diff --git a/2016-09-17-lecture01.md b/2016-09-17-lecture01.md index d75b58b..391332e 100644 --- a/2016-09-17-lecture01.md +++ b/2016-09-17-lecture01.md @@ -38,11 +38,12 @@ REASON YOU CANNOT ENROLL: * Navigate: arrow keys and `spacebar` * Menu: `m` * Fullscreen: `f` -* Overview: `o` or `esc` or pinch (touch screen) -* Zoom object: double–click +* Overview: `o` or `esc` +* Zoom: `alt-click` or two-finger multi-touch (touch screens/trackpads) + * Zoom-scroll: two-finger drag (touch screens/trackpads while zoomed in) * Print: `...lecture.html?print-pdf` -Recommend browser is Chrome on a laptop/PC. Some features (e.g. full screen, zoom) may not work on tablet/touch screen devices. +Recommend browser is Chrome on a laptop/PC. Some features that only have keyboard bindings (e.g. fullscreen, overview) may not work or be disabled on tablet/touch screen devices. --- @@ -147,7 +148,7 @@ sinew
* We are now in a gene-centric “post-genomic” phase of neuroscience -* Most genes are expressed in the brain, either during development or in the adult. It is the spatial and temporal regulation of these genes and an organisms interaction with the environment that builds a nervous system. +* Many genes are expressed in the brain, either during development or in the adult. It is the spatial and temporal regulation of these genes and an organisms interaction with the environment that builds a nervous system. * Neuroscience therefore encompasses many fields, including genetics, cell biology, physiology, and development biology. @@ -498,7 +499,7 @@ Note: Glia : greek for 'glue' -: outnumber neurons 10-50 fold (higher mammals) +: outnumber neurons 10-50 fold (higher mammals) : structural support for neurons : remove debris and maintain a functional nervous system environment diff --git a/2016-09-28-lecture03.md b/2016-09-28-lecture03.md index 1ee0ceb..87fc501 100644 --- a/2016-09-28-lecture03.md +++ b/2016-09-28-lecture03.md @@ -9,9 +9,7 @@ Note: -So, how do neurons convey information over long distances that results in information transfer to other neurons at synaptic connections? It through electrical signaling that neurons are able to generate and transmit information. And this electrical signaling is possible because of a combination of… - - +So, how do neurons convey information over long distances that results in information transfer to other neurons at synaptic connections? It is through electrical signaling that neurons are able to generate and transmit information. And this electrical signaling is possible because of a combination of… - voltage-dependent membrane permeability - which in turn requires special membrane proteins called ion channels and transporters @@ -41,9 +39,9 @@ Note: To understand the basis of electrical excitability in neurons, we first need to understand that neurons, like other excitable cells, have a difference in electrical potential across the cell membrane when it is at rest. -To learn this neuroscientists stick electrodes inside of neurons. This electrode is hooked up to a voltmeter and another electrode sits outside the cell as a ground or reference electrode to complete the circuit. The difference in voltage between the inside of the cell and the outside of the cell is monitored over time and displayed on an oscilloscope. +To learn this physiologists stick electrodes inside of cells, including neurons. This electrode is hooked up to a voltmeter and another electrode sits outside the cell as a ground or reference electrode to complete the circuit. The difference in voltage between the inside of the cell and the outside of the cell is monitored over time and displayed on an oscilloscope. -When you do this, one finds a negative resting membrane potential of the neuron with respect to the outside. You can see in this plot at the bottom right that we’ve inserted the electrode and then over time we measure a negative membrane potential of about -70 mV. Recall that volts are a unit of electrical potential energy —> +When you do this such as with this model neuron shown here, one finds a negative resting membrane potential of the neuron with respect to the outside of approximately -70 mV.Recall that volts are a unit of electrical potential energy, where 1 Volt is defined as the amount of energy that will drive 1 coulomb of elementary charge or 6x10^18 electrons or protons through a resistance of 1 ohm in 1 second —> --- @@ -70,38 +68,54 @@ Flow rate ~ Current (amperes) = `I` Note: -And voltage is related to the resistance and current in an electrical circuit as described by Ohm’s law. This analogy of a water pump/water wheel circuit helps us understand these relationships better. +And recall from physics that voltage is related to the resistance and current in an electrical circuit as described by Ohm’s law. This analogy of a water pump/water wheel circuit helps us understand these relationships better. -Voltage = is the potential difference, or electromotive force measured across the conductor in units of volts. Imagine a hand pump that you use to do some work and introduce pressure in a water system, that pressure or potential difference is the voltage. +Voltage +: is the potential difference, or electromotive force measured across the conductor in units of volts. +: So imagine a hand pump that you use to do some work and introduce pressure in a water system, that pressure or potential difference is the voltage. -* - Volt is defined as the difference in electric potential between two points of a conducting wire when an electric current of one ampere dissipates one watt of power between those points -* - voltmeter, ammeter +*Volt is defined as the difference in electric potential between two points of a conducting wire when an electric current of one ampere dissipates one watt of power between those points* +*voltmeter, ammeter* + +Current +: measured in amperes is the flow of electric charge across a surface at the rate of one coulomb per second. Used to express the flow rate of electric charge. +: So imagine the rate of water flow in this water pump as the the flow of electric charge across a cell membrane. What is the charge that is moving for a cell? Monovalent and divalent atoms like Na⁺, K⁺, Cl⁻, and Ca²⁺. + +*1A equivalent to one coulomb (roughly 6.241×10^18 times the elementary charge) per second* +*coulomb = charge (symbol: Q or q) transported by a constant current of one ampere in one second. 1C equivalent to a charge of approximately 6.242×10^18 protons or electrons.* +*elementary positive charge: This charge has a measured value of approximately 1.6021766208×10^−19 coulombs* + +Resistance +: is the difficulty to pass a current through a conductor measured in ohms. +: Image the diameter of a pipe or a valve that you can regulate to be the resistance +: inverse of resistance is conducance *g* measured in siemens (S) +: for studying neuronal excitability rewriting Ohm's law as I = g(Vm-Ex) is most useful. g = conductance, no. of open channels. (Vm-Ex) = driving force causing either positive or negative current. -Current: measured in amperes is the flow of electric charge across a surface at the rate of one coulomb per second. Used to express the flow rate of electric charge. Imagine the rate of water flow in this water pump as the the flow of electric charge across a cell membrane. What is the charge that is moving for a cell? Monovalent and divalent atoms like Na⁺, K⁺, Cl⁻, and Ca²⁺. +**Ohm’s law** from physics class relates these quantities together as V = IR, and rearranging this equation and reading it as I = V/R or Current = Voltage divided by Resistance gives you a better intuitive feel for these relations. **Notice that when you have 0 voltage or potential difference you have no current.** -* - 1A equivalent to one coulomb (roughly 6.241×10^18 times the elementary charge) per second -* - coulomb = charge (symbol: Q or q) transported by a constant current of one ampere in one second. 1C equivalent to a charge of approximately 6.242×10^18 protons or electrons. -* - elementary positive charge: This charge has a measured value of approximately 1.6021766208×10^−19 coulombs +Avogadro constant +: (symbols: L, NA) +: is the number of constituent particles, usually atoms or molecules, that are contained in the amount of substance given by one mole. +: Avogadro’s constant = 6.022×10^23 and is dimensionless. -* Resistance: is the difficulty to pass a current through a conductor measured in ohms. Image the diameter of a pipe or a valve that you can regulate to be the resistance -* Ohm’s law from physics class relates these quantities together as V = IR, and rearranging this equation and reading it as I = V/R or Current = Voltage divided by Resistance gives you a better intuitive feel for these relations. Notice that when you have 0 voltage or potential difference you have no current. -* Avogadro constant (symbols: L, NA) is the number of constituent particles, usually atoms or molecules, that are contained in the amount of substance given by one mole. Avogadro’s constant = 6.022×10^23 and is dimensionless. -* mole = It is defined as the amount of any chemical substance that contains as many elementary entities, e.g., atoms, molecules, ions, electrons, or photons, as there are atoms in 12 grams of pure carbon-12 (12C). This number is expressed by the Avogadro constant +mole +: it is defined as the amount of any chemical substance that contains as many elementary entities, e.g., atoms, molecules, ions, electrons, or photons, as there are atoms in 12 grams of pure carbon-12 (12C). +: this number is expressed by the Avogadro constant --- ## Electrical signals -* Can be generated by changing the resting potential of the neuron +* Can be generated by changing the membrane potential of the neuron * Receptor potentials can be generated from the activation of sensory receptors, from touch, light, sound, and heat * Synaptic potentials are transmitted from one neuron to another at the synapse * Action potentials are the booster system to propagate electrical signals a long distance Note: -Signals in neurons can be generated by changing the resting membrane potential. +Signals in neurons can be generated by changing the membrane potential. This includes receptor potentials inside your body’s sensory neurons for touch, heat, light, and sound. @@ -124,9 +138,9 @@ This figure shows these 3 types of neuronal signals. - Here is a synaptic potential recorded in a postsynaptic neuron. -- Here is an action potential in a motor neuron. Look as the y-axes here— the action potential has a much larger amplitude change than receptor or synaptic potentials. - +- Here is an action potential in a motor neuron. **Look as the y-axes here**— the action potential has a much larger amplitude change than receptor or synaptic potentials. +To understand the basis of these electrical signals we first need to learn about how this baseline membrane potential is generated, which is the neurons membrane potential while it is at rest. We will spend most of today's class learning about the neurons resting membrane potential and which will lead into how the action potential is generated that we'll continue with next class. --- @@ -148,11 +162,11 @@ This figure shows these 3 types of neuronal signals. Note: -I said that the resting membrane potential is more negative on the intracellularly than extracellularly– this is because of the lipid bilayer and its transmembrane proteins which together make a functional cell membrane +I said that the resting membrane potential is more negative inside the neuron with respect to its extracellular space– this is because of the lipid bilayer and its transmembrane proteins which together make a functional cell membrane -We can that the cell, a bit like American politics, is polarized with one side more negative and the other being more positive +We can think of the cell, a bit like American politics, is polarized with one side more negative and the other being more positive -This polarization results in a potential difference across the membrane (remember our water pump example) of about -70 mV +This polarization of the cell results in a potential difference across the membrane (remember our water pump example) of about -70 mV And its there is a concentration gradient in ions (which are charged atoms like sodium, potassium, and chloride) that results in this difference in distribution of charge across the neuron’s membrane @@ -212,15 +226,15 @@ Note: So we insert the microelectrode into the cell and find that this neuron is resting at -65 mV. - Then we inject a small amount of negative current (less than 1 nA) so that we hyperpolarize the cell and we see that the membrane responds passively, meaning that the membrane potential changes and recovers with an exponential relationship. -* 1-(1/e) = 63% Vm and 1/e (37%) of Vm +* 1-(1/e) = 63% (rise) Vm and 1/e (37%) (decat) of Vm If we depolarize the cell membrane from rest by injecting pulses of positive current we get corresponding passive responses with exponential rises and decays of membrane potential– **unless that cell is a neuron and we’ve exceeded the threshold potential (shown by the red dotted line) for generating an action potential in that neuron. Notice if we inject stronger current pulses, we get more action potentials, also known as a higher spiking or firing rate, rather than different action potential amplitudes. If the depolarization is sufficient to generate an AP, that AP amplitude stays largely the same within each individual neuron. +We will go over more detail each of these components later on... --- @@ -231,7 +245,7 @@ Notice if we inject stronger current pulses, we get more action potentials, also Note: -All electrical signals are the due to the flow of charge, positive or negative. In this case of neurons we the charge is due to the movement cations such as Na and K and anions such as Cl and neuronal membranes are selectively permeable to some of these ions giving the rise to the flow of charge or current across the cell membrane. +All electrical signals are the due to the flow of charge, positive or negative. In this case of neurons the charge is due to the movement cations such as Na and K and anions such as Cl and neuronal membranes are selectively permeable to some of these ions giving rise to the flow of charge or current across the cell membrane. --- @@ -243,9 +257,7 @@ All electrical signals are the due to the flow of charge, positive or negative. Note: -How do ions get across the lipid cell membrane bilayer? Remember there are proteins in the cell membrane. Some of these are selective ion transporters, remember the Na-K ATPase from cell biology. These work to create concentration gradients. - - +So how do ions get across the lipid cell membrane bilayer? Remember there are proteins in the cell membrane. Some of these are selective ion transporters, remember the Na-K ATPase from cell biology. These work to create concentration gradients. There are also ion channels that form pores in the cell membrane that are selectively permeable for certain kinds of ions to cross the membrane. These allow ions move across the membrane @@ -279,9 +291,9 @@ Here is one these ion transporters— the Na-K pump that moves 3 Na out of the c Note: -Ion channels span the membrane and act as pores. They can open and close, often in a voltage-dependent fashion as we will learn thursday. They show selectively such that there are different types of Na, K channels as well as others. +Ion channels span the membrane and act as pores. They can open and close, often in a voltage-dependent fashion as we will learn thursday. And ion channels even show selectively such that there are different types of Na, K channels as well as others. -And they can be additionally regulated or ‘gated’ by different mechanisms including voltage or binding of ligands such as neurotransmitters as we will learn in subsequent classes. +And they can be additionally regulated or ‘gated’ by different mechanisms including voltage or binding of ligands such as neurotransmitters. We will learn much more about the selectivity and function of ion channels a couple lectures from now. --- @@ -306,7 +318,7 @@ So again there are active ion transporters like the Na-K ATPase and there are io Note: -We actually can predict what the resting membrane potential by knowing the concentrations of ions inside and outside the cell and knowing the relative permeability of these ions to move across the cell membrane. +We actually can predict what the resting membrane potential is by knowing the concentrations of ions inside and outside the cell and knowing the relative permeability of these ions to move across the cell membrane. If a cell membrane is largely permeable to just one ion species, we can use the Nernst equation to predict the membrane potential for all kinds of cells. @@ -315,14 +327,14 @@ If a cell membrane is permeable to more than one ion, we can use the Goldman equ We will come back to these in a minute. -*Walther Nernst (1864-1941)* +*Walther Nernst (1864-1941), West Prussia, 1920 Nobel Prize in chemistry* --- ## Electrochemical equilibrium -
orange dots K⁺, green dots Cl⁻
Neuroscience 5e Fig. 2.5
+
orange dots K⁺, green dots Cl⁻. This simulated membrane is only permeable to K⁺
Neuroscience 5e Fig. 2.5
@@ -334,7 +346,7 @@ Imagine the following experiment. We have a cell and record intracellular membra If this membrane is only permeable to K⁺, and KCl concentration is the same inside and outside the cell, there is no net flux of K⁺ -If KCl is more concentrated inside the cell, initially there is a net flux of positively charged K⁺ from inside to outside the cell due to the chemical concentration driving force leaving the membrane hyperpolarized until the this chemical force is balanced by the electrical driving force from the positively charged K⁺ being repelled by the more positive environment now outside the cell. This is called electrochemical equilibrium, and the potential at which this occurs is called the equilibrium potential for that ion. +If KCl is more concentrated inside the cell, initially there is a net flux of positively charged K⁺ from inside to outside the cell due to the chemical concentration driving force which leaves the membrane hyperpolarized because of the net movement of postive charge to the outside until the this chemical force is balanced by the electrical driving force from the positively charged K⁺ being repelled by the more positive environment now outside the cell. This is called electrochemical equilibrium, and the potential at which this occurs is called the equilibrium potential for that ion. --- @@ -395,8 +407,7 @@ So I stated that the Nernst equation is how we can calculate the equilibrium pot And here is the Nernst equation is: -Where Ex is: - +Where Ex is... Gas constant R @@ -453,10 +464,14 @@ T = 20+273 ==>58.26427 +
+
+ --- ## Examples +* Calculate the following equilibrium potentials at room temperature: * Outside 10 mM KCl, Inside 1 mM KCl membrane only permeable to K⁺ ? * EK+ = (58/1)log10(10/1) ==> +58 * Outside 1 mM KCl, Inside 100 mM KCl membrane only permeable to K⁺ ? @@ -479,8 +494,6 @@ Since the Nernst equation is really just a linear equation of the form y = mx, y ## Electrochemical equilibrium - -
Neuroscience 5e Fig. 2.5
@@ -521,7 +534,7 @@ Note: ## Membrane potential influences ion fluxes -
Neuroscience 5e Fig. 2.6
+
Simulated cell at room temperature
Neuroscience 5e Fig. 2.6
Note: @@ -535,7 +548,6 @@ If our hypothetical battery holds the membrane at -58 mV, the equilbrium potenti At more negative membrane potentials than the nernst equilbrium potential we get net inward flow due to the stronger electrical driving force which in the case of potassium here is causing it to move against its chemical gradient. - --- ## Membrane potential influences ion fluxes @@ -564,9 +576,9 @@ Note: So to summarize, remember that both the direction (inward vs outward) and magnitude of charge flow or current depends on membrane potential. -Just remember Ohm’s law, I = V/R +Just remember Ohm’s law, I = V/R and rewrite it as Ix = (Vm - Ex)/R -And we can experimentally vary... +And we as scientists can experimentally vary... --- @@ -624,7 +636,7 @@ For a typical neuron at rest, pK : pNa : pCl = 1 : 0.05 : 0.45. Note that becaus Note: -And as we will soon learn, the resting membrane potential and action potential voltage is mostly due to changes in K permeability and Na permeability across the neuronal membrane. As you can see in this figure, the resting membrane potential for a neuron is close to the EK eq potential due to much greater permeability for K. During an action potential Na permeability initially increases, until the Vm approaches the ENa and then Na permeability decreases until the Vm again approaches the resting membrane potential and Pk increases. +And as we will soon lecarn, the resting membrane potential and action potential voltage is mostly due to changes in K permeability and Na permeability across the neuronal membrane. As you can see in this figure, the resting membrane potential for a neuron is close to the EK eq potential due to much greater permeability for K. During an action potential Na permeability initially increases, until the Vm approaches the ENa and then Na permeability decreases until the Vm again approaches the resting membrane potential and Pk increases. --- @@ -745,7 +757,7 @@ To answer this Hodgkin and and Katz measured the membrane potential while induci They observed that the Vm approached ENa during an AP... -They reason they hypothesized is that during an AP... +They reasoned that during an AP... Their experiment was to lower Na concentrations in the extracellular medium— @@ -793,6 +805,8 @@ As you can see on the left here changing extracellular [Na] changes the action p * During depolarization membrane becomes super permeable to Na⁺ * There must be Na⁺ channels that are closed during rest but become open during an action potential, and closed again at the end of an action potential +
Neuroscience 5e Fig. 2.7
+ Note: So a summary of the Hodgkin and Katz experiment conclusions... diff --git a/2016-10-03-lecture04.md b/2016-10-03-lecture04.md new file mode 100644 index 0000000..858b67a --- /dev/null +++ b/2016-10-03-lecture04.md @@ -0,0 +1,593 @@ +## Voltage dependent membrane permeability + +
+
+ +* Hodgkin and Huxley hypothesis– Action potential can be explained by **voltage-gated ion channels** +* Experiment– Measure ion permeability at varying membrane potentials +* Problem– Difficult to systematically vary the cell potential and also measure ion permeability +* Solution– Voltage clamping. Fix membrane potential in a cell without triggering an action potential while measuring ion permeability + +
+ +
Neuroscience 5e Fig. 2.7
+ +Note: + +We learned last time that the experiments of Hodgkin, Huxley, and Katz showed that the Vm during an AP approaches ENa. And they thought that this might be due to changes in permeability for Na in the cell membrane that changes during the course of an action potential. Thus Hodgkin and Huxley hypothesized that APs can be explained by ion channels that change their permeability due to voltage— that these channels are voltage-gated. + +Alan Hodgkin and Andrew Huxley began this work in the late 1930s, and quickly finished one paper before helping with the British war effort during WWII. Indeed Hodgkin said that he lost all interest in neurophysiology during those dark years as one might imagine. But as things calmed down after the war they renewed their collaboration and got back to the business of neuronal excitability. + +So they needed to proved that ion permeability changes according to membrane potential but there was an issue— how to vary the membrane potential in a systematic way and also measure the ion permeabilities? + +The solution was to build an electrophysiological recording apparatus with feedback circuitry such that you can fix or clamp the voltage across the cell membrane. + +[http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-7793/homepage/celebrating_the_work_of_alan_hodgkin_and_andrew_huxley.htm](http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-7793/homepage/celebrating_the_work_of_alan_hodgkin_and_andrew_huxley.htm) + +-- + +## Action potential summary video + +
Neuroscience 5e Animation 2.3
+ +Note: + +Summary of last time… + +-- + +## More Vm examples + +
+
+ +* Given a cell with intracellular: 1 mM NaCl, 10 mM KCl; extracellular: 10 mM NaCl, 1 mM KCl +* What is the resting potential of the cell at room temperature (20ºC + 273 = 293 K) if the membrane is only permeable to K⁺? + * `(58/1)*log10(1/10) = -58 mV` +* Only permeable to Na⁺? + * `(58/1)*log10(10/1) = +58 mV` +* Only permeable to Cl⁻? + * `(58/-1)*log10(11/11) = 0 mV` +* Equally permeable to K⁺ and Na⁺? + * `Pk = 0.5; Pna = 0.5; Pcl = 0; kOut = 1; kIn = 10; naOut = 10; naIn = 1; clIn = 11; clOut = 11` + * `(58)*log10( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) ) = 0 mV` + +
+ +Note: + +1. (58/1)*log10(1/10) = -58 mV +2. (58/1)*log10(10/1) = +58 mV +3. (58/-1)*log10(11/11) = 0 mV + +4. 0 mV: +* Pk = 0.5; Pna = 0.5; Pcl = 0; kOut = 1; kIn = 10; naOut = 10; naIn = 1; clIn = 11; clOut = 11 +* (58)*log10( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) ) = 0 mV + +* 0 mV: +* Pk = 1; Pna = 1; Pcl = 0; kOut = 1; kIn = 10; naOut = 10; naIn = 1; clIn = 11; clOut = 11 +* (58)*log10( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) ) + +* -59 mV (room temp and low Pna): +* Pk = 1; Pna = 0.001; Pcl = 0.5; kOut = 1; kIn = 10; naOut = 10; naIn = 1; clIn = 1; clOut = 11 +* (58)*log10( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) +* + +-62 mV (body temp and low Pna): + +* R = 8.3; F = 9.6e4; T = (273+37) +* Pk = 1; Pna = 0.001; Pcl = 0.5; kOut = 1; kIn = 10; naOut = 10; naIn = 1; clIn = 1; clOut = 11 +* ((R*T)/F)*log( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) ) + + +-69 mV (body temp and low Pna and physiol concentrations): + +* R = 8.3; F = 9.6e4; T = (273+37) +* Pk = 1; Pna = 0.05; Pcl = 0.45; kOut = 5; kIn = 140; naOut = 145; naIn = 5; clIn = 5; clOut = 110 +* ((R*T)/F)*log( (Pk*kOut + Pna*naOut + Pcl*clIn) / (Pk*kIn + Pna*naIn + Pcl*clOut) ) + + +Calculate the total concentration of all ions for these solutions. For every one NaCl that dissolves, two ions are produced (one Na⁺ and one Cl¯). Thus for 10 mmol/L NaCl outside there are (10 mmol/L)x(1 total Cl ions/NaCl) = 10mM. And for 1mM KCl outside there are (1 mmol/L)x(1 total Cl ions/KCl) = 1mM. Thus the total number of Cl⁻ ions per liter is 11mmol/L = 11mM + + +--- + +## The voltage clamp method + +
Neuroscience 5e Box 3A
+ + +Note: + +This is an illustration of the voltage clamp recording method. + +One internal electrode measures membrane potential and is connect to the voltage clamp amplifier. + +voltage clamp amplifier compares membrane potential to the desired command potential + +When Vm is different from the command potential the clamp amplifier injects current ion the axon through a second electrode. This feedback arrangement causes the membrane potential to become the same as the command potential. + +The current flowing back into the axon and thus across its membrane can be measured. + +**This electronic feedback circuit holds the membrane pot at the desired level, even in the face of permeability changes that would normally alter the membrane potential. (such as those generated during the action potential). Most importantly, the device permits the simultaneous measure of the current needed to keep the cell at a given voltage. This current is exactly equal to the amount of current flowing across the neuronal membrane, allowing direct measurement of these membrane currents. + + +>An amplifier, electronic amplifier or (informally) amp is an electronic device that can increase the power of a signal. It does this by taking energy from a power supply and controlling the output to match the input signal shape but with a larger amplitude. + +>A differential amplifier is a type of electronic amplifier that amplifies the difference between two input voltages but suppresses any voltage common to the two inputs.[1] + +>An operational amplifier (often op-amp or opamp) is a DC-coupled high-gain electronic voltage amplifier with a differential input and, usually, a single-ended output.[1] In this configuration, an op-amp produces an output potential (relative to circuit ground) that is typically hundreds of thousands of times larger than the potential difference between its input terminals.[2] + + +--- + +## Hodgkin and Huxley 1952 + +* Do neuronal membranes have voltage-dependent permeability? +* Which ions are changing their permeability? +* Experiment– Change potential to make neuron membrane potential more negative (hyperpolarize). No currents need to be injected into cell to maintain that potential. Therefore no current is moving from inside and outside of cell +* Change potential– Depolarize cell, now see both inward and outward currents between the inside and outside of cell + +Note: + +Hodgkin and Huxley published a series of seminal papers in 1952 that summarized their investigations using this voltage clamp method to examine voltage dependent ion flux. + +They asked… + +So the experiment was to hold the membrane potential at different voltages and measure charge flux into or out of the cell... —> + + +--- + +## Electric current flow across a squid axon membrane during voltage clamp + +
negligible current (except for a capacitive transient)
Neuroscience 5e fig. 3.1
+ +
inward and outward currents
Neuroscience 5e fig. 3.1
+ +Note: + +And so here are the results from this type of voltage clamp experiment. + +If you command that the cell membrane potential be hyperpolarized, you get very little or negligible current flowing across the membrane except for a very brief capacitive current that you always see in these voltage clamp experiments. + +This is because the cell membrane essentially acts as a parallel RC circuit where a resistor and a capacitor are connected in parallel and to a constant current source. Ion channels are resistors, lipid bilayer with the extracellular and intracellular environments act as capacitor, storing charge in the form of ions accumulating near the surface of the membrane. When a switch is turned on in an RC circuit current flows from the battery to the capacitor until the capacitor is charged to a voltage that is same as the battery. + +However when Hodgkin and Huxley depolarized the membrane, a transient inward current occurs followed by a slow outward current. + + +*A capacitor (originally known as a condenser) is a passive two-terminal electrical component used to store electrical energy temporarily in an electric field. Consists of two parallel conductors. Lipid membrane with the inner and outer cellular environment acts as this. The membrane capacitance per unit areas is mostly constant at about 1 µF/cm2.* + +* When the voltage is constant, the current through the capacitative pathway is zero because the capacitor has acquired the charge Q (coulombs) according to the relationship Q=CV. C is capacitance (farads) Ic is capacitive current. Ic = C(dV/dt) +* as long as V is changing with time, there will be a current flowing towards the capacitor. +* if V is constant in time, there is no capacitive current. +* product of resistance and capacitance has the unit of time and is called the time constant. Time constant defines how quickly capacitors charge or discharge over time. + +[http://nerve.bsd.uchicago.edu/med98c.htm](http://nerve.bsd.uchicago.edu/med98c.htm) + +--- + +## Current produced by different membrane depolarizations during voltage clamp + +
Neuroscience Fig. 3.2
+ +Note: + +This show several different voltage steps (with the brief capacitive current omitted for clarity) + +...Notice as we approach ENa the inward current disappears. + +--- + +## Relationship between current amplitude and membrane potential + +
External Na⁺ 440 mM, internal Na⁺ 50 mM, therefore Nernst says **ENa = 55 mV**
Neuroscience 5e Fig. 3.3
+ + +Note: + +This summarizes the peak magnitude of these these two currents at different Vm + +--- + +## How do we prove the inward current is sodium? + +* Prediction– If you could change the Na⁺ concentrations in the system, for example have less sodium outside than inside (instead of the normal high outside low inside), Nernst equation would predict an early outward current instead of an early inward current +* Experiment– Change the Na⁺ concentration in the bath. Normally 440 mM NaCl outside & 50 mM inside for squid axon, now make it 50 mM inside & 0 mM outside + +Note: + +So it seems like this inward current may be carried by Na ions. + +--- + +## Dependence of the early inward current on sodium + +
Neuroscience 5e Fig. 3.4
+ +
Squid giant axon voltage clamping
+ + +Note: + +>Choline is a water-soluble nutrient. It is usually grouped within the B-complex vitamins. Choline generally refers to the various quaternary ammonium salts containing the N,N,N-trimethylethanolammonium cation. (X− on the right denotes an undefined counteranion.) + +*The cation appears in the head groups of phosphatidylcholine and sphingomyelin, two classes of phospholipid that are abundant in cell membranes. Choline is the precursor molecule for the neurotransmitter acetylcholine, which is involved in many functions including memory and muscle control.* + +--- + +## Voltage clamp method summary + +
Neuroscience 5e Animation 3.1
+ +Note: + +--- + +## Pathways of the two currents are distinct + +* Question– Do Na⁺ and K⁺ go through the same channels? Or do they have distinct channels? + * Experiment– Add tetrodotoxin (TTX) to block inward current but not outward current + * Experiment– Add tetraethylammonium (TEA) to block outward current but not inward current +* TTX inactivates Na⁺ channels, TEA blocks K⁺ channels + +Note: + + +-- + +## Neurotoxins as pharmacological tools + +
+
+ +* Fugu (puffer fish or blow fish) +* TTX concentrated in their livers (don’t eat it) +* TTX blocks voltage-gated Na⁺ channels + +
+ +
puffer fish
+
Simpsons poison tasty fish
+ +Note: + +Its mechanism of action, selective blocking of the sodium channel, was shown definitively in 1964 by Toshio Narahashi and professor John W. Moore at Duke University, using the sucrose gap voltage clamp technique (Narahashi et al, J Gen Physiol 1964) + +--- + +## Pharmacological separation of inward and outward currents into Na⁺ and K⁺ dependent components + +
Neuroscience 5e Fig. 3.5
+ + +Note: + +Tetramethylammonium chloride is one of the simplest quaternary ammonium salts. + +[https://en.wikipedia.org/wiki/Tetramethylammonium_chloride](https://en.wikipedia.org/wiki/Tetramethylammonium_chloride) + +TTX and TEA experiments from Moore 1967 J Gen Physiol; Armstrong and Binstock, 1965 J Gen Physiol + + +--- + +## Voltage dependent membrane conductances of Na⁺ and K⁺ + +
+
+ +* Another way of describing permeability is using membrane conductance (*g*). Conductance (measured in siemens, *S*) is the reciprocal of resistance + * *g = 1/R* +* Ohm’s law: + * *I = V/R* + * *I = gV* + * For an ion *x*, + * *Ix* = ionic current flow, *Ex* = equilibrium potential + * The membrane potential (*Vm*) minus the equilibrium potential (*Ex*) is the electrochemical driving force acting on an ion, thus *V = Vm - Ex* + * *Ix = gx* + * *Ix = gx(Vm - Ex)* +* Solve for *g*: + * *gx = Ix/(Vm - Ex)* +* *Ix* determined from measurement of current changes plus or minus ion (or during pharmacological inhibition) +* *Ex* calculated from Nernst equation using concentrations of inside and outside ions + +
+ +Note: + +For our purposes, we can consider conductance to be another way of describing permeability. + +technically conductance is the degree to which an object conducts electricity, calculated as the ratio of the current that flows to the potential difference present. It deals with the movement of charge, whereas permeability refers to the ability of a specific ion to move across the cell membrane. + +* [http://www2.montana.edu/cftr/ion_channel_glossary.htm](http://www2.montana.edu/cftr/ion_channel_glossary.htm) + +Ohms law= Voltage = Current times resistance. + +Can use this to calculate the dependence of Na and K conductances vs. time and membrane potential. + + +--- + +## Membrane conductance changes are time and voltage dependent + +
Neuroscience Fig. 3.6
+ + +Note: + + + +--- + +## Depolarization increases Na⁺ and K⁺ conductances of the squid giant axon + +
Neuroscience Fig. 3.7
+ + +Note: + +Determine the peak conductance of ions at different membrane potentials. + +--- + +## Description of an action potential using Na⁺ and K⁺ conductances + +
+
+ +* At rest (-70 mV), voltage-gated Na⁺ and K⁺ channels are closed. Non voltage-gated K⁺ channels are open and dictate the resting potential, together with the distribution of ions across cell membranes +* A stimulus raises the membrane potential in the cell. Depolarization causes voltage-gated Na⁺ channels to open, which allows Na⁺ to rush in the cell which increases the membrane potential, which causes more Na⁺ channels to open, which causes more Na⁺ to rush in which causes higher membrane potential (a positive feedback loop). As membrane potential is approaching ENa, the further depolarization causes Na⁺ channels to inactivate which prevents more Na⁺ from from flowing through these channels +* Depolarization also opens voltage gated K⁺ channels, which causes K⁺ to flow out, thus lowering the membrane potential + +
+ +Note: + + +--- + +## Ion conductances underlying the action potential + +
Neuroscience 5e Fig. 3.8
+ +Note: + + + + +--- + +## Properties of action potentials explained + +
+
+ +* Question– Why do APs exhibit an all-or-nothing threshold? + * Answer– When membrane potential (Vm) is below threshold there is not enough Na⁺ channels open to raise Vm high enough to open more channels. When Vm is above threshold the action potential cycle is activated. +* Question– Why to APs exhibit an undershoot? + * Answer– During the AP voltage-gated K⁺ conductance slowly increases (delayed activation of voltage-gated K⁺ channels) and during the falling phase these K⁺ channels are still open and active whereas voltage-gated Na⁺ channels are inactivated… as Vm approaches Ek there is briefly more K⁺ flowing out than at rest and the hyperpolarization inactivates voltage-gated K⁺ channels. K⁺ leak channels and ion transporters bring back cell to resting potential. + +
+ + +Note: + +The threshold is a point of criticality in the system like trying to balance on a knifes edge. Just imagine any self-organized phenomena in nature: a snow field suddenly turning into an avalanche, liquid water turning into gas or solid forms, videos of cats or korean pop stars suddenly going viral. The point at which the states of these systems veer on the edge of order or disorder is the point of criticality also known to physicists as a phase transition. + +--- + +## Properties of action potentials explained + +* Action potential propagation and directionality? +* Refractory periods? +* What does myelin do? + +Note: + +Next we will look at the following properties of APs such as: + +--- + +## Action potential propagation + +* Charge flowing in through Na⁺ channels can diffuse inside the axon. This passive current cannot diffuse very far because of current leakage. Potentials below threshold taper out fast (like passive conduction of subthreshold depolarizations). +* Potentials above threshold cause increased depolarization (due to more Na⁺ channels open). Now there is enough current to diffuse laterally and still be above threshold for a new set of Na⁺ channels. + +Note: + +First let’s talk about AP propagation. + +During an action potential, inward current through Na⁺ channels + +--- + +## Passive current flow in an axon + +
subthreshold changes diffuse rapidly
Neuroscience 5e Fig. 2.3
+ + +Note: + +bottom graph shows the peak Vm + +--- + +## Propagation of an action potential + +
suprathreshold depolarizations propagate down the axon
Neuroscience 5e Fig. 2.3
+ +Note: + +bottom graph shows the peak Vm + +--- + +## Action potential conduction requires both active and passive current flow + +
Neuroscience 5e Fig. 3.10
+ +
Neuroscience 5e Fig. 3.10
+ +
Neuroscience 5e Fig. 3.10
+ + + + +Note: + +Active and Passive current flow. + +* Na+ chan locally open in response to stimulus, generating an AP +* Depolarzing current passively flows down axon +* Local passive depolarization causes nearby Na chan to open and another AP is generated +* Na chan upstream inactivate and K chan open. Vm repolarizes and is refractory to further AP generation upstream +* Process repeated downstream, propagating AP along the axon + +--- + +## Why is there a refractory period? + +
+
+ +* Remember during the falling to undershoot phase of an action potential K⁺ channels are still open but Na⁺ are channels inactivated (decreased gNa), leading to temporary hyperpolarization more negative than the resting membrane potential +* Therefore (1) inactivation of Na⁺ channels and (2) slow K⁺ channel kinetics are responsible for the refractory period +* This makes it harder to initiate a new AP either from a new stimulus or for an AP to propagate backwards +* Different axons will have different refractory periods (and thus different maximal firing rates) depending on the particular subtypes of Na⁺ and K⁺ channels they express + +
+ +
+ +Note: + + +-- + +## Voltage-gated channel states during an action potential + +
Neuroscience 5e fig. 4.3
+ + + +Note: + +--- + +## What does myelin do? + +* Rate of action potential formation limits the flow of information +* How to speed up AP conduction? + * Increase the diameter of the axon– bigger axon diameters have less resistance (decreased resistance to passive current flow) + * Myelin **insulates the axon**, reducing current leak. Example AP conduction velocities for axons: unmyelinated 0.5–10 m/s, myelinated 150 m/s + +Note: + + +--- + +## Nodes of Ranvier + +* Can’t insulate the whole axon because transmembrane current flow is required to generate the action potential +* Current from one action potential flows passively to next node where a new action potential is made +* Action potentials have saltatory conduction– meaning from node to node + +Note: + + + +--- + +## Nodes of Ranvier + +
Neuroscience 5e Fig. 3.11
+
Neuroscience 5e Fig. 3.10
+ +Note: + +saltatory action potential condution along a myelinated axon. + +red indicates imaged expression of voltage gated Na channels. green indicates a protein (Caspr) associated with the nodes of Ranvier. + + +--- + +## Speed of action potential conduction in unmyelinated versus myelinated axons + +
+ + +Note: + +figure comparing action potential propagation speed in an unmyelinated and myelinated axon. + +action potential genaration occurs only at specific points, the nodes of Ranvier, along the myelinated axon + +--- + +## The Nobel Prize in Physiology or Medicine (1963) + +>"for their discoveries concerning the ionic mechanisms involved in excitation and inhibition in the peripheral and central portions of the nerve cell membrane" + +
+ +Alan Lloyd Hodgkin + +
+ +
+ +Andrew Fielding Huxley + +
+ + +Note: + + +--- + +## Painless dentistry + +* Lidocaine blocks some types of Na⁺ channels +* Blocks action potentials in sensory axons +* Pain signals do not reach the brain + +
+ +
voltage-gated sodium channel
Basic Neurochemistry 6e Fig. 6.6
+ + + + +Note: + + + +--- + +## Multiple sclerosis + +* Disease caused by myelination defects and loss of neurons +* Seems like an autoimmune disease +* 1/750 of population in US get multiple sclerosis (MS) +* 1/40 risk if a parent has it +* 1/3 if an identical twin gets it +* Genetic and environmental risk factors + +Note: + +onset between ages 20-40. + +blindness, motor weakness, paralysis. + +ultimate cause of MS remains unclear. Immune system contributes to damage and is key component. Immune cells in CSF and injection of myelin in animals can cause EAE. Autoimmune disorder. Or persistent infection with a human retrovirus? + + +* women to men ratio 3/2 +* Genetic component is likely the effect of multiple genes + +--- diff --git a/2016-10-03-lecture05.md b/2016-10-03-lecture05.md new file mode 100644 index 0000000..b469773 --- /dev/null +++ b/2016-10-03-lecture05.md @@ -0,0 +1,1277 @@ +## Ion channels underlie action potentials + +* Predictions about the nature of ion channels from Hodgkin and Huxley: +* Because conductances are large, channels must be able to pass ions at high rate. +* Channels must be gated by the membrane potential. +* Different channels for Na+ and K+. +* Problem– Voltage clamping cannot look at individual channels…its measuring the aggregate current flowing through a whole bunch of channels at once. What does an individual channel look like? How does it work? +* Solution– Patch Clamping + +2016-01-19 17:18:07 + +Note: + +Today we will take a closer look at how ion channels are able to exhibit their remarkable properties and enable action potentials and all forms of electrical signaling in the nervous system. + +Now we know from our previous classes covering the work by HH, that there are some predictions we make concerning the nature of ion channels: + + +--- + +## Question + +* During the rising phase of the action potential: +* a. All sodium channels are closed. +* b. Some of the sodium channels are closed +* c. All potassium channels are open. +* d. All sodium channels are open. +* e. The membrane potential is hyperpolarizing. + + During the rising phase of the action potential: + +a. All sodium channels are closed. + +b. Some of the sodium channels are closed + +c. All potassium channels are open. + +d. All sodium channels are open. + +e. The membrane potential is hyperpolarizing. + +Note: + +So a quick question + +--- + +## The Nobel Prize in Physiology or Medicine (1991) + +“for their discoveries concerning the the function of single ion channels in cells” + +Erwin Neher + +Bert Sakmann + + + +[http://nobelprize.org/nobel_prizes/medicine/laureates/1991/press.html](http://nobelprize.org/nobel_prizes/medicine/laureates/1991/press.html) + +
+ +
+ +
+ +Note: + + + +--- + +## Title Text + +* What ions are permeable at rest? +* What ions are permeable at peak? +* What ions are permeable during overshoot? + +
+ +
+ +Note: + +How would the shape of the action potential change if the extracellular Na concentration was lowered, what if the K+ was raised + +--- + +## Patch clamp method: + +## Neher and Sakmann + +cell-attached recording + +
+ +
+ +Note: + + + +--- + +## Patch clamp + +* Allows one to look at currents flowing through a single channel. +* Pipette with small opening makes a tight seal with the membrane. +* Currents are amplified and measured +* Can be adapted to do whole cell recordings, inside out recordings or outside out recordings. + +Note: + + + +--- + +## The patch clamp method + +* Can measures ion flow through a single channel + +
+ +Note: + + + +--- + +## The patch clamp method + + + +Pipette is continuous with cytoplasm. Can measure potentials and currents from the entire cell and also can introduce things into the cytoplasm + +Makes it easy to introduce things to the cytoplasmic side of the channel + +Makes it easy to introduce + +things to the extracellular + +side of the channel + +
+ +
+ +
+ +Note: + + + +--- + +## Patch clamping Na+ channels + +* Block K+ channels with Cs+ or with tetraethylammonium (TEA). +* Brief depolarizations cause small inward currents that disappear right away. +* Each inward current is the opening of one Na+ channel. +* About 1-2 pA of current == thousands of ions/ms +* Stochastic opening, biased at the beginning of a pulse. +* Probability of opening varies with membrane potential. +* If you remove Na+ from medium, do not see currents. +* Tetrodotoxin (TTX) blocks currents. + +Note: + + + +--- + +## Measurements of ionic currents flowing through single Na+ channels + +Small inward currents + +Open at beginning of pulse + +Close quickly + +Neuroscience5e Fig. 4.1 + +
+ +Note: + +Patch a piece of membrane and block K currents. Do a bunch of short recordings while clamping the membrane at depolarized potential. e.g. here is 7 trials. Notice the amplitude is discrete— it is unitary. If you were recording from lots of… + + + +Transient channel opening in Na+ channels (inward current). + + + +--- + +## Measurements of ionic currents flowing through single Na+ channels + +Neuroscience5e Fig. 4.1 + +Summed current from many single channels looks like macroscopic currents seen in voltage clamping + +Probability of opening increases as a function of membrane potential + + + + + +Note: + +You’d expect this macroscopic current— + + + +Average the microscopic currents together and you get something very similar. + + + +Sum these microscopic inwa + +--- + +## Patch clamping K+ channels + +* Add tetrodotoxin (TTX) to block Na+ channels +* Depolarization pulses cause outward currents. +* Once a channel opens (usually with a delay) it remains open for the duration of the pulse. +* The probability of channel opening depends on the membrane potential. +* Sensitive to TEA. + +Note: + + + +--- + +## Measurements of ionic currents flowing through single K+ channels + +Early delay in opening + +Once open stay open + +Neuroscience5e Fig. 4.2 + +
+ +Note: + +Sustained channel opening in K+ channels (outward current). + +--- + +## Measurements of ionic currents flowing through single K+ channels + +Summed current from many single channels looks like macroscopic currents seen in voltage clamping + +Probability of opening increases as a function of membrane potential + + + + + +Neuroscience5e Fig. 4.2 + +Note: + +Sum a bunch of these microscopic channel currents and you get this top curve and which looks very similar to the macroscopic current curve as we’ve seen previously. + +--- + +## Functional states of voltage-gated Na+ and K+ channels + +Neuroscience5e Fig. 4.3 + +
+ +Note: + +Shown here is a model of the functional states for these channels. Notice a few states for Na vs two for K. + +--- + +## Conclusions from patch clamp experiments + +* Allowed the direct observation of ionic currents flowing through single ion channels. +* Both Na+ and K+ channels are voltage gated. +* Thus there must be a voltage sensor in these channels. +* Depolarization inactivates Na+ channels but not K+ channels. + +Note: + +So the conclusions are… + +--- + +## Title Text + +[http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-01ThePatchClampMethod.mov](http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-01ThePatchClampMethod.mov) + +
+ +Note: + + + +--- + +## Many genes encode ion channels + +* There are hundreds of genes encoding ion channels (e.g. about 100 K+ channel genes). +* They have common properties (similarities in amino acid sequence and protein topology). +* They also have variations (differences in ion selectivity, how they are gated, inactivation mechanisms). + +Note: + +Now everything going on in our nervous systems depends on the function of ion channels. And there are lots of them. + +--- + +## Different ways to gate ion channels + +
+ +Note: + +Different classes of gated ion channels. + + + +voltage gated ion channels, such as we’ve been discussing over the last couple classes. + + + +ligand gated channels such as those that bind neurotransmitters, will talk about more later and next class. + +others are ligand gated channels sensitive to chemical signals arising in the cytoplasm of neurons such second messengers like Ca2+, cyclic nucleotide cAMP and cGMP. + + + +--- + +## Even within a family of channelsthere is huge variation + +* Voltage gated– Na+, K+, Cl-, and Ca2+ channels. +* Approximately 10 different genes for Na+ channels, 16 Ca2+, 3–5 Cl- and 100 K+ channels. +* Different genes may give rise to channels with different properties– e.g. different inactivation times, probability of opening at a given voltages, gating mechanisms. +* Can also be multiple splice variants of the same gene. +* Creates huge diversity of channels. +* How to characterize all these channels? + +Note: + + + +--- + +## Lets say you have a gene for a channel, how do you determine its properties + +* Need an experimental system where you can express gene of interest functionally and away from other channels. +* Xenopus oocytes have been a historical way to do this. + +Note: + +frog germ cells + +--- + +## Xenopus oocytes + +* Large (1 mm in diameter) cell that contains lots of protein synthesis machinery. +* Can inject RNA into it and it will express protein encoded by RNA. +* Works great for ion channels, can voltage clamp and determine properties of a given channel. +* Can make specific mutations in genes and see what happens to function of protein. + +Note: + + + +--- + +## Xenopus oocytes for ion channel physiology studies + +Channel gene (DNA) + + + +Transcribe in vitro + +mRNA + + + + + + + + + +Oocyte makes protein + + + + + +Whole cell record or + + patch clamp channel + + + + + +Note: + + + +--- + +## Xenopus oocytes for ion channel physiology studies + +Ion channel mRNA + + + + + + + + + +Oocyte makes protein + + + + + +Whole cell record or + + patch clamp channel + + + + + +Note: + + + +--- + +## Xenopus oocytes for ion channel physiology studies + +
+ +Note: + +shows voltage clamp experiment results after expression of a K channel in an oocyte. + +--- + +## Diverse properties of K+ channels + +Neuroscience5e Fig. 4.5 + +Similar to action potential + +K+ channels + +Inactivates like Na+ channels + +Same conductance vs voltage profiles, different inactivation properties. + + + + + +
+ +
+ +Note: + +- Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization + +- Kv4.1 channels inactivate during a depolarization. + +HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization + +inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials + +Ca activated K channels open in response to intracellular Ca ions + +2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH + + + +--- + +## Diverse properties of K+ channels + +More current flows when + +hyperpolarization conditions + +More current flows if Ca2+ is + +added intracellularly + +pH sensitive channel + +Neuroscience5e Fig. 4.5 + +
+ +
+ +Note: + +- Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization + +- Kv4.1 channels inactivate during a depolarization. + +HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization + +inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials + +Ca activated K channels open in response to intracellular Ca ions + +2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH + + + +--- + +## Molecular structures of ion channels + +* Multiple membrane spanning domains +* K+ channels 4 subunits come together, (each with 6 transmembrane helices). +* Na+ channels 1 protein with 24 transmembrane helices +* Center has an opening that makes a pore for the ion to flow through +* Contains selectivity filter +* Voltage-gated ion channels also contain a voltage sensitive transmembrane domain + +Note: + +We’ve learned from biophysical structure studies that in general ion channels have 24 transmembrane peptide domains with… + + + +We can also guess a few characteristics of their structure from the classic voltage clamp and patch clamp studies we’ve discussed over the past couple classes… + + + +from wikipedia: + +>X-ray crystallography is a tool used for identifying the atomic and molecular structure of a crystal, in which the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. By measuring the angles and intensities of these diffracted beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined, as well as their chemical bonds, their disorder and various other information. + + + + + +--- + +## Molecular structures of ion channels + +* Voltage-gated cation channels consist of four subunits, each of which has 6 transmembrane segments and a pore loop. In sodium and calcium channels, the four subunits are part of the same molecule. In potassium channels, they are different molecules. + +The resulting channel has four-fold symmetry + + + +
+ +
+ +Note: + + + +--- + +## Molecular structures of ion channel proteins + +
+ +Note: + +- Kv2.1 show little inactivation and are closely related to the delayed rectifier K channels involved in AP repolarization + +- Kv4.1 channels inactivate during a depolarization. + +HERG channels inactivate so rapidly that current flows only when inactivation is rapidly removed at end of a depolarization + +* human Ether-à-go-go-Related Gene), best known for its contribution to the electrical activity of the heart that coordinates the heart's beating, mediates the repolarizing IKr current in the cardiac action potential). + +inward rectifier K channels allow more K current to flow at hyperpolarized potentials than at depolarized potentials + +Ca activated K channels open in response to intracellular Ca ions + +2-P K channels usually respond to chemical signals rather than changes in membrane potential. These are primarily responsible for the resting membrane potential of neurons. e.g. TASK channels can by regulated by extracellular pH + + + +--- + +## Channel selectivity + + + + + + + +can get through a Na channel + +Can’t + +
+ +Note: + + + +--- + +## Potassium channel with four subunits + +Crude understanding of structure– 4 subunits come together to + +make a pore. + +
+ +Note: + + + +--- + +## Structure of the bacterial K+ channel + +* Bacteria have K+ channels that are very similar in structure to mammalian K+ channels. Main difference is that they are not gated by voltage. +* Could be crystallized in the bacterial membrane. +* 3D structure tells us a lot about function. +* Roderick Mackinnon Nobel Prize in Chemistry 2003 + +“for structural and mechanistic studies of ion channels” + +Note: + +prokaryotic + +eukaryotic + + + +--- + +## Structure of the bacterial K+ channel + +3D structure of bacterial K channel. Yellow is the K channel, + +white are phospholipids, purple Na, green K. + +
+ +Note: + + + +--- + +## Structure of a bacterial K+ channel determined by crystallography + +helps dehydrate + +K+ ions + +inside + +outside + +Neuroscience5e Fig. 4.7 + +
+ +Note: + +Simplified model of bacterial K channel, showing you the pore and selectivity filter. + + + +helical domains of channel point negative charges towards cavity allwing K ions to become dehydrated and then push through selectivity filter through electrostatic repulsion. + + + +--- + +## Structure of the bacterial K+ channel + +Each subunit has 2 + +transmembrane domains, 4 subunits make a channel + +Neuroscience5e Fig. 4.7 + +
+ +Note: + + + + + +(Doyle et al, Science 280:69, 1998) + +--- + +## Structure of the bacterial K+ channel + +A space-filling model of the KcsA channel, showing the pore. Ions (green balls) tend to occupy three sites in the channel, two in the selectivity filter and one in a pool of water in the center of the channel. + +red – charge; blue + charge; yellow hydrophobic + +(Doyle et al, Science 280:69, 1998) + +
+ +Note: + + + + + + + +(Doyle et al, Science 280:69, 1998) + +--- + +## Selectivity filter of the K+ channel + + + + + + + + + + + + + +Up to 4-6 water molecules form hydration shells around both Na+ and K+ ions + +Ions move with their hydration shells + +To pass through the potassium channel, an ion must remove most of its surrounding water molecules (dehydrated) + +K+ is dehydrated by the K+ channel selectivity filter (leaving just two water molecules– one at front and one at back) + +Na+ has a more stable water shell, binding H2O more strongly and thus has a larger effective diameter— would require more dehydration energy than K channel pore region can provide. + + + + + + + + + + + + + + + + + + + +H2O + +K+ + + + + + + + + + + + +K+ + + + + + + + +dehydrate, move through filter + +filter + +
+ +
+ +Note: + +Larger cations cannot traverse the pore region, smaller cations like Na cannot enter the pore because the walls are just too far apart to stabilize a dehydrated Na ion long enough to pass through. + + + +Na is the most hydrated ion with 4 to 6 water molecules in the first shell. Binds water strongly, making a stable hydration shell and moving together with the cation. Any sodium movement is followed by H2O movement (water retention, excretion). + + + +Potassium ion is larger, having 8 more electrons shielding positively charged nucleus, thus K+ makes transient associations with water rather than a discrete hydration layer. Helps explain higher permeability across cell membrane for K+. + + + +ion | ion diameter (nm) free | ion diameter hydrated + +--- | ---------------------- | ------------------- + +Na | 0.19 | 0.52 + +K | 0.27 | 0.46 + + + + + +[http://web-books.com/MoBio/Memory/Channel.htm :](http://web-books.com/MoBio/Memory/Channel.htm) + +>To pass through the potassium channel, an ion must remove most of its surrounding water molecules, leaving only two - one at the front and another at the back. + +The selectivity filter of the sodium channel is slightly larger than that of the potassium channel. It may accommodate a Na+ ion attached with three water molecules, but not enough for a K+ ion attached with three water molecules. + + + +>In the sodium channel, the Na+ ion is more permeable than the K+ ion. This is because the selectivity filter of the sodium channel is slightly larger than that of the potassium channel. It is large enough to accommodate a Na+ ion attached with three water molecules, but not enough for a K+ ion attached with three water molecules. Therefore, to pass through the sodium channel, the Na+ ion needs to remove only three, but the K+ ion has to remove four, water molecules from its first hydration shell. The required dehydration energy for the K+ ion is greater than the Na+ ion. + + + + + +>In calcium channels, the permeability of monovalent cations (Na+ and K+) is about three orders of magnitude smaller than the Ca2+ permeability. This ion selectivity does not seem to involve hydration, because Ca2+ is more heavily hydrated than Na+, and the unhydrated diameters of Ca2+ and Na+ are almost identical. Then, how could calcium channels select Ca2+ over Na+? + + + +>Although the permeability of monovalent cations in the calcium channel is quite small at normal ionic concentrations, large monovalent cationic current can be observed in the absence of Ca2+ and other divalent cations. This suggests that the calcium channel is basically permeable to both divalent and monovalent cations, but the selectivity arises from competition between ions. The calcium channel may contain a negatively charged binding site to facilitate ion conduction. The monovalent cations simply cannot compete with Ca2+ for this binding site. This idea has been confirmed experimentally. In the calcium channel, if a negatively charged glutamate residue in the pore-lining region is mutated into a positively charged lysine, the calcium channel becomes more permeable to Na+ than Ba2+ + + + +This explains the selectivity but not the voltage sensor + + + + + +Atomic masses + + + +1H < 2He + +3Li < Be < B < 6C < N < 8O < 9F < 10Ne + +11Na < 12Mg < Al < 14Si < P < S < 17Cl < 18Ar + +19K < 20Ca + +37Rb < 38Sr + +55Cs < 56Ba + +--- + +## Structure of the subunit of a voltage gated + +## channel protein + +
+ +Note: + +N-terminus (amino terminus). Start of a protein or polypeptide. Translation from mRNA occurs from N—>C. Often occurs targeting signals. + + + +C-terminus (carboxyl terminus), carboxyl group. Often contains retention signals for protein sorting (such as keeping protein in the ER and out of the secretory pathway) + +--- + +## Structure of a mammalian voltage-gated K+ + +Neuroscience5e Fig. 4.8 + +top view + +side view + +
+ +
+ +Note: + +Now we know from what we’ve learned over the past couple classes that neurons have K+ channels that are gated by voltage + + + + + +[http://web-books.com/MoBio/Memory/Channel.htm :](http://web-books.com/MoBio/Memory/Channel.htm) + +>There are many types of potassium channels. The one involved in the generation of action potentials is composed of four subunits, each is homologous to the Shaker protein (Fig. 3.2). The hydrophobicity profile indicates that it contains six hydrophobic segments, designated as S1 - S6. These segments are likely to be the transmembrane domains. Other experimental results suggests that the P-region is lining the channel pore. + +--- + +## Structure of a mammalian voltage-gated K+ + +Neuroscience5e Fig. 4.8 + +
+ +Note: + + + +--- + +## Topology of the principal subunits of voltage-gated Na+ channels + +
+ +Note: + +Yellow are voltage sensing tm domains + +--- + +## Topology of the principal subunits of voltage-gated Ca2+ channels + +
+ +Note: + +Yellow are voltage sensing tm domains + +--- + +## Topology of the principal subunits of voltage-gated K+ channels + +
+ +Note: + +K channels are more diverse + + + +Yellow are voltage sensing tm domains + +--- + +## How do Na+ channels inactivate? + +* Contains an activation gate that binds to the channel in the intracellular region and blocks the channel +* Activation gate changes conformation (closes/swings shut) to block channel only during the channel’s open state +* Therefore, at resting Vm channel is closed and activation gate is open. +* After depolarization, the channel opens and Na+ ions go through. After a little bit of time (~ 1 ms) the activation gate swings shut to block channel. + +[http://www.nature.com/nature/journal/v475/n7356/full/nature10238.html](http://www.nature.com/nature/journal/v475/n7356/full/nature10238.html) + +Note: + + + + + +The theory is that the inactivation gate “swings” shut, turning off the channel + + + + + +The physical structure of voltage gated Na channels has only recently begun to be solved, with the results so far fitting the models for Na channel opening and inactivation. + + + + + +--- + +## Sodium channel inactivation cycle + +Lodish, Mol Cell Bio + + + + + + + +
+ +Note: + + + + + +Figure 21-13 Lodish 4th edition OR Figure 7-33 Lodish 5th edition. Structure and function of the voltage-gated Na+ channel. + + + +[http://www.amazon.com/Molecular-Cell-Biology-Lodish/dp/0716776014](http://www.amazon.com/Molecular-Cell-Biology-Lodish/dp/0716776014) + + + + + + + +--- + +## Sodium channel inactivation + +The theory is + +that the inactivation gate + +“swings” shut, turning off + +the channel + +
+ +
+ +
+ +
+ +Note: + +The theory is that the inactivation gate “swings” shut, turning off the channel + +--- + +## Toxins that poison ion channels + +prolongs Na+ currents + +by messing up channel inactivation + + + +AP profile reflects + +the shift in Na+ + +conductance. + +[http://www.nature.com/news/rodent-immune-to-scorpion-venom-1.14014](http://www.nature.com/news/rodent-immune-to-scorpion-venom-1.14014) + +
+ +
+ +Note: + +already learned about tetrodotoxin from puffer fish. blocks voltage gated Na channels underlying the AP + + + +saxitoxin similar (homologue) to ttx, produced by dinoflagellates and possible effects from ‘red tide’ or eating shellfish that have injested these dinoflagellates. + + + +scorpions paralyse prey by injecting alpha-toxins (left panels). Slow inactivation of Na channels, prolonging the AP and messing up information flow in CNS. Beta-toxins in scorpion venom shift the voltage dependence of Na channel activation (right panel), causing Na channels to open at potential much more negative than normal inducing uncontrolled AP firing. + + + +Some alkaloid toxins (batrachotoxin, produced by S. American frogs) do both of these mechanisms. + + + +Similar toxins from plants (aconitine from buttercups, veratridine from lilies) and insecticidal toxins (pyrethrins) produced by chrysanthemums and rhododendrons. + + + +dendrotoxin from wasps affects K channels + +apamin from bees K channels + +charybdotoxin from scorpions K channels + +--- + +## Toxin binding sites + +
+ +Note: + +Blue diamonds- persistent activation + + + +Picture from sigma catalog of blockers + +--- + +## Diseases caused by altered ion channels + +EA1: episodic ataxia type 1 (abnormal limb movements and severe ataxia) + + + +BFNC: benign familial neonatal convulsion. Frequent brief seizures starting in first postnatal week then disappearing in a few months. Mutation mapped to two K+ channel genes + +
+ +Note: + +ataxia: greek for ‘without order’ or ‘incoordination’. Movement coordination problems. + + + +paralysis: muscle weakness + +myotonia: muscle contraction + + + +--- + +## Diseases caused by altered ion channels + +FHM: familial hemiplegic migraine; CSNB: congenital stationary night blindness, rod photoreceptors nonfunctional (resulting in decreased acuity, myopia, nystagmus, strabismus); EA2: episodic ataxia type 2 (abnormal limb movements and severe ataxia); Paralysis: muscle weakness + + + +
+ +Note: + + + + + +nystagmus: involuntary eye movements (dancing eyes) + +strabismus: eyes not directed towards same fixation point, disruption of binocular vision, depth perception, resulting in amblyopia when present in children + +amblyopia: greek for ‘blunt vision’, decr vision through an eye because of a developmental pathophysioloy of the brain (e.g. visual cortex), 1-5% of population + + + +--- + +## Diseases caused by altered ion channels + +GEFS: generalized epilepsy with febrile seizures, begins at infancy and continues through puberty. Mapped to two mutations, one on an alpha Na channel subunit and one on a beta subunit. Cause slowing of sodium channel inactivation; Myotonia: muscle contractions; Paralysis: muscle weakness + + + +
+ +Note: + + + +--- + +## Diseases caused by altered ion channels + +
+ +Note: + + + +--- + +## Epilepsy can result from mutated Na+ channels + +
+ +Note: + +You can see the the slower inactivation kinetics in this figure here in patch clamp recordings from normal and a number of different Na channel mutants. This slowing of Na inactivation is just enough to mess up spike patterns in single neurons and elicit hyperexcitability that results in seizures in networks of connected neurons. + + + +GEFS: generalized epilepsy with febrile seizures + + + +--- + +## Impaired inactivation of Na+ channels underlie hyperkalemic periodic paralysis disease + +* Normally Na+ channels open and close rapidly due to inactivation. +* Na+ channels from hyper periodic paralysis close more slowly and reopen. +* Mutant myotubes (Met 1592 Val) + +Cannon et al, Neuron 1991 + +
+ +Note: + +autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+ + +whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current + + linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation + +patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels + + + + + + + + + +Muscle fibers generally form from the fusion of myoblasts into multi-nucleated fibers called myotubes + + + +--- + +## Channelopathies + +* Diseases can be linked to defective ion channels + +
+ +Note: + + + +--- + +## Ion transporters + +Neuroscience5e Fig. 4.9 + +
+ +Note: + + + +--- + +## Title Text + +[http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-02TheSodiumPotassiumPump.mov](http://courses.pbsci.ucsc.edu/mcdb/bio125/Animation04-02TheSodiumPotassiumPump.mov) + +
+ +Note: + + + +---