Table of Contents
- In this Types of Patch Clamp Technique post we have briefly explained about patch-clamp technique general principle, variants of the patch-clamp technique, and patch-clamp technique applications.
Patch-Clamp Technique, which allows you to observe the functions of individual ion channel in different cell types, revolutionized the study and practice of cellular biology. Sakmann and Neher invented the patch clamp technique in 1976. They were awarded the Nobel Prize in Physiology and Medicine in 1991.
The idea behind Patch-Clamp Technique is simple. To make contact with a tiny area of the cell membrane, a glass pipette is used with a very small opening. The contact between membrane and pipette becomes tighter after a little suction is applied to the back. All ions that flow through a single channel must flow into the pipette. Even though the electrical current is small, it can be measured using an ultra-sensitive electronic amplifier attached to the pipette. This arrangement is often called the cell-attached patch Clamp recording due to the geometry.
- To form cell-attached mode, place a pipette tip on the cell’s surface. This will create a low resistance seal (seal), with the membrane. A tight seal is formed by slight suction at the top end of the pipette. This seal is known as a ‘giga seal’. It has a resistance of around gigaohms. The cell-attached mode records from the membrane under the pipette while the inside of the cell is intact. The metal electrode in the glass pipette contains a salt solution that is similar to the fluid found inside the cell. Another one in the bath solution acts as the ground. Because of their weak polarization and low junction potentials, Pt and Ag/AgCl are popular electrodes. The tight seal between pipette and cell membrane isolates the membrane patch electrically, which means that all ions fluxing the membrane patch flow into the pipette and are recorded by an electrode connected to a highly sensitive electronic amplifier. A bath electrode is used to set the zero level.
- The current clamp mode and voltage clamp can both be used for patch clamp. The majority of patch-clamp measurements can be performed in a voltage clamp mode. The voltage clamp allows the experimenter to “clamp” the cell membrane potential (voltage), at a selected value. This allows you to measure the voltage-specific activity of lon channel channels. This mode is used to study the voltage-gated channels. Patch clamp has been used to refer to voltage clamping of membrane patches since 1976 with the introduction of the patch clamp method by Neher and Sakmann. This technique was used to record the tiny (pico Amperes) ions currents in single channels within cell membranes.
- To control the voltage of the membrane, the voltage-clamp mode can be used. This mode uses a patch-clamp amplifier that allows you to maintain (clamp) a specific membrane voltage while simultaneously measuring the membrane’s current. The electronic feedback system of an amplifier measures the voltage across the membrane and compares it with a preset voltage. The voltage of the membrane changes when a current is applied. A current of equal magnitude, but in the opposite direction, is injected into the pipette to compensate for the change and return the voltage to its pre-set value.
General principle of patch-clamp recordings. A glass pipette containing electrolyte solution is tightly sealed onto the cell membrane and thus isolates a membrane patch electrically. Currents fluxing through the channels in this patch hence flow into the pipette and can be recorded by an electrode that is connected to a highly sensitive differential amplifier. In the voltage-clamp configuration, a current is injected into the cell via a negative feedback loop to compensate changes in membrane potential. Recording this current allows conclusions about the membrane conductance. Image Source: www.leica-microsystems.com
- Current clamp measures membrane potential by injecting a current pulse into the cell, and then measuring the potential changes in response. This technique can be used to examine how cells respond to electrical current. This mode monitors cell activity in different ways, including action potentials, excitatory, inhibitory post-synaptic potent and changes in membrane potentials resulting from activation of electrogenic membrane carriers.
Types of Patch Clamp Technique
- Here, we describe the different types of patch clamp techniques and when you would use each.
- Whole-Cell Patch Clamping
- Cell-Attached Patch Clamping
- Inside-Out Patch Clamp
- Outside-Out Patch Clamping
- Loose Patch Clamping
1. Whole-Cell Patch Clamping
The whole cell patch clamp measures the currents throughout the cell membrane. The whole cell patch clamp works in the same way as cell-attached patch clamping but with more suction to break the membrane. The tube can then be inserted into the intracellular space where drugs or ligands can be added. A large current pulse can also be used to rupture the membrane.
2. Cell-Attached Patch Clamping
Cell-attached patch clamping uses a pipette that is attached to the cell membrane to measure current through the ion channels. The cell membrane is intact. Attaching to the membrane only on the exterior has the advantage that the cell structure remains intact and intracellular mechanisms work normally.
3. Inside-Out Patch Clamp
By attaching a piece cell membrane to the tube glass, the inside-out patch clamp exposes its cytosolic surfaces. This allows you to access the surface via the electrolyte solutions bath. This is useful when there are changes at the intracellular surface ion channels.
4. Outside-Out Patch Clamping
Outside-out clamping is an alternative to inside-out patch-clamping. It uses a whole-cell technique. The membrane is broken and the electrode is pulled out of the cell. A bulb of membrane will protrude from it. This is when a small amount of the membrane comes off the cell and forms a smaller chamber. You can now see the original membrane on the inside. This allows you to study the inner membrane surface and even move it to another solution bath.
5. Loose Patch Clamping
Loose clamping is similar in principle to attached cell clamping. However, the tube is held to the cell by a lighter seal. Loose seal clamping has the advantage that the pipette is easily removed and the membrane can still be intact. This allows for repeat measurements. The downside is that the resistance between membrane and pipette is lower. Current may leak through the seal and reduce resolution for small currents.
- The vast majority of physiological questions can be addressed using patch-clamp experiments, and not just in neuroscience. Patch-clamp recordings have become increasingly important in the study of ion channels within non-excitable cells over the past two decades. This is a vital method for medical research as many diseases can be attributed to malfunctioning definite ion channel channels. Automated patch-clamping is used in pharmacological research to screen for potential ion channel changes.
- You can combine patch-clamp recordings with live-cell imaging techniques such as Ca2+ imaging. The patch pipette is used to apply a fluorescent dye that is Ca2+-sensitive to the cell. Both the membrane current and fluorescence changes are simultaneously recorded. Similar experiments can also be done with Cl- and pH-sensitive dyes.