BISC 307L 2nd Edition Lecture 6Current LectureRecording from Single K+ ChannelsThis is the Patchclamp method, and it measures current flowing through a single ion channel. The basic definition of current = movement of charge through space. So ions carrying their +/-, and moving through aqueous solution, are a current. The way it works: Take a glass pipette with a polished tip and press it against the membrane of aneuron or any cell. The binding of the charges in the glass to the charges in the membrane will be very tight, and if there happens to be an ion channel in the patch of membrane underneath the open tip of the pipette, and if the seal between the glass and membrane is good, then any current flowing through the channel will follow into the electrode and ultimately into an amplifier where it is measured. im = measure of current through a single channel.From the recordings, Patchclamp was able to plot data graphs of current vs. time. Most of the time, there was 0 current flowing, but at random intervals, a small current of 10 pA occurred rapidly(instantaneous – faster than our current technology can measure) and then shut off just as quickly. There abrupt transitions represent the opening and closing of a single ion channel.They observed that the time at which the channel opens is random, but you can determine the probability of it being in an open or closed state. Depolarization affects Single Channel CurrentCurrent = i, average current that flows when the channel is open. With this apparatus, they could utilize a depolarization on a patch of membrane, and they foundrecordings like the one on the bottom. The channel opens more frequently (still random though,but probability has increased) and the individual single current amplitude is bigger(i is bigger). In general, this directly demonstrates that this channel is voltage gated. The second effect can be understood by looking at Ohm’s Law, which relates current to voltage to resistance. Rate at which charge moves past a point in space = voltage divided by resistance. Current is movement of ions through channels. Lipid bilayer membrane itself has a very high resistance. Ions are hydrated in real life, surrounded by a shell of water. Lipid bilayer has a high, close to infinite resistance, BUT open ion channels give it a finite resistance.For a single channel current, I = (Vm – Eion)/R. The net force making an ion move is not the membrane potential, but the difference between the membrane potential and the equilibrium potential of the ion (the driving force).Conductance = inverse of resistance. Conductance = 1/R. Describes the ability of a material to allow current to flow through it (or permeability). There is always more than one ion channel existing in a membrane in a cell. Some are for the same ion. Most cells have a K selective channel that is always open (leakage channel). Right nextto that could exist a voltage gated potassium channel (several types – some that when you depolarize, they open either slowly or fast.) Can open due to increase in intracellular calcium. And there is an interesting one in the heart, the inwardly rectifying one that is one that closes when you depolarize – probability of opening goes down when you depolarize. Electrical Properties of Axons Action Potentials – long distance electrical signals that are essential for allowing neurons to communicate with each other.Shown on the top of the figure above is a segment of an unmyelinated axon. On either side of its plasma membrane are the ECF and the ICF, which are electrolyte solutions that conduct electricity wellOn the bottom of the figure is the electrical model for the axon.-Though the ECF and ICF are good conductors, they still have resistance. The ECF has a very low resistance, and the ICF or axoplasm, has a more moderate resistance. -The bigger the axon, the bigger the radius, the lower the resistance. In a tiny axon with small radius, Raxoplasm would be high. -The membrane has resistance as well, almost infinite resistance except for the open channels ithas. It also has another interesting quality called capacitance, the measure of a system to store charge. We have two conductors(ECF, ICF), separated by a thin insulator(lipid bilayer). Because the membrane has resistance and capacitance, if charges are going to it, they can go through the resistance of the membrane or through the capacitance of the membrane. The resistance for a membrane in an unmyelinated axon where channels are uniformly distributed is inversely proportional to the surface area of the axon because more surface area = more open channels in direct proportion to the surface area = less resistance. -The driving force making the charge go through the circuit is Vm – Ena.Important rule: *Direction of current movement is assigned to cation movement. Opposite in the case of anions. For example, an inward chloride current would mean the ions are going out of the cell. *Negative signs = inward current. Positive sign = outward current.So current is being driven through the axon, and it reaches the intersection – here, it can go out of the cell or it can continue down the Raxoplasm until it hits another branch point. What’s actually physically going down the axoplasm? The sodium ions that came in? Probably not. What happens is that Na comes in, displaces a positive charge that was there, and that positive charge moves away OR it attracts a negative charge that was next to it, and where the negative moved from, a positive is left behind. Actual ion carrying current down is dependent on whatever ion is there and available at that moment. Actual charge going down the right can be any cation or any anion going the other way. Eventually, it gets out and flows through the ECF, which has a big volume, and the density of current drops off exponentially with distance from the axon. Application to real life: When you want to measure ECG of patient, put some conductive paste on them and put electrodes on the skin and measure the blips, which are the current(AP) of cardiac muscles. Flow out of the cell all the way to the skin, which is why electrodes can measure ECG. Sharks have electroreceptors in snouts, and could detect your EKG from a quarter/half mile away. Spread of Graded PotentialsFigure above explains a local, graded potential spreading down the axon. Notice the 4 sharp microelectrodes impaling the axon. They are glass pipettes, hollow inside and with a tiny, tiny