1. Ion Channels

2. Active Transporters: The proteins that created and maintain ion gradients
Ion channels : give rise to selective ion permeability changes

3. ION CHANNELS
Ion channels are transmembrane proteins that contain a specialized structure, THE PORE that that allow particulars ions to cross the membrane.
Some ion channels contain voltage sensor ( voltage gated channels) that open or close the channel in response to changes in voltage.
Other gated channels are regulated by extracellular chemical signals such as neurotransmitter or by intracellular signals as a second messengers.

4. ACTIVE TRANSPORTERS
Membrane proteins that produce and maintain ion concentration gradients.
For example the Na+ pump which utilizes ATP to regulate internal concentration of Na+ and K+.
Transporters create the ionic gradient that drive ions through open channels, thus generating electric signals

5. What is the mechanism for ion movement across the membrane?
K+ and Na+ currents were distinct, suggesting distinct mechanisms
Mechanism is voltage dependent (must sense voltage)
Voltage clamp recordings showed that ions move across membrane at high rates (~ 600,000 /s) – inconsistent with an ion pump mechanism
Ion selectivity of Na+ and K+ currents – size dependent permeability suggests pore of certain diameter.
Armstrong (1965-6) – TEA block could be overcome by adding excess K+ to the extracellular fluid and stepping to hyperpolarized potentials (K+ comes into cell) suggesting that K+ ions dislodge TEA from pore
Acetylcholinesterase – 5000 molecules/sec
Introduction of TTX in 1964 – showed that Na current could be blocked while leaving voltage activated K current and leak K current intact. Indicates that distinct mechanisms are responsible for different currents. Similar but opposite effects of TEA (blocks voltage dependent K but not leak)
Binding studies of labeled TTX (or STX) provided estimates of density of Na sites – knowing density and current, can estimate # of ions coming in from each site
TTX also only binds when added to extracellular surface rather than intracellular surface.

6. Ion channels share several characteristics
The flux of ions through the channel is passive .
The kinetic properties of ion permeation are best described by the channel conductance (g) that is determinate by measuring the current flux (I) that flows through the channel in repose to a given electrochemical driving force. (Electrochemical driving force is determinate by difference in electric potential across membrane and gradient of concentration of ions) .
At the single channel level, the gating transitions are stochastic. They can be predicted only in terms of probability.

7. Ion channels share several characteristics
In some channels the current flow varies linearly with the driving force ( channels behave as resistors)
In other channels, current flow is a non-linear function of driving force ( Rectifiers)
High conductance (γ)
I (pA)
V (mV)
Low conductance (γ)
Ohmic channel
Rectifying Channel
( I=Vm/R)

8. Ion channels share several characteristics
The rate of ion flux (current) depends on the concentration of the ions in solution ( At low concentrations the current increases linearly with the concentration, at higher concentrations the current reach a saturation point ) .
The ionic concentration at which current flow reaches half its maximum defines the dissociation constant for ion binding.
Some ion channels are susceptible to occlusion by free ions or molecules

9. The Opening and closing of channels involve conformational changes
In all channel so far studied, the channel protein has two or more conformational states that are relatively stable. Each stable conformation represents a different functional state.. Each channel has an open state and one or two closed states. The transition between states is calling gating.

10. The Opening and closing of channels involve conformational changes
Three major regulatory mechanisms have evolved to control the amount of time that a channel remains open and active.
Under the influence of these regulators ,channels enter one of three functional states: closed and activable (resting), open (active) or closed and nonactivable ( refractory).
The signal that gate the channel also controls the rate of transition between states.

11. The Opening and closing of channels involve conformational changes
Ligand -gated and voltage gated channels enter refractory states through different process. Ligand-gated channels can enter refractory state when the exposure to ligand sis prolonged (desensitization)
Voltage-gate channels enter a refractory state after activation. The process is called inactivation.
Activation is the rapid process that opens Na+ channels during a depolarization.
Inactivation is a process that closes Na+ channels during depolarization. The membrane needs to be hyperpolarized for many milliseconds to remove inactivation.

12. The Opening and closing of channels involve conformational changes
Exogenous factors such as drugs and toxins can affect the gating control sites.

13. Structure of Ion Channels
Ion channels are composed of several subunits. They can be constructed as heterooligomers from distinct subunits, as homooligomers from a single type of subunit o from a single polypeptide chain organized into repeated motifs.
In addition to one or more pore forming unties, which comprise a central core, some channels contain auxiliary subunits which modulate the characteristics of the central core

14. Structure of voltage gated ion channels
Repeated series of 6 TM a helices
S4 helix is voltage sensor
Loop between S5 & S6 composes selectivity filter

16. Gating currents
Movement of + charges in S4 segment produces small outward current that precedes ion flux through channel

17. Role of auxiliary subunits
Auxiliary (non pore) subunits affect:
• Surface expression
• Gating properties

18. Voltage gated sodium channels
A large alpha subunit that forms the core of the channel and its functional on its own. It can associate with beta subunits
Blocked by: TTX, STX, *cain local anesthetics

19. Persistent (non-inactivating) Na+ currents are produced by an alternative channel gating mode

20. Functions of voltage-gated Na+ channel alpha subunits
Protein name
Gene
Expression profile
Associated channelopathies
NaV 1.1
SCN1A
Central and peripheral neurons and cardiac myocites
Febrile epilepsy, severe myclonic epilepsy of infancy, infantile spasms, intractable childhood epilepsy, familial autisms
Nav1.2
SCN2A
Central and peripheral neurons
Febrile seizures and epilepsy
Nav1.4
SCN4A
Skeletal muscle
Periodic paralysis, potassium agravated myotonia
Nav1.5
SCN5A
Cardiac myocites, skeletal muscle, central neurons
Idiopathic ventricular fibrillation
Nav1.7
SCN9A
Dorsal root ganglia, peripheral neurons. Heart, glia
Insensitivity to pain.

21. Voltage gated Ca2+ channels
Gene Product Cav Cav2.1 Cav2.2 Cav2.3 Cav
Tsien Type “L” “P/Q” “N” “R” “T”
Characteristics High voltage Mod voltage High voltage Mod voltage Low voltage
activated, activated, activated, activated activated
slow inactivation moderate moderate fast fast
(Ca2+ dependent) inactivation inactivation inactivation inactivation
Blocked by dihydropiridines Agatoxin Conotoxin SNX 482 Mibefridil
(nimodipine) IVA GVIA High Ni2+
Form by different subunits:α1, α2δ,β and γ. The α1 subunit forms the pore, the other subunits modulate gating.

22. Ca2+ dependent Ca2+ channel inactivation
CaM
Ca2+ -
Ca2+
Ca2+
Ca2+

23. Potassium Channels Inwardly rectifying Voltage gated 2 pore (“leak”)
Ca2+ activated

24. Inwardly-rectifying and “leak” K+ channels
2 pore “leak”
Inwardly-rectifying channels
•  subunits: Kir 1.X - 7.X
• Rectifying character due to internal block by Mg2+ and polyamines
• Roles:
• Constitutively active resting K+ conductance (eg. Kir1, Kir2)
• G-protein activated (Kir3)
• ATP sensitive (Kir6)
2 pore “leak” channels
• many different  subunits, nomenclature still argued
• Outwardly rectifying due to unequal [K+] across the membrane
• Constitutively active resting K+ conductance
• pH sensing
• Mechanosensitive
• Thermosensitive
• Second messenger sensitive (cAMP, PKC, arachadonic acid)

25. Voltage gated K+ channels
Gene Product Kv1.X (1-8) Kv2.X (1-2) Kv3.X (1-4) Kv4.X (1-4) Kv7.X (1-5)
“D type” “Delayed “Delayed “A type” “M current”
rectifier” rectifier”
Characteristics Low voltage High voltage High voltage Low voltage Low voltage
activated (~50 mV), activated (0 mV), activated (-10 mV), activated (-60 mV) activated (-60 mV)
fast activation mod activation fast activation fast activation slow activation
(< 10 ms) (>20 ms) (10-20 ms) (10-20 ms) (>100 ms)
slow inactivation very slow very slow fast no
inactivation inactivation inactivation inactivation
fast deactivation
Blocked by 4-AP (100 µM) TEA (5-10 mM) TEA ( mM) 4-AP (5 mM) XE991
dendrotoxin 4AP (1-5 mM) 4AP (0.5-1 mM)
BDS (50 nM)
Kv4
(“A type”)
Kv1
(“D type”)
Kv2
(“DR type”)
Kv3
(“DR type”)

26. Ca2+ activated K+ channels - role in repolarization following APs
Spike frequency accommodation
Voltage response
currents mediating AHP
Role of IKCa in burst duration

27. Ca2+ activated K+ channels
Channel Type BK SK sAHP
“maxi K, IC fAHP” “mAHP” “sAHP”
Gene product slo 1-3 SK1-3 ????
Voltage dep? Yes No No
[Ca2+] to activate 1-10 µM µM µM
Ca2+ binding direct to subunit calmodulin hippocalcin?
Single channel pS 5-20 pS 5-10 pS
Conductance
Blocked by charybdotoxin apamin TEA (> 20 mM)
TEA (< 1 mM) TEA (> 20 mM)

28. Many drugs and toxins act on voltage gated ion channels

29. Effect of drugs and toxins
Many toxins block ion channels directly either from the outer (TTX) or inner (lidocaine) surface of the channel
Other toxins change the properties of the channel without blocking it
Delaying inactivation
Shifting voltage dependence
FUGU
TTX LA 29

30. Modulation of Ion Channels
Example, enhancement of Ca2+ channels in cardiac myocytes by NE

31. Dendritic ion channels participate in synaptic amplification and integration
The answer to that question is that within the past decade or so there is a lot of evidence that has shown that Na+ channels, particularly dendritic Na+ channels, have a lot of other interesting roles in the way dendrites process information.

32. Channelopathies Condition Channel type Paramyotonia congenita
Vgated Na+ channel
Hemiplegia of childhood
Na+/K+ ATPase
Congenital hyperinsulinism
IR K+ channel
Cystic fibrosis
Cl- Channel
Episodic ataxia
Vgated K+ channel
Erythromegalia
Generalized epilepsy with febrile seisures
Hyperkalemic periodic paralysis
Malignant hyperthermia
L gated Ca2+ channel
Myasthenia Gravis
Lgated Na+ channel
Neuromyotonia

33. Recommended Readings:
Kandel. Principles of Neural Science, 4 th Edition chapter: 6
Hille. Ion Channels of Excitable Membranes. 3 ed. Edition.