level of manual intervention required to make the measurements, which severely restricts the measurement throughput. Typically, an experienced technician can patch and make measurements from 10-20 individual cells per day. This level of measurement throughput is far below that required for secondary (hundreds to thousands / day) or primary (thousands to tens of thousands / day) pharmaceutical drug screening and has limited the ability to screen ion channel targets with high fidelity.
Essen Technology (IonWorks™ HT)
The goal of the high-throughput electrophysiology instrumentation developed at Essen is to create a technology that couples the high-fidelity measurements made by a standard patch-clamp device with the measurement throughput required to perform high-throughput screening. Achieving this goal requires more than just the automation of existing patch-clamp techniques; it requires the development of an entirely new paradigm for making electrophysiological measurements.
As shown in the figure, the Essen technology is based on positioning a cell on a small pore separating two isolated fluid chambers in a manner that requires no manual intervention or micromanipulation.
In order to make "whole-cell" electrophysiological measurements within this geometry, two criteria must be met. First, a high-resistance seal must form between the cell membrane and peripheral region of the substrate pore. As in the case of patch-clamp electrophysiology, this insures that the current measured between the two electrodes passes through the cell membrane. Second, in order to be able to control the cell’s membrane potential, a low-resistance electrical pathway must form through the cell wall that covers the pore. This latter requirement, in effect, places the associated electrode at the interior of the cell and allows one to clamp the membrane potential over the rest of the cell membrane. Once these criteria have been met, and assuming no manual intervention, it is then possible to conceive of a parallel format where many wells can be measured simultaneously. The current IonWorks™ HT system being developed at Essen allows for the measurement of approximately 200 whole-cell currents in parallel along with the required fluidics and system integration necessary for a full-fledged screening instrument.