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1. Cell & Molecular Pharmacology (6BBM0314)
Advanced Cellular Pharmacology (7BBM0014)
Professor Ian McFadzean
Pharmacology & Therapeutics
Life Sciences & Medicine
Molecular pharmacology of voltage-
operated ion channels

2. Learning outcomes
By the end of this lecture you should be able to
◼ Describe what is meant by the terms “open channel” and “foot in the door” block and
illustrate these mechanisms by reference to selected drugs
◼ Understand what is meant by activation and inactivation curves and the information that
can be gained from them
◼ Compare and contrast the actions of isoprenaline and BAYK8644 on L-type voltage-
dependent calcium channels
◼ Apply the ideas discussed in the lecture to other drugs for example the antiarrythmic drug
ivabradine
Note; the terms “voltage-operated”, “voltage-dependent”, “voltage-gated” are used interchangeably to
mean channels that are opened (gated) by changes in membrane potential

3. Drugs can inhibit (voltage-operated) ion channel
function by two overlapping mechanisms
◼ Physical “plugging” of the pore – channel block
❑ Quaternary ammonium ions on voltage-operated potassium channels
❑ Local anaesthetics on voltage-operated sodium channels
◼ By binding to allosteric sites on the channel protein and altering channel gating
characteristics
❑ Dihydropyridine acting on L-type voltage-operated calcium channels
❑ Local anaesthetics on voltage-operated sodium channels

4. A bit of revision; for any drug;
Drug + Binding site DB
k1
k-1
Units for k1 = M-1s-1
i.e. the forward rate dependent upon [D], the concentration of drug
Units for k-1 = s-1
i.e. the backward rate is independent of [D]
So, when we add a drug, the rate at which the system approaches equilibrium is
dependent upon the concentration of drug; the higher the concentration the more
rapidly the system approaches equilibrium

5. Let’s apply this to ion channels – “open channel block”
◼ Many channel blocking drugs act through “open channel block”, where the drug must
wait for the channel to open before gaining access to its binding site deep within the
pore of the channel
◼ One of the earliest examples was the blockade of voltage-dependent potassium
currents by internally applied quaternary ammonium ions such as TPeA (KD  10-5 M)
External
Internal

6. The effects of internal TPeA on IK from squid giant axon
The “inactivation” time course
shows the onset of block as the
drug molecules enter the open
channels and the system reaches
equilibrium .
The rate the system approaches
equilibrium (the time course of
current decay) is concentration-
dependent.
From “Ion Channels of Excitable Membranes”, B.Hille, Sinauer press
Drug + Binding site DB
k1
k-1

7. A simplified “state model” for blockade of IK by internal QA ions
Resting Open
O*
R*
QA
QA
unblocked
* blocked
The drug molecules get trapped inside the ion channel if the gate closes when
they are in there. They will not “escape” until the channel opens again.

8. Open channel block mimics “N-type” ball and chain
inactivation (see VOC Structure and function lecture)
Activation gate closed
Inactivation gate open
Activation gate open
Inactivation gate open
Binding site for “ball” exposed
Activation gate open
Inactivation gate closed
Resting Open Inactivated
- -
+ +
+

9. A slight variation on open channel block – the “foot in
the door” model
◼ The drug has still to wait for the channel to open BUT
◼ When the drug binds, the gate cannot close behind it, so there is no “trapping” of the
drug
External
Internal

10. A simplified state diagram for the foot in the door model
Resting Open
O*
drug
drug
unblocked
* blocked

11. Local anaesthetics
◼ Block voltage-dependent Na+ channels
◼ In additional to blocking the pore these drugs also alter the gating of the channel
◼ Most are tertiary amines with pKa values around 8.0
Lignocaine (lidocaine)
R-NR2 + H+ RNH+R2
base acid
hydrophobic hydrophilic
H+

12. Recall; activation, inactivation and deactivation
R O I
activation inactivation
recovery
from inactivation
deactivation

13. The blocking action of QX-314 (a quaternary analogue
of lignocaine, always positively charged)
◼ Only effective when applied intracellularly,
so QX-314 is not clinically useful
◼ Acts by open channel block
◼ The drug gets trapped in the channel when
the gates close
◼ Blocking action shows marked use-
dependence (the guarded receptor
hypothesis)
◼ This same mechanisms applies to the
charged from of lignocaine (RNH+R2) and is
called the hydrophilic pathway for local
anaesthetic action, but…….
R O I
R* O* I*
QX314
blocked
unblocked
QX314

14. …the blocking action of lignocaine is more complex
◼ Lignocaine blocks resting, open and inactivated channels
◼ Shows less profound use-dependence
◼ The hydrophobic (uncharged) form enters and leaves the channel, and the cell, via
the membrane. This is called the hydrophobic pathway, and this pathway is not
use-dependent (it does not require the channel to be open)
◼ Extracellular pH affects blocking action in a complex way; low extracellular pH
 reduces the availability of the lipid soluble, non-ionised form of the drug
 slows down leakage of drug from closed channels as the drug is ionised in the channel
pore and the pH of the pore seems to be governed by the extracellular pH

15. Blocking action of lignocaine
R O I
R* O* I*
blocked
unblocked
All other things being equal this
blocking of the pore by the local
anaesthetic will produce a
concentration-dependent
reduction in the peak sodium
current
hydrophobic hydrophobic
hydrophilic +
hydrophobic

16. Before moving on, make sure you can answer the
following questions
◼ Can you explain what is meant by “open channel block”?
◼ Why does a drug that acts by open channel block appear to make channels
“inactivate”?
◼ What is the “foot in the door” mechanism?
◼ Why is QX314 not clinically useful?
◼ Can you distinguish between the hydrophobic and hydrophilic pathways through
which local anaesthetics reach their site of action?
◼ What is meant by “use dependence” when applied to local anaesthetic action

17. Activation and inactivation curves
◼ Once in the inactivated state, the channels cannot open again without cycling back through the
resting state
◼ At any given value of membrane potential, the proportion of channels in the resting state
(available to open) vs the inactivated state (unavailable to open) will vary
◼ The relationship between membrane potential and what proportion of channels are available is
described by an inactivation curve
Available to open Unavailable to open
R O I
Open only transiently
Membrane depolarisation

18. A typical inactivation curve
100%
0%
50%
membrane potential (mV)
Proportion
of channels
available to open
-90mV 0 mV
The inactivation curve gives information on the
relative proportions of channels in the resting
(available to open) and inactivated (unavailable
to open) states at a given membrane potential
All channels resting; none inactivated
All channels inactivated; none resting

19. We can also construct activation curves
100%
0%
50%
membrane potential (mV)
Proportion
of available
channels that will
open
-90mV 0 mV
The activation curve gives information on the proportion
of the available channels that will open when the
membrane is depolarised to a new value of membrane
potential
All available
channels will
open
None of the
available
channels will
open

20. Let’s consider activation and inactivation curves together
❑ By considering both curves together we
can estimate how many channels will open
(ie how big the ion current might be) when
the membrane potential depolarises from
one value to the another, for example, a
step in membrane potential from -70 mV
to -20 mV
❑ The inactivation curve tells you what
proportion of channels are available to
open at -70 mV
❑ The activation curve tells you what
proportion of the available channels will
open when you step the membrane
potential to -20 mV
100%
0%
50%
membrane potential (mV)
Proportion
of channels
-90mV 0 mV
Inactivation
Activation

21. Try this little exercise
◼ The diagram on the right shows an
activation curve and an inactivation curve
for a voltage dependent potassium
current.
◼ Consider a hypothetical cell with 1000
such channels.
◼ How many channels would open during
the following step changes in membrane
potential?
 -100 to +60 mV
 -100 to -30 mV
 -100 to +17 mV
 -50 to +60 mV
 -50 to -30 mV
 -50 to +17 mV
-100 -80 -60 -40 -20 0 20 40 60 80
0.0
0.2
0.4
0.6
0.8
1.0
Inactivation curve
Activation curve
Fractional
conductance
Membrane Potential
membrane potential (mV)
Proportion
of
channels

22. Back to local anaesthetics
◼ In addition to blocking the channel
pore, local anaesthetics bind with
higher affinity to, and consequently
stabilise, the inactivated state of
the channel
◼ Presumably due to a binding “site”
that changes its conformation (low
to high affinity) when the channel
inactivates
◼ The effect is seen as a negative
shift in the inactivation curve and
slowed recovery from inactivation
◼ This contributes to the inhibition
they produce by making it more
likely the channel will be
inactivated
100%
0%
50%
membrane potential (mV)
Proportion
of channels
available to
open
-90mV 0 mV
In presence
of local
anaesthetic
Control

23. The “Modulated Receptor Hypothesis” of local
anaesthetic and anti-arrhythmic action
◼ Conceptually we can consider the sodium
channel as representing (at least) three
separate local anaesthetic “receptors”
equivalent to the Resting, Open and
Inactivated states of the channel.
◼ Anything that alters the likelihood of the
channel existing in one or other state will
alter the affinity of the drug for the channel.
Thus, strong hyperpolarisations reduce the
affinity of the local anaesthetic for the
channel; opposite for depolarisation
◼ This may also contribute to use dependence
R O I
Increasing affinity for LA

24. Pharmacology of “L-type” CaV channels
◼ L-type calcium currents are affected by three distinct classes of drugs
 dihydropyridines
“antagonists” e.g. nifedipine
“agonists” eg BAYK8644
 phenylalkylamines e.g. verapamil
 benzothiazepines e.g. diltiazem
◼ Phenylalkylamines and benzothiazepines act in much the same way as local
anaesthetics on sodium channels
◼ Dihydropyridines on the other hand alter channel gating in a very distinctive
way

25. A comparison of the effects of isoprenaline and BAYK8644
on L-type calcium channels in ventricular myocytes
◼ Both isoprenaline (a -receptor agonist) and BAYK8644 (a dihydropyridine) increase the
size of the voltage-operated calcium current in ventricular myocytes
◼ They do so in subtly different ways, revealed by single channel recordings
◼ The following experiments were carried out using cell-attached patches from guinea-pig
ventricular cells
◼ The patch of membrane was depolarized repetitively from -40 mV to +70 mV and single
channel currents through L-type calcium channels recorded as downward deflections on
the current trace
◼ After several hundred such depolarisation sweeps, the experimenters also looked at what
fraction of the sweeps were blank (i.e the channel in the patch didn’t open). This is a
measure of the number of “functional channels” in the patch when it depolarises
◼ Finally, for each individual sweep, the measured what fraction of the time the channel was
open for; this is called the open probability (Po)

26. Isoprenaline increases the number of functional
channels
◼ Responses to repetitive
membrane depolarisations from
-40mV to +70 mV
◼ Under control conditions,
depolarisation often didn’t
evoke channel openings (6/10
“sweeps” were blank, as shown
by arrows)
◼ However, in the presence of
isoprenaline the channel almost
always opened (only 1/10
sweeps were blank)
control + isoprenaline
channel
opening
blank “sweep”
-40mV
+70mV

27. Isoprenaline had no effect on the “open probability
(Po)”, measured during individual sweeps
◼ Although isoprenaline has
increased the chances that a
channel will open on membrane
depolarisation, once open, it
behaves as under “control“
conditions, with a
Po of around 0.3
◼ This effect is due to cAMP-
dependent kinase-mediated
phosphorylation of the channel.
Only phosphorylated channel can
open in response to membrane
depolarisation
open probability
control isoprenaline

28. Unlike isoprenaline, BAYK8644 had no effect on the
number of functional channels
◼ When the membrane was
depolarised, the number of
channels opening was the
same in the absence and
presence of BAYK8644
◼ In the example shown here,
under control conditions 3/10
sweeps were blank (arrowed)
in both the absence and
presence of the drug
control + BAYK8644

29. BAYK8644 massively increased the “open probability
(Po)”, measured during individual sweeps
◼ Although BAYK8644 didn’t increases
the chances of channel opening, once
the channel did open, it stayed open for
longer (i.e. increased Po)
◼ BAYK8644 puts the channel into a
“mode” of gating characterised by long-
duration openings
open probability
control BAYK8644

30. “Modal model” for dihydropyridine action on L-type
channel gating
◼ Mode 2 characterised by long
openings. Occurs rarely in drug-free
conditions but stabilised by DHP
agonists eg BAYK8644
◼ Mode 1 characterised by short
openings. Most common in drug-free
conditions
◼ Mode 0 characterised by no openings
i.e. channel non functional. Stabilised
by DHP antagonists eg nifedipine

31. And finally
◼ Test your understanding of the
concepts discussed in this lecture by
reading the articles below concerning
ivabradine – a drug licensed for the
treatment of angina – that blocks HCN
channels (also called the “funny
channel”) in the cardiac atria.
 Bucchi et al (2007) Heart rate reduction via
selective “funny” channel blockers”.
Current Opinion in Pharmacology, 7, 208-
213
 Postea O.& Biel M. (2011) Exploring HCN
channels as novel drug targets. Nature
Reviews Drug Discovery 10 (12), 903-914

32. Learning outcomes
By the end of this lecture you should be able to
◼ Describe what is meant by the terms “open channel” and “foot in the door” block and
illustrate these mechanisms by reference to selected drugs
◼ Understand what is meant by activation and inactivation curves and the information that
can be gained from them
◼ Compare and contrast the actions of isoprenaline and BAYK8644 on L-type voltage-
dependent calcium channels
◼ Apply the ideas discussed in the lecture to other drugs for example the antiarrythmic drug
ivabradine
Note; the terms “voltage-operated”, “voltage-dependent”, “voltage-gated” are used interchangeably to
mean channels that are opened (gated) by changes in membrane potential