ELM 12 Electrophysiology Flashcards
What is the basic principle behind measuring electrical activity in cells?
Electrical activity in cells is measured by recording the potential difference (voltage) between the inside and outside of the cell. This is achieved using electrodes to measure voltage or current, allowing for versatile measurements on tissues, organs, and cells.
What are the advantages of using electrical recording techniques?
Electrical recording techniques allow measurement of events on a microsecond timescale, such as the activity of single ion channels. They provide good spatial resolution and insights into how channels work, making them versatile tools for studying cellular physiology.
Describe the three main techniques for electrical recording: extracellular, intracellular, and patch clamp.
Extracellular recording involves placing an electrode outside the cell to measure the activity of groups of cells, while intracellular recording uses a sharp glass electrode to penetrate the cell membrane and measure activity inside a single cell. Patch clamp recording, invented by Bert Sakmann and Erwin Neher, involves sealing a glass pipette to the cell membrane to achieve excellent spatial and temporal resolution, allowing recording of single ion channels.
What are the challenges associated with patch clamp recording, and how is it performed?
Patch clamp recording involves sealing a glass pipette to the cell membrane, requiring precise techniques to create and manipulate the electrode. The process involves heating a glass tube to create a fine-tip electrode, coating it with resin, and beveling the glass to ensure smoothness. Patch clamp rigs, including an amplifier, microscope, micromanipulator, and other equipment, are used to perform the recordings, which require careful control and shielding from electrical noise and vibrations.
What are the components of a patch clamp rig, and why is it necessary for patch clamp recordings?
A patch clamp rig includes essential components such as a fluorescence microscope, air table, Faraday cage for shielding, patch amplifier, AD board, PC, and micromanipulator. This setup, which can cost around £50,000, is necessary for performing patch clamp recordings due to their high precision and sensitivity requirements.
What is cell-attached patch clamp recording, and how is it performed?
In cell-attached patch clamp recording, an electrode is placed onto the surface of a cell with gentle suction, trapping channels under the pipette tip. This configuration allows for recording currents through a limited number of active channels at the cell surface. If drugs need to be applied, they must be introduced into the electrode, as the seal prevents the exchange of substances once the recording begins.
Describe whole-cell patch clamp recording and its applications.
Whole-cell patch clamp recording involves further suction to disrupt the membrane trapped under the electrode, making the inside of the electrode continuous with the cell membrane. This configuration allows for recording currents through active channels in the entire cell and is suitable for studying cell currents in response to drugs or regulation of channels by the cell itself. Changes in solutions can be made relatively quickly in whole-cell recordings.
What is perforated patch clamp recording, and how does it differ from whole-cell recording?
Perforated patch clamp recording involves adding an antibiotic into the electrode, which acts as a wide-diameter pore through the membrane, maintaining the seal while allowing ion flow. This configuration is a variation of whole-cell recording and is useful for studying currents through active channels while preserving the integrity of the cell membrane.
Explain inside-out patch clamp recording and its applications.
In inside-out patch clamp recording, the membrane faces the bath solution, with its inner face exposed. This configuration allows for recording currents through single active channels away from the cell and is suitable for studying agents that modulate channels by working at their intracellular face.
What is outside-out patch clamp recording, and when is it used?
Outside-out patch clamp recording involves the outer face of the membrane facing the bath solution. This configuration allows for recording currents through single active channels away from the cell and is useful for studying agents that modulate channels by working at their extracellular face. Rapid changes in solutions can be made during outside-out recordings.
What are the key properties of ion channels at the single-channel level?
Ion channels exhibit multiple conformational states influenced by agonists, including closed, closed with agonist bound, open/active, and desensitized with agonist. Activation mechanisms involve transitions between closed and open states, resulting in brief current blips. The rate constants (k+1 and k-1) governing these transitions can be influenced by agonists, affecting the duration of channel opening.
How do pore properties of ion channels affect current flow, and what factors influence ion channel conductance?
Ion channel conductance, which determines the likelihood of ions passing through, is influenced by ion concentration, membrane potential, and pore properties. Conductance is measured experimentally and reflects the size of the current blip observed. Membrane potential affects current size, with steeper gradients having a greater impact.
What is the significance of whole-cell recording in electrophysiology, and how does it differ from single-channel recording?
Whole-cell recording involves measuring the sum of currents from all channels in a cell, providing insights into cell behavior as a whole. While not as precise as single-channel recording, it is useful for studying agonist concentration relationships and overall cellular responses to stimuli.
Describe the two-electrode voltage clamp (TEVC) technique and its application in electrophysiology experiments.
The two-electrode voltage clamp (TEVC) technique involves inserting two electrodes into a cell, one to measure membrane potential and the other to inject current for clamping. This method is commonly used in oocytes of Xenopus laevis for electrophysiology experiments, allowing researchers to study ion channel function and drug effects.
How is patch clamping combined with single-cell reverse transcription PCR (RT-PCR), and what information does it provide?
Patch clamping can be combined with single-cell reverse transcription PCR (RT-PCR) to measure levels of individual mRNA molecules and infer expression levels of specific proteins. After patch clamp recording, the contents of the cell are collected into the patch clamp electrode and transferred for RT-PCR. This allows researchers to correlate ion channel function with the expression of particular proteins, providing insights into channel regulation and drug effects.
