LECTURE 2 – NERVOUS SYSTEM PHYSIOLOGY

Excitability and ionic transport

Assoc. Prof. Ana-Maria Zagrean MD, PhD Physiology & Fundamental Neuroscience Division [email protected], [email protected]

Excitability and ionic transport

1) input Excitability

2) integration 3) output

The capacity / condition for a live system to recognize and respond to specific signals, as a form of updated information, necessary for its adaptive and continuous organization.

physical

stimulus

chemical ?

Receptor

Interaction stimulus - receptor

response

The excitability of neurons is based on ion gradients across the cell membrane, and on transport properties of the cell membrane.

Premises Interrelation between cell membrane properties and the complex characteristics of biological systems.

acquiring excitability

identity

Selective membrane permeability

adaptability

coordinating disseminating evolution

Selective membrane permeability: The lipid barrier of the cell membrane and cell membrane transport proteins

Chemical compositions of extracellular and intracellular fluids.

“Diffusion” Versus “Active Transport” Diffusion - random molecular movement of substances molecule by molecule, either through intermolecular spaces in the membrane or in combination with a carrier protein. The energy that causes diffusion is the energy of the normal kinetic motion of matter. Active transport - movement of ions or other substances across the membrane in combination with a carrier protein in such a way that the carrier protein causes the substance to move against an energy gradient, such as from a low-concentration state to a high-concentration state; requires an additional source of energy (ATP) besides kinetic energy.

Cell membrane and its selective permeability TRANSPORT OF SUBSTANCES THROUGH THE CELL MEMBRANE

1.Diffusion -Simple diffusion: - lipid-soluble subst. (O2, CO2, alcohols) through intermolecular spaces of the lipid barrier - through a membrane opening - protein channels (e.g., water, lipid-insoluble molecules that are water-soluble and small enough):  selective permeable channels  non-gated OR gated (open/closed by gates) voltage-gated ligand-gated (chemical-gated) -Facilitated diffusion = carrier mediated diffusion e.g. transport of most of aminoacids and glucose Driving force of diffusion and net diffusion depends on: -Substance availability, kinetic energy, membrane permeability -Concentration difference/gradient -Membrane electrical potential effect on diffusion of ions 2.Active transport - Primary active (pumps) - Secondary active (co- and counter-transport)

Simple diffusion through protein channels: • Pores/channels are integral cell membrane proteins that are always open • Pore diameter, its shape and its internal electrical charge/chemical bonds provide selectivity

Aquaporins = water channels (13 different types) - protein pores which permit rapid passage of water through cell membranes but exclude other molecules (a narrow pore permits water molecules to diffuse through the membrane in single file). The pore is too narrow to permit passage of any hydrated ions. ! Density of aquaporins (e.g., aquaporin-2) in cell membranes is not static but is altered in different physiological conditions.

Diffusion: simple and facilitated

Effect of concentration of a substance on rate of diffusion through a membrane by simple diffusion & facilitated diffusion. Note that facilitated diffusion approaches a maximum rate Vmax.

Postulated mechanism for facilitated /carrier mediated diffusion (e.g. glucose or amino acids transport)

Diffusion and the cell membrane potential: What determine ions diffusion? A. Net Diffusion Rate Is Proportional to the Concentration Difference Across a Membrane. B. Effect of Membrane Electrical Potential on Diffusion of Ions—The “Nernst Potential.” C. Effect of a Pressure Difference Across the Membrane.

Diffusion and the cell membrane potential Effect of membrane electrical potential on diffusion of ions: the “Nernst Potential”

The concentration of (-) ions is initially the same on both sides of the membrane, but a (+) charge applied to the right side of the membrane and a (-) charge to the left, creates an electrical gradient across the membrane, moving the ions. When the concentration difference rises high enough, the two effects balance each other.

EMF (mV) = -RT/zF log C inside/C outside EMF = +/-61 log C inside/C outside at 37°C for any univalent ion (z=1), as Na+ or K+ R = gas constant, F = Faraday constant, z = valence, T = temp; C = ion conc. (+) for negative ions / (-) for positive ions, diffusing from inside to outside.

Diffusion and the cell membrane potential

=10-1

B, Establishment of a "diffusion potential" when the nerve fiber membrane would be permeable only to Na+: the membrane potential rises high enough within msec. to block further net diffusion of Na+ to the inside; however, this time, in the mammalian nerve fiber, the potential is ~ 61 mV positive inside the fiber. Internal membrane potential is negative when K+ diffuse and positive when Na+ diffuse because of opposite concentration gradients of these two ions.

Diffusion and the cell membrane potential

A, Establishment of a "diffusion potential" across a nerve fiber membrane, caused by diffusion of K+ from inside the cell to outside through a membrane that is selectively permeable only to K+: within ~1 msec. diffusion potential becomes great enough to block further net K+ diffusion to the exterior, despite the high K+ concentration gradient. For a normal nerve fiber, the potential difference is ~ 94 mV, with negativity inside the fiber membrane.

Calculation of the diffusion potential when the membrane is permeable to several different ions – Goldman equation

(Actual potential)

Calculation of the diffusion potential when the membrane is permeable to several different ions – Goldman equation The membrane is permeable to several different ions and the diffusion potential that develops depends on three factors: (1) the polarity of the electrical charge of each ion, (2) the permeability of the membrane (P) to each ion, (3) the concentrations (C) of the respective ions on the inside (i) and outside (o) of the membrane.  Goldman /Goldman-Hodgkin-Katz (GHK) equation gives the calculated membrane potential (Vm) on the inside of the membrane when two univalent positive ions, sodium (Na+) and potassium (K+), and one univalent negative ion, chloride (Cl-), are involved.

Electro-neutrality principle

The membrane maintains a separation of charges, as an electrical dipole layer. Electrostatic forces between charges keeps them in close proximity to the membrane. To establish the normal "resting potential" of -90 mV inside the nerve fiber, only about 1/3,000,000 to 1/100,000,000 of the total (+) charges inside the fiber needs to be transferred. Also, an equally small number of (+) ions moving from outside to inside the fiber can reverse the potential from -90mV to as much as +35 mV within 0.1 msec ! Rapid shifting of ions in this manner causes the nerve signals.

Relation between Cell Membrane Potential and Membrane Ionic Transport System (MITS) Proteins & phosphates are negatively charged at normal cellular pH (7.2). These anions attract positively charged cations that can diffuse through the membrane pores. Membrane more permeable to K+ than Na+.

Membrane ionic transport system (MITS) 1 - Ion channels 2 - Ion pumps 3 - Ion exchangers, carriers, co/counter transporters

Components of membrane ionic transport systems H+

H+

K+

3Na+

Ca2+

CARRIERS

PUMPS

K+ Cl-

CHANNELS

K+

Cl-

Ca2+

AA Na+ Cl-

1. Ion channels Gated (active) Ion Channels

- Voltage gated - Ligand gated - Mechanic gated Non-gated (passive) Ion Channels The diversity of ion channels is significant, especially in excitable cells of nerves and muscles. Of the more than 400 ion channel genes currently identified in the human genome, about 79 encode potassium channels, 38 encode calcium channels, 29 encode sodium channels, 58 encode chloride channels, and 15 encode glutamate receptors. The remaining are genes encoding other channels such as inositol triphosphate (IP3) receptors, transient receptor potential (TRP) channels and others.

Gated (active) Ion Channels

Gated (active) Ion Channels VOLTAGE-GATED ION CHANNELS: ion selective pore, voltage sensor, activation/inactivation gate

3

1 2

Transport of Na+ and K+ through protein channels

Conformational changes in the protein molecules open or close "gates" guarding the channels: voltage or ligand gated

Gating of protein channels to control channel permeability VOLTAGE GATED: -for Na+ channel: strong negative charge on the inside of the cell membrane  outside Na+ gates remain tightly closed when the inside of the membrane loses its negative charge, these gates would open suddenly and allow tremendous quantities of dehydrated Na+ to pass inward through the strongly negatively charged sodium pores (0.3-0.5 nm in diameter)  selective permeability for Na+. -for K+ channel:  gates are on the intracellular ends of the K+ channels, and they open when the inside of the cell membrane becomes positively charged.  channel pores are up to 0.3 nm in diameter, but their inner surfaces are not negatively charged  the smaller hydrated K+ can pass easily through the channel, whereas the larger hydrated sodium* ions are rejected, thus providing selective permeability for K+. carbonyl oxygens line the walls of the selectivity filter, forming sites for transiently binding dehydrated K+. The interaction of the K+ with carbonyl oxygens causes them to shed their bound water molecules, permitting the dehydrated K+ to pass through the pore

*K+ are slightly larger than Na+, but Na+ attracts far more water molecules…

K+ are slightly larger than Na+, but Na+ attracts far more water molecules…

The structure of a potassium channel.

- four subunits, each with two transmembrane helices. - a narrow selectivity filter formed from the pore loops, with carbonyl oxygens lining the walls

The open potassium channel, with the potassium ion shown in purple in the middle, and hydrogen atoms omitted. When the channel is closed, the passage is blocked.

Voltage-gated K+ channel

At rest, negative cellular potential keep voltage-gated K+ channels closed. Depolarization  cell potential becomes positive K+ channel is activated and can conduct K+ ions (yellow arrow, right). The conformation change from the closed to the open state is driven by the movement of the positively-charged amino acids (orange "+" symbols) located in a region of the protein called the voltage sensor surrounding the central ionic pore.

The voltage-gated Na+ channel - used in the rapid electrical signaling

- components: - ion selectivity filter for Na+: Na+ discard the water molecules associated with them in order to pass in single file through the narrowest portion of the channel - activation gate that can open and close, as controlled by voltage sensors, which respond to the level of the membrane potential - inactivation gate limits the period of time the channel remains open, despite steady stimulation.  a subunit: polypeptide chain of >1800 am.ac. embedded in cell membrane. * Nonpolar side chains coil into transmembrane alpha-helices and face outward where they readily interact with the lipids of the membrane. * By contrast, the polar peptide bonds face inward, separated from the lipid environment of the membrane.  b subunit: anchor the channel to the plasma membrane

- activation: - at resting membrane potential (-90÷-70 mV) the channel is closed; - the voltage sensor moves outward and the gate opens if any factor depolarize the membrane potential sufficiently (threshold ~ -50 mV).

Voltage gated Na+ channel

From Basic Neurochemistry, 7th Edition

An experimental strategy to study the ionic currents passing through the membrane, is one using agents that specifically block either the voltage-gated Na+ channels or the voltage-gated K+ channels. Tetrodotoxin (TTX) is a highly potent toxin that inhibits voltage-gated Na+ channels. The source of TTX is the puffer fish (‘fugu’), that is a delicacy in some countries. Even minute quantities of ingested TTX are fatal!

Local anesthetics / nerve blocking agents such as lidocaine (Xylocaine®) and procaine (Novacaine®) prevent the generation of APs by inhibiting voltage-gated Na+ channels of sensory neurons. Thus, depolarization elicited by sensory stimulation does not lead to the generation of action potentials that can travel to the CNS. Tetraethyl ammonium (TEA) is a chemical agent that inhibits the voltage-gated K+ channels. Blockers such as TTX and TEA have been instrumental in revealing the workings of ion channels and their roles in neuronal function.

Voltage-gated Na+ channel

Gated (active) Ion Channels Ligand-gated ion channels : ionotropic vs metabotropic – Ionotropic - directly gate ion channels – Metabotropic - indirectly gate channels via 2nd messengers

Ligand-gated ion channels - glutamate receptors: - NMDA & AMPA ionotropic receptors - metabotropic group I & II receptors (G-prot. coupled)

PCP- phenylciclidine

Gated (active) Ion Channels Mechanic gating ion channel extracellular

Anchoring situs

Cell membrane

intracellular Fibrillary protein

gate

Non-gated (passive) Ion Channels K+ leak channels

2. Ion Pumps Functional particularities: -active transport of ions and organic molecules against concentration gradient

- involve enzymatic reactions, ATP consume -decreased transport rate

Ex: Na+/K+ pump, H+ pump, Ca2+ pump...

Ion Pumps - Na+/K+ pump Na+-K+ ATPase pump can run in reverse: If the electrochemical gradients for Na+ and K+ are experimentally increased enough so that the energy stored in their gradients is greater than the chemical energy of ATP hydrolysis, these ions will move down their concentration gradients and the Na+-K+ pump will synthesize ATP from ADP and phosphate.

The phosphorylated form of the Na+-K+pump can either donate its phosphate to ADP to produce ATP or use the energy to change its conformation and pump Na+ out of the cell and K+ into the cell. The relative concentrations of ATP, ADP, and phosphate, as well as the electrochemical gradients for Na+ and K+, determine the direction of the enzyme reaction. For nerve cells, 60 to 70% of the cells’ energy requirement may be devoted to pumping Na+ out of the cell and K+ into the cell.

The Na+/K+ Pump - The Na+-K+ Pump is important for resting membrane potential and for controlling cell volume. Inside the cell are large numbers of proteins and other organic molecules that cannot escape from the cell. Most of these are negatively charged and therefore attract large numbers of potassium, sodium, and other positive ions as well. All these molecules and ions then cause osmosis of water to the interior of the cell. Unless this is checked, the cell will swell indefinitely until it bursts. The normal mechanism for preventing this is the Na+-K+ pump.  Na+-K+ ATPase pump initiates osmosis of water out of the cell - Electrogenic Nature of the Na+-K+ Pump: 3Na+ out /2K+ in -Inhibitors: gangliosides (digoxin – inotropic positive drug), ouabaine; effects on Ca2+ transport through the Na+-Ca2+ cotransporter Endogenous Na-K Pump Inhibitor – human plasma contains an endogenous ouabain-like steroid that inhibits Na-K pumps in a wide variety of cells. Levels of this natural Na-K pump inhibitor increase with salt loading and it is present in high levels in patients with hypertension

Calcium Pump The Ca pump and the Na-Ca exchanger keep intracellular [Ca2+] four orders of magnitude lower than extracellular [Ca2+]

Ca Pump (SERCA) in Organelle Membranes Ca pumps (ATPases): are present on the membranes that surround various intracellular organelles, such as the sacroplasmic and endoplasmic reticulum

Ca Pump (PMCA) on the Plasma Membrane The plasma membranes of most cells contain a Ca pump that plays a major role in extruding Ca2+ from the cell

3. Ion Exchangers/ Carriers/Cotransporters - Na/Ca - Na/H - Na/HCO3 - Na/ aa, Na/G - Cl/HCO3 - Na/K/2Cl - K/Cl, etc

Ion gradients, channels, and transporters in a typical cell (Boron, 2009)

- Cell membrane potential Resting Membrane Potential The basis for the resting membrane potential: Na+-K+ pump [Na+]

[K+]

Out

142

4

In

14

140

Ratios Na+ In:Out = 0.1 K+ In:Out = 35.0

K+-Na+ “leak” channels Membranes are 100X more permeable to K+, as there are more leakage channels for K+ (see no. of genes…)

diffusion

2

K+

3 Na+

Factors that influence the resting membrane potential

The Na+ /K+ pump contributes to resting membrane potential in 2 ways: • Pumping Na+ & K+ ions in a 3:2 ratio  contribute to internal electronegativity • Maintaining a high K+ concentration in the cell’s interior

The membrane conductance to K+ far exceeds that to Na+ : • K+ leakage results in internal electronegativity

How is membrane potential measured?

When the neuron is inactive, the membrane is said to be at rest and has a resting membrane potential When the neuron is active, the flow of information is from soma to axon terminal action potentials (AP).

A Motor Neuron

Membrane responses to stimulus current Hyperpolarizing currents produce responses 1 and 2. A small depolarizing current produces response 3. These are all graded local responses which dissipate locally. A sufficiently large current (threshold) produces an action potential (4), which can be propagated along the axon. Animation at http://www.sumanasinc.com/webcontent/animations/neurobiology.html

-A stimulus initiates a membrane electrical change that depend on the passive properties of the neuronal membrane -Electrical signal /potentials are initiated by local current flow -Local potentials then spread electrotonically over short distances, and decay with distance from their site of initiation (as some of the ions leak back out across the cell membrane and less charge reaches more distant sites); Considering the Ohms law and a stable membrane resistance, the diminished current with distance away from the source results in a diminished voltage change.

- When the potential is equal/over threshold, it propagates over a long distance - at the axon hillock level, the potential initiates an action potential (AP) that propagates without changing its amplitude - APs depend on a regenerative wave of channel openings and closings in the membrane

Action Potential (AP)

• nerve impulse = action potential: cycle of depolarization & repolarization • needs no direct energy • all-or-none principle

The action potential is essential to our understanding of nervous system function. Its shape, velocity of conduction, and propagation fidelity are essential to the timing, synchrony, and efficacy of neuronal communication. G. J. Kress and S. Mennerick / Neuroscience 158 (2009) 211–222

Action Potential -The necessary actor in causing both depolarization and repolarization of the nerve membrane during the action potential is the voltage-gated Na+ channel -A voltage-gated K+ channel also plays an important role in increasing the rapidity of repolarization of the membrane. -These two voltage-gated channels are in addition to the Na+-K+ pump and the K+-Na+ leak channels.

Na+ permeability increases 500-5000 x

The nerve action potential Profile of a Nerve Action Potential

Threshold -Occurs when Na+ entering exceeds K+ leaving -A rise in potential of 15-30 mV is required The “All-or-None” principle An action potential will not occur until the initial rise in membrane potential reaches threshold. However any larger stimulus produces no greater response than that produced by the threshold stimulus, i.e., the threshold stimulus produces the maximal effect  the action potential.

The nerve action potential Resting Stage Depolarization Stage Result of Voltage-gated Na+ channels

•Membrane is polarized i.e., a –90 mV membrane resting potential present •Membrane becomes very permeable to Na+ ions •Influx of Na+ ions •Polarized state is neutralized • Potential merely approaches in smaller CNS fibres • Membrane potentials overshoots beyond zero in large fibres

Repolarization

•Na+ channels get inactivated •Permeability to K+ increases

After-Hyperpolarization

K+ channels remain open after repolarization

Cation conductances during an action potential action potential

Ion conductance

Na+ conductance increases faster and lasts for a shorter duration. K+ conductance is delayed, increases slowly and lasts longer

The Action Potential and the positive feedback of the Na+ channels activation START

+ feed-back

Roles of other ions than Na+ and K+ during the AP •Impermeant anions inside the nerve axon… •Calcium Ions: - calcium pump pumps calcium ions from the interior to the exterior of the cell membrane (or into the endoplasmic reticulum of the cell), creating a calcium ion gradient of about 10,000-fold (internal cell conc ~10-7 molar). - voltage-gated calcium channels slightly permeable to sodium ions as well as to calcium ions; when they open, both calcium and sodium ions flow to the interior of the fiber = Ca++-Na+ channels. The calcium channels are slow to become activated (slow channels), requiring 10 -20 x as long for activation as the sodium channels •Increased permeability of the Na channels when there is a deficit of Ca2+ - extracellular Ca concentration effect on the voltage level at which the Na channels become activated: a deficit of Ca2+ of ~50% determine Na channels to become activated by very little increase of the membrane potential from its normal, very negative level  nerve fiber becomes highly excitable, sometimes discharging repetitively without provocation rather than remaining in the resting state  spontaneous discharge in peripheral nerves, often causing muscle "tetany" (lethal when triggering tetanic contraction of the respiratory muscles). -mechanism: Ca2+ appear to bind to the exterior surfaces of the Na channel protein molecule. The positive charges of Ca in turn alter the electrical state of the channel protein itself, in this way altering the voltage level required to open the sodium gate.

Membrane Refractoriness

Refractoriness = non-responsive state •Involves Na channel inactivation •Absolute refractory period (ARP)membrane is not responsive to any stimulation •Relative refractory period (RRP) membrane is responsive to supra-threshold stimuli

Distribution - function relation for different types of channels on nerve cell membrane -Nongated ion channels - throughout the neuron -Ligand-gated channels - more at sites of synaptic contact (dendritic spines, dendrites, somata); also, at non-synaptic sites. -Voltage-gated channels - predominantly on axons and axon terminals

A spinal motor neuron. Sodium channels (red); Microtubuleassociated protein 2 - MAP2 (green) (Shrager Lab)

Na channels distribution and generation of AP in axon hillock The soma membrane has few Na+ channels it is harder to have sufficient Na+ influx to change membrane potential to the threshold potential (-45 mV). A voltage change up to +30 mV is required Axon hillock membrane has 7x more Na+ channels than the soma membrane and the threshold potential is lower (a voltage change of only +10÷+20 mV is required to bring the membrane potential to threshold) = trigger zone for AP Action potentials in postsynaptic neurons are initiated at the axon hillock.

Simultaneous recording of action potentials from different parts of a neuron. A, an excitatory synapse on a dendrite is stimulated, and the response near that dendrite is recorded in the soma and at the initial segment. The excitatory postsynaptic potential (EPSP) attenuates in the soma and the initial segment, but the EPSP is large enough to trigger an action potential at the initial segment. B, The threshold is high (-35 mV) in regions of the neuron that have few Na+ channels but starts to fall rather steeply in the hillock and initial segment. Typically, a stimulus of sufficient strength triggers an action potential at the initial segment. C, The density of Na+ channels is high only at the initial segment and at each node of Ranvier.

AP generation and conductance along the axon - initial depolarization at the axon hillock +f.b. for Na+ channels  critical membrane potential = threshold (all-or-none response)

-AP: depolarization and repolarization, followed by afterhyperpolarization, as Ca2+-dependent K+ channels remain open and membrane permeability for K+ is higher - propagation of AP to the axon terminals  synapses - also backpropagation in the soma & dendrites, without regenerating in the somal membrane, as somal membrane has too few Na+ channels to regenerate APs; also, inactivation of Na channels at axon hillock (here, refractory period).

-Speed of propagation depends on axon diameter & presence of myelin sheath

-in unmyelinated axons, Na & K voltage-gated channels are uniformly distributed  AP as a traveling wave… -large diameter axons allow a grater flow of ions  grated length of the axon to be depolarized  increase of the conduction velocity -in myelinated axons, myelin sheath insulate the axon membrane…  generation of AP between the myelinated segments, at the nodes of Ranvier  saltatory conduction

Propagation of impulses from the axon hillock

Once the action potential begins, the potential travels forward along the axon and usually also backward toward the soma. However it does not regenerate in the soma membrane. Why is regeneration impossible in the soma membrane? • EPSPs arrive and an AP is generated at the axon hillock. The AP is regenerated forward to the axon, depolarization spreads backwards to soma and dendrites, but impulse potential decays “dies” because the somal membrane has too few Na+ channels to regenerate APs.

Saltatory Conduction

• current flows electronically to the next node • action potentials are regenerated only at nodes • action potential ‘jumps’ from node to node

Propagation of an Action Potential

Action Potential travels along the membrane as a wave of depolarization. Directional propagation of an AP

Speed of propagation depend on the presence of myelin sheath

AP generation and conductance in the sensory neurons: trigger zone is near the peripheral target.

Neural activity (step 1)  rise in [K+]BECF (step 2) depolarize astrocytes (step 3). This depolarization promotes electrogenic Na/HCO3 influx (step 4), simultaneously raises pHi and lowers pHBECF (step 5). The low pHBECF inhibits the neuronal Na+-driven Cl-HCO3 exchanger (step 6)  causes neuronal pHi to fall (step 7). The decreases in both pHBECF and neuronal pHi complete the feedback loop by inhibiting voltage-gated channels and ligand-gated changes, thereby decreasing neuronal excitability (step 8).

Low pH appears to reduce neuronal activity in experimental models of epilepsy