Foundations I Fall, 2016 Voltage-gated Ion Channels Na+ Channel - - PowerPoint PPT Presentation

Voltage-gated Ion Channels Foundations I Fall, 2016

Most Na+ channels are blocked by TTX in nM concentrations

Na+ Channel

300 nM TTX

Hille, 2001

Ogata and Ohishi, 2002

TTX resistant Na Channels

DRG cells- nocioception

Blair and Bean, 2002

TTX resistant Na Channels

Blair and Bean, 2002

Na+ (and other VGCs ) only relatively selective

K currents blocked pharmacologically

Hille, 2001

TTX, STX scorpion toxins pronase The selectivity filter is based on size of the hydrated ion. For Na channel, Na~=Li>Tl>K>Rb>Cs

Sodium Channel

Density in range of 35-500 channels/µm , except at nodes of Ranvier where it may be as high as 23,000/ µm

2 2

Single channel conductance ~4-18 pS

Hille, 2001

The barrier to movement of a heavily hydrated ion into a narrow pore is the energy required to dehydrate the ion. Maximum channel conductance is limited by (among many other variables) the length of the pore. Water forms shells (usually at least 3) around ions. When an ion enters a pore it is dehydrated and the pore walls solvate the ion. If the diameter of the crystal radius of the ion plus one water molecule is equal to the pore size, the channel is permeable to that ion.

see Hille 3rd Ed. 2001

4 transmembrane repeat domains in the alpha subunit similar basic structure for voltage gated Na+, Ca++ and K+ channels each channel made from one alpha and 2 beta subunits (beta 1, 2, 3)

Voltage-gated Na+ channel (Na 1.1-1.9)

v

pore modulatory sites inactivation gate voltage sensor

The voltage-gated Na+ channel is very similar among different species and tissues

Hille, 2001

Mechanisms of Inactivation

Ball and chain model

Kay et al., 1998

All Ca++ currents blocked What is this?

Text

Persistent Na+ Current

Potassium Channels

Lower density than Na channels - ~7-240 channels/µm

2

up to 11,000/µm at nodes

2

single channel conductance ~ 2.4-230 pS Highly diverse - much more so than Na+ channels

Alpha subunits of several different K+ channels were first identified in different Drosophila mutants (1987-1990)

Kv1 Kv2 Kv3 Kv4

Hille, 2001

Ogata and Ohishi, 2002

Multiple K+ genes are always coexpressed in mammalian neurons

Potassium Channels each K+ channel is comprised of 4 subunits

Song, 2002

Outward Rectifier

blocked by mM TEA or 4-AP, or Cs+ or Ba+ ions

40 mV 1.6 nA 20.0 ms

• 1.10 nA

1.300 nA 30.00 mV

• 70.0 mV

spike repolarization limits depolarization

(Delayed Rectifier, I )

K

I Aactivates on depolarization I Ainactivates with depolarization and is inactivated at rest

A-current

not a single molecular entity

Hille, 2001

The voltage dependence of both activation and inactivation is very steep and the curves only overlap over a narrow range of membrane

• potential. Thus only conducts in a narrow window of membrane

potential from about -65 mV to -40 mV. I A “Window Current”

Hille, 2001

Hille, 2001

A-currents allow for slow repetitive firing

Rudy and MacBain, 2001

Kv 3.x channels

Kv 3.x channels

Kv 3.x channels activate at very depolarized membrane potentials

Rudy and MacBain, 2001

Kv3.X is associated with fast-spiking interneurons

Many fast-spiking interneurons express the calcium-binding protein, parvalbumin

Rudy and MacBain, 2001

Kv3.1 Kv1.1

HEK293 Cells

Kv3.x is blocked by µM 4-AP

hippocampal basket cells in TTX

Inward Rectifier

K+ channel that opens when hyperpolarized

Text

Rectification mechanism appears to involve intracellular polyamines (spermine) plus a Mg++ block K 1.x - 5.x

ir

Wilson, 1993

Command steps of 10 mV from -60 mV to + 60 mV Inward Rectifier

Calcium-activated potassium conductance

BK

• r

maxiK

3 families

SK

(SK1, SK2, SK3)

IK

(non-neuronal)

BK

Calcium-activated potassium conductance

BK

large conductance - 200 - 400 ps

Sah and Faber, 2002

blocked by TEA in low µM and scorpion toxins (charybdotoxin, iberiotoxin) Tetramer

Calcium-activated potassium conductances

BK

Ca++ and voltage dependent - requires depolarization β subunit shifts voltage dependence and increases Ca++ sensitivity

Calcium-activated potassium conductance

SK

small conductance - 2 - 20 ps unaffected by TEA in low concentrations very sensitive to apamin (bee venom toxin)

Sah and Faber, 2002

calcium sensor is external - calmodulin

Sah and Faber, 2002

3 components of AHP

fAHP, mAHP, sAHP fAHP is voltage dependent, is blocked by µM TEA, iberiotoxin and paxilline BK channel

I K(Ca) and the AHP

Shepard and Bunney, 1991

rat DA neuron

Sah and Faber, 2002

mAHP

mAHP is voltage-insensitive and is blocked by apamin SK channel

Hallworth et al., 2003

rat STN

Sah and Faber, 2002

sAHP

voltage-insensitive, not blocked by apamin or TEA underlying channel unknown -SK with unknown β subunit?

• not a true calcium-activated K+ channel?

blocked by several neurotransmitters

Hille, 2001

Cortical Pyramidal Neuron Madison and Nicoll, 1986

I h

mixed cation channel

• pens slowly under hyperpolarization

blocked by Cs+ and Rb+, not strongly blocked by Ba++ E between -20 and -40 mV

rev

I h

rat substantia nigra dopaminergic neuron

10 mV 0.2 nA 40 ms

I h participates in rhythmic firing and rebound from strong inhibition

Best characterized HVA Ca++ channel is the L-Channel L(arge) conductance L(ong-lasting) current There are two main families of voltage-gated Ca++ channels They are distinguished primarily by their activation thresholds, and are termed Low Voltage Activated (LVA) and High Voltage Activated (HVA)

Voltage-gated Calcium Channels

Best characterized LVA Ca++ channel is the T-Channel T(ransient) conductance

Calcium Channels

Hille, 2001

Calcium Channels

La >Co >Mn >Ni >Mg

3+

2+ 2+ 2+ 2+

Most Ca++ channels blocked by Cd and transition metals

2+

fi fi fi

Calcium Channels

calcium-dependent inactivation Ca channels are permeable to Ca , Ba or Sr

2+ 2+ 2+ 2+

HVA Ca channels show Ca-dependent inactivation

2+ 2+

L-channel openings are prolonged by BAY K8644 L-channels are blocked by Cd+ and dihyropyridines nifedipine, nitrendipine, diltiazem

Hernandez-Lopez et al., 2000

Ca 1.x

v

Kammermeier, P. J. et al. J Neurophysiol 77: 465-475 1997

N-type calcium channel ( ) is sensitive to low µM concentrations of -conotoxin Ca 2.2

v

ω

Kammermeier, P. J. et al. J Neurophysiol 77: 465-475 1997

Spider venom toxin blocks little of the HVA in thalamic neuron but almost 100% of the HVA in a Purkinje cell The Purkinje cell HVA is called P-current and isextremely sensitive to

• Agatoxin

ω

Jahnsen and Llinas, 1984

It is Na+ -independent It can be activated upon depolarization but only from a hyperpolarized membrane potential. It is inactivated at rest. It is blocked by Co++and low Ca+

The LTS is a low voltage activated calcium conductance

The underlying conductance is a member of the Ca 3 family (Ca 3.1, Ca 3.2 and Ca 3.3) and is called a T- current

v v v v

T stands for transient - note the shorter mean open time compared to L-channels

LTS deinactivation is time and voltage dependent The LTS is blocked by low µm concentrations of Ni dopamine neuron in vitro

Kang and Kitai, 1993

CsCl, 4-AP, TTX

activation inactivation

thalamocortical neuron

relay mode bursting mode

Livingston et al., 1997

forebrain (LMAN) neuron in songbird

McCormick and Pape, 1990

• r ACh, NE, Glu

(or anything that enhances Ih)

Rhythmic bursting mediated by I and T-currents

h

Kang and Kitai, 1993

L- channel T-channel

What is the reversal potential for Ca++ ?

[Ca++]i =100nM [Ca

++]o = 2mM

ECa++ = +124

• bserved (+52 mV)

predicted (+124mV)

What is the reversal potential for Ca++ ?

[Ca++]i =100nM [Ca

++]o = 2mM

ECa++ = +124

• bserved (+52 mV)

predicted (+124mV)

What is the reversal potential for Ca++ ?

[Ca++]i =100nM [Ca

++]o = 2mM

ECa++ = +124

ρK

+ /ρC

a

++ =1:1000

the channel is also permeable to K+ with there is so much more K inside than Ca that it exits when the channel is open and the

• bserved reversal

potential is a mixture

• f the two Nernst

equilibrium potentials

Cl- Channels

Verkman & Galietta, 2009

Calcium-activated chloride channel (CaCC) in hippocampal pyramidal neuron CaCC are selectively blocked by niflumic acid (NFA).

Huang et al, 2012

sub threshold oscillations in striatal PLTS interneurons independent of Na+ channels but requires Ca++ channels

Song and Wilson, 2016

sub threshold oscillations in striatal PLTS interneurons are blocked by NFA sub threshold oscillations in striatal PLTS interneurons are caused by CaCC

Song and Wilson, 2016