‘Can inherited genetic diseases tell us anything about the structure and function of ion

channels?’

Introduction
An inherited genetic disease is any disease caused by an abnormality in an individual’s
genome. When this abnormality occurs in the genes encoding ion channels, the resulting
disease is termed a channelopathy. These channelopathies disrupt the function of ion
channels; to allow the passive flow of ions in and out of cells, following their
electrochemical gradients. This flux generates a membrane potential, mediating a myriad of
functions including muscle contraction, hormone secretion and signal transduction. Through
analysing how channelopathies work, one can glean an insight into both the structure and
function of ion channels.
For example, the fact that channelopathies exist as inherited genetic diseases indicate that
ion channels are proteins, as the genome exclusively encodes protein. Furthermore,
channelopathies provide evidence for the multi-subunit nature of most ion channels
through the dominant negative effect, wherein recessive, loss of function mutations can be
inherited in a dominant pattern; as one ‘mutant’ subunit is sufficient to ‘kill’ the channel.
Thus, via binomial distribution, only 1/16th of a 4 subunit channel will have no mutant
subunit ergo will function correctly. If ion channels were not multi-subunit, this inheritance
pattern would be impossible.

Ion channel function

Channelopathies can either be loss or gain of function, providing an insight into the specific
functions of various ion channels in a manner akin to knock out and knock in experiments.
This has proven especially pertinent within sodium and potassium channels within the
context of their function in driving action potentials, as illustrated diagrammatically below.

1. Na+ in-Opening of voltage gated

sodium channels leads to
depolarisation.
2. K+ out-Closure of voltage gated
sodium channels and opening of
voltage gated potassium channels
drives repolarisation.
3. Inwardly rectifying potassium
channels maintain the resting
membrane potential.

Evidence for the role of both sodium and potassium currents in driving and curtailing the
action potential respectively can be found within long QT syndrome, where ‘QT’ refers to
the QT interval and the approximate length of the action potential. This can be caused by
either loss of function in the repolarising potassium channels, for example due to a
mutation in KCNQ1 leading to non-functional cardiac Kv7.1, or gain of function in
depolarising sodium channels, for example a mutation in SCN5A leading to slower
inactivation ergo greater Na current. This condition is potentially fatal, reflected in its
previous nomenclature as sudden death syndrome, as if heart rate increases the pacemaker
can attempt to fire an action potential before the previous has been completed, leading to
early after depolarisation, arrhythmia and heart attack.
One can also use channelopathies to gain an insight into the function of specific ion channel
classes, for example in Anderson Tawil syndrome, a loss of function mutation in KCNJ2
encoding Kir2.1; an inward rectifying potassium channel mediating regulation of the resting
membrane potential without short circuiting the action potential through voltage
dependent block by intracellular magnesium and polyamines. Without this, suffers are
unable to maintain the resting membrane potential, leading to cardiac arrhythmias and
periodic paralysis. This is a contrast to Episodic ataxia type 1, where mutations in the KCNA1
gene encoding voltage gated potassium channel Kv1.1 (expressed in the synaptic terminals
and dendrites of many brain neurons) lead to a prolonged action potential and repetitive
neuronal firing, excessive transmitter release and clinical symptoms of ataxia and
myokymia. Thus, the issue is not with maintaining the RMP, reflecting the different role of
Kv1.1 and Kir2.1 channels. This disease also acts as an example of the dominant negative
effect in multimeric subunits, with the majority of sufferers being heterozygous. This ties in
with the fact that Kv channels are composed of four alpha subunits, with the severe clinical
phenotype reflecting an ability to heteropolymerise with Kv1.2 and 1.5.
Of course, the function of an ion channel physiologically is defined by the tissue it is
expressed in, and channelopathies yet again provide abundant evidence for this. For
example, loss of function mutations in SCN9A, encoding Nav1.7 manifests as congenital
insensitivity to pain; as these channels are expressed at high levels in nociceptive neurons in
the dorsal root ganglion, preventing the formation and propagation of action potentials
carrying nociceptive information. These patients, however, often also suffer from anosmia,
illustrating the point that the function of ion channels is not only defined by the ions they
are permeable to, but the tissue that they are expressed in, with loss of Nav1.7 in olfactory
sensory neurons inhibiting sense of smell as opposed to pain. This is corroborated in
conditional null mice, which no longer display odour guided behaviours such as innate
odour recognition and avoidance (Weiss, 2011). Mice provide an excellent animal model for
olfaction; a highly evolutionarily conserved sensation regarding its mechanism due to its
reliance on fairly simple chemo-sensing.

The structures behind ion channel function

Much of what we have learnt of ion channel structure has been derived from experimental
techniques such as cryo-electron microscopy and XFEL. However, channelopathies can
provide a more dynamic insight into channel structure; for example in their gating
mechanics which regulate the transition of closed, to open to inactivated to closed
underlying channel function.

Gating: activation
A key component of the structure and function of ion channels is the gating conditions
permitting activation. This is commonly in response to voltage, with voltage gated ion
channels responsible for initiating the inward sodium current and outward potassium which
drive the action potential. Channelopathies have provided significant insight into this
process, both in terms of how it is achieved structurally and how this translates into
function. This is illustrated by Hypokalaemic periodic paralysis (HypoPP), characterised by
muscle paralysis when blood potassium is low, with the first HypoPP mutations identified in
the α subunit of the skeletal muscle L-type Ca2+ channel CaV1.1, in the arginines of the S4
segment of domain 4 (Jurkat-Roth et al 1994). This pointed at a role of the S4 residue in
channel function, a role which when compromised leads to slower activation, reduced
current density, reduced excitation and paralysis. This suggests involvement of the S4
domain in gating, confirmed by experiments mutating the individual residues of the domain
to check if it influences the voltage dependence of activation. Such studies have shown that
a reduction in the net positive charge in the S4 segment, produced by replacing positive
charges with neutral, leads to a decrease in the steepness of the voltage-dependence of
activation (Stuhmeret al,1989).
This is supported by the fact that the S4 segment is found in most voltage-sensitive cation-
selective channels so far cloned, and that its primary sequence is highly conserved, as well
as the discovery of several HypoPP causing mutations in Nav1.4, all within the S4 region.
Furthermore, the striking homology of their clinical phenotypes suggests a common
functional defect, specific to substitutions at arginine’s in S4 segments, leading to an influx
of cations and the depolarising nature of an attack of hypoPP. This provides insight into how
the S4 domain behaves structurally; with it thought that this segment moves physically
through the membrane; translocating through a narrow crevasse or ‘gating pore’. If the
arginine residues within S4 are mutated, ions (hydrogen in the case of Nav1.4 hypoPP) are
conducted through the pore. How these gating pore currents actually manifest clinically is a
matter of debate, with heterologous expression of CaV1.1 in non-muscle cells still quite
poor; impeding the ability to test with sufficient sensitivity for gating pore currents from
HypoPP mutations in the calcium channel. However, model simulations demonstrate that
gating pore currents, although small in amplitude, are sufficient to cause paradoxical
depolarization in the setting of mild hypokalaemia, providing a link between genotype and
phenotype (Carmeliet, 1982).

Gating: inactivation
Equally important to ion channel function is their ability to inactivate, with both sodium and
potassium channels displaying inactivation.
The structures behind inactivation are illustrated by specific mutations causing potassium-
aggravated myotonia (PAM) and hyperkalemic periodic paralysis (HyperPP), wherein slower
inactivation leads to sustained depolarisation and paralysis, as sodium channels are
inactivated and action potentials cannot be generated, or myotonia, wherein sustained
depolarisation leads to increased contraction and muscle tension.
PAM provides an insight into the role of the linker between repeats 3 and 4, the inactivation
ball, with single channel analysis of the G1306E mutation revealing that the mutant
channels open more frequently and for longer durations, corroborated by the fact that the
severity of myotonia in patients with mutations in G1306 is correlated with the degree to
which inactivation is slowed (Mitrovicet al.,1995). This has led to the postulation that this
region of the channel forms a latch mechanism with the IFM triplet, which is destabilised
allowing it to reopen and slowing inactivation. This role of the linker between repeats 3 and
4 as an inactivation gate was confirmed by Vassilev and colleagues (1988), whom found that
antibodies directed against the region between repeats III and IV of the Na1 channel
slowed inactivation, whereas antibodies directed at other intracellular regions had no
effect.
Hyperkalemic periodic paralysis provides an insight into the role of S5 and S6 as an acceptor
site for the inactivation ball, with mutations occurring in said site generating a small
persistent current as a result of transition into a state where the channel continues to open
and close instead of inactivating.
The location of S5 and S6 on the inner mouth of the sodium pore suggests a mechanism
wherein the ‘inactivation particle’ moves into the inner mouth of the pore where it blocks
ion flux. This is reinforced by the fact that intracellular perfusion of squid axon with the
proteolytic enzyme pronase removed inactivation (Armstronget al.,1973), indicating that
the inactivation gate was on the inside of the membrane.
The distinct phenotype of HyperPP is also thought to be a result of pathology in slow
inactivation again implicating S5 and S6, potentially through a conformational change in the
inner mouth of the sodium channel pore.
Mutations in the S4 domain also appear to impact inactivation causing a dramatic slowing of
the rate of fast inactivation and a reduction in its apparent voltage dependence. The S4
mutations are believed to slow inactivation by reducing the degree of coupling between
activation and inactivation, illustrating its importance in coupling activation to fast
inactivation physiologically.

Beyond the action potential

Functionally, the role of ion channels has been somewhat restricted in this essay to the
action potential. It is important to note that physiologically this is not the case, exemplified
by voltage gated calcium channels which can mediate an incredibly diverse range of
mechanisms through ubiquitous use of calcium as an intracellular messenger. This diversity
in function is hinted at by calcium channelopathies such as timothy syndrome, where a
mutation in CACNA1C, coding for Cav1.2, causes a multisystem disorder including lethal
arrhythmias, webbing of fingers and toes, congenital heart disease, immune deficiency etc.
Beyond the fact that these calcium channels are ubiquitous and diverse in function, these
channelopathies are less immediately useful in informing on ion channel structure and
function, with the link between genotype and phenotype less easily decipherable than their
sodium and potassium counterparts.

Conclusion
To conclude, inherited diseases in the form of channelopathies can tell us a great deal on
the function of the ion channels they effect, on both a molecular and physiological level.
Channelopathies can also be used on a codon level to resolve the functions of individual
domains of ion channels; from which potential structures can then be theorised.