Paper 12: Membrane Biophysics Module 13: Membrane potential, Transmembranes potential & its measurement by microelectrodes In all cell types, there is an electrical potential difference exits between the inside and outside of the cell resulting from the differential concentrations of sodium and potassium on either side of the membrane. This is termed the membrane potential of the cell. While this phenomenon is present in all cells, it is especially important in muscle and nerve cells, because changes in their membrane potentials are used to code and transmit information or signals. More specifically, the action potentials are electrical signals; these signals carry efferent messages to the central nervous system for processing and afferent messages away from the brain to elicit a specific reaction or movement. The membrane potential is measured in units of volts. (A volt is defined in terms of energy per unit charge; that is, one volt is equal to one joule/coulomb). Learning Objectives Understanding the membrane potential How to calculate membrane potential ? To understand how the membrane potential is generated The contribution of the Na + /K + ATPase Transmembrane Potential Resting potential Action potential Measurement of Transmembrane Potential by Microelectrodes 1. Introduction What is an electrical potential difference? An electrical potential difference exists between two locations when there is a net separation of charge between the two locations. All living cells maintain a potential difference across their membrane. This membrane potential is due to difference in concentration and permeability of sodium and potassium on either side of the membrane. Because of the unequal concentrations of ions across a membrane, the membrane has an electrical charge, inside negative. Changes in membrane potential to elicit action potentials and give cells the ability to send messages to different parts of the body. Some cells, such as nerve and muscle cells are capable of generating quickly changing electrochemical impulses at their membranes, and these impulses are used to transmit signals along the nerve or muscle membranes. In other types of cells, such as macrophages, glandular cells, and ciliated cells, local changes in membrane potentials also stimulate many of the cellular functions. The membrane potential is created due to various proteins embedded within the cell membrane, as well as the structure of the phospholipid lipid bilayer. From a physiological point of view, the membrane potential is responsible for sending messages to and from the central nervous system. It is also very important in cellular biology and shows how cell biology is fundamentally connected with electrochemistry and physiology. 2. Background Membrane potential plays an important role across multiple scientific disciplines such as, Physiology, Biology and Chemistry. In 1902, Julius Bernstein, a German physiologist, came up with a hypothesis for how neurons work. His hypothesis basically stated that the resting potential of a nerve cell was due to a concentration potential of potassium ions. Professor Bernstein hypothesized that there were three contributing factors to membrane potential; membrane permeability and difference in potassium ion (K+) concentrations, higher inside and lower on the outside of the cell. Walther H. Nernst, German physicist who is known for the development of the Nernst equation and won the 1920 Nobel Prize in chemistry, was a major contributor to the study of membrane potential. Nernst helped establish the modern field of physical chemistry and contributed to electrochemistry, thermodynamics and solid state physics. He developed the Nernst equation to solve for the equilibrium potential for a specific ion. Goldman, Hodgkin and Katz furthered the study of membrane potential by developing the Goldman-Hodgkin-Katz equation to account for any ion that might permeate the membrane and affect its potential. The study of membrane potential utilizes electrochemistry and physiology to formulate a conclusive idea of how the charges are separated across a membrane. 3. Physiology of Membrane Potential Cell membrane works as a barrier between the inner and outer surface of a cell. In discussing the concept of membrane potentials and how they function, the establishment of membrane potential is essential. The membrane that surrounds a cell is composed of phospholipids, glycolipids, cholesterol and proteins. The primary structure is a lipid bilayer. Phospholipid molecules have an electrically charged head that attracts water and a hydrocarbon tail that repels water; they line up side by side in two opposing layers, with their heads on the inner or outer surface of the membrane and their tails in the core, from which water is excluded. The other lipids affect the structural properties of the membrane. The proteins embedded in the lipid bilayer, the Na+/K+ ATPase, ion transporters, and voltage gated channels, and it is the site of vesicular transport. The membrane structure regulates entry ions and exit of ions to determine the concentration of specific ions inside of the cell. Membrane potentials in cells are determined primarily by three factors: a) the concentration of ions on the either side of the membrane; b) the permeability of the cell membrane to those ions through specific ion channels; and c) by the activity of electrogenic pumps (e.g., Na+/K+- ATPase and Ca2+ transport pumps) that maintain the ion concentrations across the membrane. Figure 1. Model of membrane lipid bilayer 3.1 Understanding membrane potential The following points important to understand how the membrane potential works The difference between the electrical and chemical gradient is important. Electrical Gradient Opposes the chemical gradient. Represents the difference in electrical charge across the membrane Chemical Gradient Opposes the electrical gradient Represents the difference in the concentration of a specific ion across the membrane. A good example is K+. The membrane is very permeable to K+ and the K+ ion concentration inside the cell is high, therefore a positive charge is flowing out of the cell along with K+. The K+ ion concentration inside the cell decreases causing the concentration gradient to flow towards the outside of the cell. This also causes the inside of the cell to become more electronegative increasing its electrical gradient. The Nernst equation can help us relate the numerical values of concentration to the electrical gradient. Leak Channels Channels that are always open Permit unregulated flow of ions down an electrochemical gradient. Na+/K+ ATPase Actively transports Na+ ions out of the cell and K+ ions into the cell. Helps to maintain the concentration gradient and to counteract the leak channels. Figure 2. Differences in the concentration of ions on both sides of a cell membrane produce a voltage difference called the membrane potential. The major contributions usually come from sodium (Na+) and chloride (Cl–) ions which have high concentrations in the extracellular side, and potassium (K+) ions, which along with large protein anions have high concentrations in the intracellular side of the membrane. 3.2 How to calculate membrane potential of a cell ? The calculation for the charge of an ion across a membrane, The Nernst Potential, is relatively easy to calculate. The equation is as follows: (RT/zF) log([I]out/[I]in) RT/F is approximately 61, therefore the equation can be written as (61/z) ln([I]out/[I]in) Derived under resting membrane conditions when the work required to move an ion across the membrane (up its concentration gradient) equals the electrical work required to move an ion against a voltage gradient. R= gas constant (8.314 jules/oK.mol) T= temperature (oK) F= Faraday constant (96, 000 coulombs/mol) z= the electric charge on the ion. For example, z is +1 for K+, +2 for Mg2+ etc. z have no unit. [I]out= ion concentration outside the cell [I] in= ion concentration inside the cell The only difference in the Goldman-Hodgkin-Katz equation is that is adds together the concentrations of all permeable ions as follows + + - + + - i (RT/zF) log([K ]o+[Na ]o+[Cl ]o /[K ]i+[Na ]i+[Cl ] ) 3.3 To understand how the membrane potential is generated Cell membranes maintain a small voltage or "potential" across the membrane in its normal or resting state. In the rest state, the inside of the cell membrane is negative with respect to the outside. The voltage arises from differences in concentration of the K+ and Na+ ions. The membrane potential of a cell derives ultimately from two factors: electrical force and diffusion. Electrical force arises from the mutual attraction between particles with opposite electrical charges (positive and negative) and the mutual repulsion between particles with the same type of charge (both positive or both negative). Diffusion arises from the statistical tendency of particles to redistribute from regions where they are highly concentrated to regions where the concentration is low 3.3a Electrical force In the selectively permeable cell membranes are ion channels which allow K+ ions to pass into the interior of the cell, but block Na+ ions. Negatively charged proteins on the interior of the cell are also denied passage across the membrane. In addition, there are active transport mechanisms at work. There is a process called the Na + /K + ATPase, which utilizes ATP to pump out three Na+ ions and pump in two K+ ions. The collective action of these mechanisms leaves the interior of the membrane about -70 mV with respect to the outside. 3.3b Membrane potentials caused by diffusion “Diffusion Potential” caused by an ion centration difference on the two sides of the membrane. The potassium concentration is great inside, but very low outside the membrane. Let us assume that the membrane in this instance is permeable to the potassium ions, but not to any other ions. Because of the large potassium concentration gradient from inside to outside, there is a strong tendency for extra numbers of potassium ions to diffuse outward through the membrane.