Lecture 2 Nervous System 2014 (1)

Lecture 2 Physiology of the Nervous System

Excitability and ionic transport

Dr. Ana-Maria Zagrean

Discipline of Physiology and Fundamental Neurosciences

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.

Excitability

1) input

2) integration

3) output

Excitability and Ionic transport

Interaction stimulus - receptor

stimulus

R e

c e

p t

o r

?

chemical

physical

response

The excitability of neurons is based on ion gradients

across the cell membrane, and on transport properties

of the cell membrane.

Interrelation between cell membrane properties and the

complex characteristics of biological systems.

identity

Selective membrane

permeability

excitability

adaptability evolution

Premises

acquiring

coordinating

disseminating

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

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

(see Nernst potential)

2.Active transport

- Primary active (pumps)

- Secondary active (co- and counter-transport)

TRANSPORT OF SUBSTANCES THROUGH THE CELL MEMBRANE

Pores/channels integral cell membrane proteins

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.

Simple diffusion through protein channels

Conformational changes in the protein molecules

open or close "gates" guarding the channels:

voltage or ligand gated

Na+ channel is only 0.3 by 0.5 nm in diameter,

the inner surfaces of this channel are strongly

negatively charged and pull small dehydrated

sodium ions into these channels selective permeability for Na+.

K+ channels are up to 0.3 nm in diameter,

but their inner surfaces are not negatively

charged K+ are not pulled away from the water molecules that hydrate them the smaller hydrated potassium ions can pass

easily through the channel, whereas the

larger hydrated sodium* ions are rejected,

thus providing selective permeability for K+.

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

Transport of Na+ and K+ through protein channels

Na +

K +

- four subunits, each with two transmembrane helices.

- a narrow selectivity filter formed from the pore loops

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.

The structure of a potassium channel.

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 Na+ to pass inward through the

sodium pores.

-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.

CHEMICAL (LIGAND) GATING:

- protein channel gates are opened by the binding of a chemical substance (a

ligand) with the protein; this causes a conformational or chemical bonding change

in the protein molecule that opens or closes the gate.

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. what limit it?

Diffusion: simple and facilitated

Postulated mechanism for

facilitated /carrier mediated diffusion

(e.g. glucose or amino acids transport)

- glucose transport by GLUT4 is increased

10-20x by insulin (but not in the brain).

A. Net Diffusion Rate Is Proportional to the Concentration Difference Across a Membrane.

B. Effect of Membrane Electrical Potential on Diffusion of IonsThe Nernst Potential.

C. Effect of a Pressure Difference Across the Membrane.

Diffusion and the cell membrane potential

Net Diffusion Rate Is Proportional to the Concentration Difference Across a Membrane.


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 37C 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.

Effect of a Pressure Difference Across the Membrane.


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.



B, Establishment of a "diffusion potential" when the nerve fiber membrane is

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.

(Actual potential)

Calculation of the diffusion potential when the membrane is

permeable to several different ions Goldman equation

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.

The passive properties of a plasma membrane

A plasma membrane has resistance, capacitance and conductance

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.

Cell Membrane Potential

Proteins & phosphates are negatively charged at normal cellular pH.

These anions attract positively charged cations that can diffuse through the membrane

pores.

Membrane more permeable to K+ than Na+.

Relation between cell membrane potential - membrane ionic transport system

1 - Ion channels

2 - Ion pumps

3 - Ion exchangers, carriers, co/counter transporters

Membrane ionic transport system (MITS)

3Na+

Ca2+

Cl-

K+

H+

H+

Ca2+

K+

Cl-

Cl-

AA

Na+

PUMPS CARRIERS

CHANNELS

K+

Components of membrane ionic transport systems

1. Ion channels

- Voltage gated

- Ligand gated

- Mechanic gated

Gated (active) Ion Channels

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 170 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.


VOLTAGE-GATED ION CHANNELS:

ion selective pore, voltage sensor, activation/inactivation gate

1

2

3

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).

From Basic Neurochemistry, 7th Edition

Voltage gated Na+ channel

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


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

Cell

membrane

gate

Fibrillary

protein

Anchoring

situs extracellular

intracellular


Mechanic gating ion channel

Non-gated (passive) Ion Channels

K+ leak channels

Functional particularities:

2. Ion Pumps

-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+ 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 Is Important 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

The Na+-K+ Pump

Calcium Pump

Ca2+

Ca2+

- Na/H

- Cl/HCO3 -

- Na/K/2Cl

- K/Cl, etc

- Na/Ca

- Na/HCO3

3. Ion Exchangers/

Carriers/Cotransporters

- Na/ aa, Na/G

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

Cell membrane potential Resting Membrane Potential

Na+-K+ pump K+-Na+ leak channels

Humans

[Na+]

[K+]

Out

142

4

In

14

140

Ratios

Na+ In:Out = 0.1

K+ In:Out = 35.0

Membranes are 100X more

permeable to K+, as there

are more leakage channels

for K+ (see no. of genes)

The basis for the resting membrane potential:

Slow rate of Na+ influx is accompanied by slow rate of K+ efflux.

Na+/K+ pump maintains the concentration gradients.

Electrochemical gradient membrane potential of ~ - 65 mV

diffusion

3 Na+ 2 K+

pump

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 Maintaining a high K+ concentration in the cells interior

The membrane conductance to K+

far exceeds that to Na+ :

K+ leakage results in internal electronegativity

How is membrane potential measured?

A Motor Neuron

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).

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) 211222

-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

Action Potential

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 Membrane is polarized i.e., a 90 mV membrane resting potential present

Depolarization Stage 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

Result of

Voltage-gated

Na+ channels

Na+ channels get inactivated Permeability to K+ increases

After-Hyperpolarization

K+ channels remain open after repolarization

Cation conductances during an action potential

Na+ conductance increases faster

and lasts for a shorter duration.

K+ conductance is delayed,

increases slowly and lasts longer

action potential

Ion conductance

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);

Microtubule-associated

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

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 axom hillock (here, refractory period).

-Speed of propagation depend 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.

Condition associated with channelopathies (congenital or acquired (often resulting from autoimmune

attack on an ion channel).

Channel type

Alternating hemiplegia of childhood Na/K-ATPase

Congenital hyperinsulinism Inward-rectifier potassium ion channel

Cystic fibrosis Chloride channel

Episodic Ataxia Voltage-gated potassium channel

Erythromelalgia Voltage-gated sodium channel

Generalized epilepsy with febrile seizures plus Voltage-gated sodium channel

Familial hemiplegic migraine various

Hyperkalemic periodic paralysis Voltage-gated sodium channel

Hypokalemic periodic paralysis Voltage-gated sodium channel

or voltage-dependent calcium channel (calciumopathy)

Long QT syndrome various

Malignant hyperthermia Ligand-gated calcium channel

Mucolipidosis type IV Non-selective cation channel

Myasthenia Gravis Ligand-gated sodium channel

Myotonia congenita Voltage-dependent chloride channel

Neuromyotonia Voltage-gated potassium channel

Nonsyndromic deafness various

Paramyotonia congenita (a periodic paralysis) Voltage-gated sodium channel

Retinitis pigmentosa(some forms) Ligand-gated non-specific ion channels

Short QT syndrome various potassium channels suspected

Timothy syndrome Voltage-dependent calcium channel

Seizure Voltage-dependent potassium channel

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Lecture 2 Nervous System 2014 (1)