Experimental protocols to derive the biophysical properties of a given

component of the macroscopic ionic current

I-V (Ipeak vs Vt) if Erev > activation threshold

Instantaneous I-V (Itail vs Vrep at constant Vt) if Erev < activation threshold

 Erev

I-V (Ipeak vs Vt);

Itail vs Vt

 Steady state activation curve: Po (V)

G(V)

Po (V, t) ?
 Kinetics of G changes (kinetics of Po changes) during a step depolarization reflect
the kinetics of the conformational changes between the different states of the channel
(closed, open, inactivated) )  kinetics of channel activation and inactivation

GNa(V, t) = INa(V, t) / (V-ENa) GK(V, t) = IK(V, t) / (V-EK)

9 ms long step
depolarizations
at different V
4 ms long step
depolarizations
at different V The maximal fraction of
channels that open at
each V increases with
increasing V (cf
activation curves).
The kinetics with which
this maximal value is
reached become faster
with increasing V.
After reaching the
maximal value, the
fraction of open K+
channels remain constant
for the entire duration of
T=6.3 °C the depolarization at each
Hodgkin and V (real steady-state).
Huxley, 1952
 Kinetics of G changes (kinetics of Po changes) during a step depolarization reflect
the kinetics of the conformational changes between the different states of the channel
(closed, open, inactivated) )  kinetics of channel activation and inactivation

GNa(V, t) = INa(V, t) / (V-ENa) GK(V, t) = IK(V, t) / (V-EK)

9 ms long step
depolarizations
at different V
4 ms long step
depolarizations
at different V After reaching the
maximal value, the
fraction of open Na+
channels decreases and
goes to 0 with voltage-
dependent kinetics (faster
with increasing voltage).
The maximal value only
approximates the steady-
state.

T=6.3 °C
Hodgkin and
Huxley, 1952
 Kinetics of G changes (kinetics of Po changes) during a step depolarization reflect
the kinetics of the conformational changes between the different states of the channel
(closed, open, inactivated)  kinetics of channel activation and inactivation
GNa(V, t) = INa(V, t) / (V-ENa) GK(V, t) = IK(V, t) / (V-EK)
9 ms long step
depolarizations
4 ms long step
at different V
depolarizations
at different V Note also the much faster
kinetics of activation of
Na+ compared to K+
channels
(cf rapid upstroke of action
potential mediated by Na+
channels, despite similar st-
st- voltage dependence of
Na+ and K+ channels)

T=6.3 °C Hodgkin and Huxley, 1952

 Kinetics of G changes (kinetics of Po changes) during a step depolarization reflect
the kinetics of the conformational changes between the different states of the channel
(closed, open, inactivated)  kinetics of channel activation and inactivation
GNa(V, t) = INa(V, t) / (V-ENa) GK(V, t) = IK(V, t) / (V-EK)
9 ms long step
depolarizations
4 ms long step
at different V
depolarizations
at different V

T=6.3 °C Hodgkin and Huxley, 1952

 KINETICS
The velocity with which G varies following a change in V depends on the velocity
with which the channel changes conformation (and reaches the new C=O
distribution), which is described by a 1° order kinetics.

α(V)
C O
β(V)

Po(t) = Po,∞ - (Po,∞ - Po,0) exp (-t/τ(V))

with τ(V) = 1 / (α+β)

For t∞, dPo/dt = 0
Po,∞(V)= α /(α + β) steady-state channel open probability
 GNa = m3h GNamax
With Po,0 =0
m(t) = m∞ (1 - exp (-t/τm))
Po(t) = Po,∞ (1 - exp (-t/τ) )
con m =0 0

h(t) = h0 exp (-t/τh)

con h∞ = 0

For t=τ m3h = probabilita’ di trovare canale

Po(τ) = Po,∞ (1- exp (-1)) = 0.63
Na+ P
aperto
o,∞ (o frazione di
di canali Na+ aperti in popolazione).
Considering a realistic model of an ion channel,
the kinetic equations become very complex.

α (V)

β (V)
k1(V)
k2(V) γ
k1
k2

Hence, we still use the simplified formalism of

Hodgkin and Huxley to describe the kinetics of
voltage-gated ion channels c
 Models of voltage-gated K+ and Na+ channels,
proposed by Hodgkin and Huxley
The K+ channel has only one gate that needs The Na+ channel has two gates that need to
to be open to allow ion permeation: the be open to allow ion permeation: the
activation gate activation gate and the inactivation gates

Hodgkin and Huxley, 1952: the open or closed state of the gates depends on the position of one (or more
than one) charged particle within the membrane, which can move between two states (permissive the
gate opens; non-permissive the gate closes) in response to voltage changes. The equilibrium distribution
of permissive (open) and non-permissive (closed) states can be describred by the Boltzmann equation
 GK GNa

G = Gmax/ (1+ exp (-(V-V1/2)zF/RT)

G varies from 0 for V--> -∞ to Gmax for V-->+∞

This equation can be derived from the Boltzmann equation of

statistical mechanics, which describes the equilibrium distribution of
independent charged particles in an electrical field.
 K+ channels GNa = m3h GNamax

In the terminology of Hodgkin and Huxley, m(t) = m∞ (1 - exp (-t/τm))

n is the probability of finding a single charged
con m0 =0
particle controlling the K+
channel gate in the permissive state;
h(t) = h0 exp (-t/τh)
con
If opening of the gate is controlled by a single h∞ = 0
charged particle in the
permissive state, then
m3h = probabilita’ di trovare canale
Po,K = n Na+ aperto (o frazione di
Po,K(t) = n(t) = n∞ (1- exp (-t/τndi))canali
with n0Na
=0+ aperti in popolazione).

τn(V) = 1 / (αn+βn) n∞(V) = αn / (αn+βn)

where αn is the rate constant of transition of the charged particle controlling the activation
gate from the non permissive to the permissive state and βn is the rate constant of
transition of the charged particle from the permissive to the non-permissive state

If opening of the gate is controlled by N charged particles in the permissive

state, then
Po = nN
 G = m 3h G
GK(t) = NK gK Po,K(t) = GKmax
NaP (t)
o,K
Namax

m(t) = m∞ (1 - exp (-t/τm))

Po,K(t) = n(t) = n∞ (1- expcon
(-t/τ ))
m0n=0
h(t) = h0 exp (-t/τh)
con h∞ = 0
To be able to fit the kinetics of GK (G
mK 3(t)h )=it probabilita’
is necessary di to
trovare canal
assume that 4 charged particles have +to be in the permissive
Na aperto (o frazione di
state to open the K+ channel activation gate +
di canali Na aperti in popolazione).

GK = n4 GKmax
n(t) = n∞ (1- exp (-t/τn))
with n0 =0
GK
n4 = open probability of the K+
channel (= fraction of open K+
channels in population)
 Na+ channels
To be able to fit the kinetics of GNa (GNa (t) ) it is necessary to
assume that 3 charged particles have to be in the permissive
state to open the Na+ channel activation gate and one particle
has to be in the permissive state to open the inactivation gate
h(t) = h0 exp (-t/τh)
GNa
con h∞ ==0m3h GNamax
3h = probabilita’ di trovare canal
mm(t) = m∞ (1 - exp (-t/τm))
Na +
withaperto
m0 =0(o frazione di
di canali Na+ aperti in popolazione).
GNa τm(V) = 1 / (αm+βm)
m∞(V) = αm / (αm+βm)
G = n4 G
K Kmax

m3h = open probability of the h(t) = h0 exp (-t/τh)

Na+ channel (= fraction of open with h∞ = 0
Na+ channels in population).
th (V) = 1 / (αh+βh)
 From fitting the experimental GNa and GK
as a function of time during
depolarizations at different V one derives:

τn(V)
τm(V)
τh(V)
n∞(V)
m∞(V)
at voltages higher than the
threshold for opening the channel
activation gate (activation
threshold)

Hodgkin and Huxley, 1952

T=6.3 °C
 The values of τn, τm, τh
at voltages lower than
the channel activation
threshold are derived
from the kinetics of
deactivation, i.e.
closing of the channel
activation gate upon
repolarization (τn, τm)
and the kinetics of
recovery from
inactivation, i.e.
reopening of the
channel inactivation
gate upon
repolarization (τh).

Upon repolarization:
n(t) = exp (-t/τn) with n0 =1 and n∞= 0
Kinetics of deactivation
m(t) = exp (-t/τm) with m0 =1 and m∞= 0
Kinetics of recovery from
h(t) = 1 - exp (-t/τh) con h0= 0 e h∞= 1 inactivation
τn(V), τm(V) τn(V), τm(V)
at V > Vthreshold at V < Vthreshold
are derived from are derived from
fitting the activation fitting the decay
kinetics of the (deactivation)
currents obtained kinetics of the
with protocol A1 currents obtained
(varying V1) with protocol A2
τh(V) is derived from (varying V2)
fitting the kinetics of
inactivation using
the same protocol
 to measure the kinetics of recovery from inactivation

Vm
V= -75 mV

Τh(V) at voltages lower

than the activation threshold
is measured from the
kinetics of recovery from
inactivation at different
repolarization voltages in
two pulses experiments at
the same Vt applied at
increasing time distances
from each other.
m∞(V) = αm(V) / (αm(V)+βm(V)) αm(V) = m∞(V) / τm(V)

τm(V) = 1 / (αm(V)+βm(V)) βm(V) = (1-m∞(V)) / τm(V)

αm and αn increase with increasing V in a nearly exponential manner.

βm and βn decrease with increasing V in a neraly exponential manner.

αm and βm are both much larger than αn and βn (much faster kinetics of activation and
deactivation of NaV compared to KV).

αh decreases with increasing V (as shown later); βh increases with increasing V.

βh is smaller than αm ( the Na+ channel first activates and then inactivates, but since
βh and αm are not very different, the activation and inactivation kinetics a little overlap
αh = 0 except at very low V (once arrived in the non permissive state the inactivation
particle can move to the permissive state only at very low V).
T = 6.3 °C

The values of the rate constants depend very much on temperature

Q10 = 3-4
Q10 = rate (T+10°C) / rate (T).

For an arbitrary T interval ∆T:

Q∆T = (Q10 )∆T/10
Po, Na (V, t) = m(V,t)3h(V,t)

Po, K (V, t) = n(V,t)4

Po (V, t)
G(V, t) = N Po(V, t) g
I (V, t) = G(V, t)(V-E)

I (V, t) = ∑iGi(V, t)(V-Ei) + Ileak + C (dV/dt)

I = IK(V,t) + INa(V,t) + Il + C(dV/dt)

I = m(V,t)3h(V,t)GNamax(V-ENa) + n(V,t)4GKmax(V-EK)
+ Gl (V-El) + C (dV/dt)

dn/dt = αn(V)(1-n) - βn(V) x n

dm/dt = αm(V)(1-m) - βm(V) x m

dh/dt = αh(V)(1-h) - βh(V) x h

This system of 4 equations was used by HH to simulate the action potential

of the squid giant axon.

The 1st equation (with dV/dt=0) was used to simulate the ionic currents in
voltage-clamp
V
Secondary structure
of pore forming α1 NaV
subunit of V-gated
ion channels

CaV

KV
The four highly-charged S4 segments are the voltage
sensors of V-gated ion channels
 (considering 4 equivalent and independent charged particles as
in the Hodgkin and Huxley model)

C1 C2 C3 C4 O
Incorrect assumptions in Hodgkin-Huxley model:

1. In many voltage-gated ion channels the 4 voltage sensors are

not equivalent and hence the rate constants of the transitions
C1-C2-C3-C4-O are not related to each other as in the HH
model

2. Activation and inactivation of voltage-gated ion channels

are not independent processes as in the HH model
 Inactivation

α (V)

β (V)
k1(V)
k2(V) γ
k1
k2

fraction of channels that can open from closed state

Steady-state inactivation curve (h∞(V))

 3. How do brain cells communicate?

Mechanisms of intracellular
neuronal communication

-- Specific action potential waveforms and specific

patterns of action potential firing in different types of
neurons and their mechanism of generation

--Electrophysiological methods:
experimental protocols to measure the biophysical
properties of voltage-gated ion channels and to
determine the specific ion channels underlying the
different firing properties of different neurons
To understand the mechanisms underlying the specific
action potential and firing properties of a given neuron
we need to characterize the biophysical properties of
the voltage-gated ion channels involved in the
generation of the action potential.

This is done by performing voltage-clamp (patch-

clamp) experiments and measuring in isolation the
whole-cell current carried by the different channels
(different pharmacological or ionic components of the
mascoscopic current)
Experimental voltage-clamp protocols to derive the biophysical properties of
a given component of the macroscopic ionic current

I-V (Ipeak vs Vtest), Instantaneous I-V (Itail vs Vrep)  Erev

I-V, Itail vs Vtest  st. st. activation curve: Po (V)

I vs Vhold  st.st. inactivation curve.: Pc (V)

I(t) a different Vt  activation kinetics: act(V)

inactivation kinetics:  inact(V)

Itail(t) at different Vrep  deactivation kinetics: deact(V)

I2/I1 of paired pulses vs t

at different Vrep  recovery from inactivation kinetics: rinact(V)
Do the different ionic components of the whole-cell current (I)
having the biophysical properties obtained in voltage clamp
experiments in a given neuron underlie the specific action
potential and the firing properties of that neuron?

We need
-a kinetic model for the gating of the ion channels underlying the ionic
components, from which we derive Po(V,t) as a function of the rate constants
of the transitions between the different states in the model
-to be able to obtain the voltage dependence of these rate constants from the
voltage dependence of the experimental parameters (act(V), deact(V), inact(V),
Po(V), Pc(V)) obtained by fitting the experimental data in voltage-clamp
experiments.
-solve a system of differential equations to derive the voltage changes as a
function of time upon depolarization of the membrane above threshold.

If the obtained V changes well simulate the specific action potential recorded
in the neuron, we can conclude that the action potential is generated by ion
channels having the biophysical properties we have characterized in V-clamp
experiments.
If not, other ion channels are involved in the generation of the action potential.
The experimental parameters (act(V), deact(V), inact(V), Po(V), Pc(V))
depend on the rate constants of the model according to equations that
depend on the specific kinetic model.

For realistic kinetic models of ion channels in which activation and

inactivation are not independent, these equations are complex and one
cannot derive the voltage dependence of the rate constants from the
voltage dependence of the experimental parameters (act(V), deact(V),
inact(V), Po(V), Pc(V)) obtained by fitting the experimental data in
voltage-clamp experiments.

Only in the case of the simple equations of the HH model, in which

activation is independent of inactivation, one can derive the voltage
dependence of the different rate constants from fitting of the data
obtained in whole-cell voltage-clamp experiments.
Even condidering the simplest 3-states model of an ion channel,
the kinetic equations become very complex.

α (V)

β (V)
k1(V)
k2(V) γ
k1
k2

Hence, we still use the simplified formalism of Hodgkin and

Huxley to describe the kinetics of voltage-gated ion channels
c
I = IK(V,t) + INa(V,t) + Il + C(dV/dt)

I = Po,Na(V,t) GNamax(V-ENa) + Po,K(V,t)GKmax(V-EK)

+ Gl (V-El) + C (dV/dt)

dPo,Na/dt = α Pc,Na - β Po,Na- γ Po,Na

dPC,Na/dt = β Po,Na + k2 PI,Na - α Pc,Na – k1 Pc,Na
dPI,Na/dt =

dPo,K/dt =
dPc,K/dt =
dPI,K/dt =
 The values of the different time constants  describing the gating
kinetics depend on all the rate constants of the kinetic model.

I(t) a different Vt  activation kinetics: act(V)  mainly α, β

inactivation kinetics:  inact(V)  , α, β

Itail(t) at different Vrep  deactivation kinetics: deact(V)  mainly α, β

I2/I1 of paired pulses vs t

at different Vrep  recovery from inactivation kinetics: rinact(V)
 k 1, k 2