1. What are channelopathies?

In order to understand channelopathies, it is necessary to review the physiology of ion channels. They constitute a class of proteins that generates and orchestrates the myriad of electrical signals that pass through the brain, heart and muscle each second throughout life. Ion channels are classified according to the type of ion they allow to pass - sodium, potassium, calcium or chloride. They are gated by extracellular ligands, changes in transmembrane voltage or intracellular second messengers.

Ion channels are macromolecular protein tunnels that span the cell membrane. The direction of ionic movement is governed by electrical and chemical concentration gradients.

Most ion channel proteins are composed of individual subunits or groups of subunits assembled around a central ion-selective pore. Each subunit contains four domains (l, ll, lll and lV), which, in turn, each contain six helical transmembrane units (S1 – S6). Sodium and calcium channels have their domains covalently linked. Potassium channels are also tetrameric but their domains are not covalently linked. Alpha subunits appear to be the most important of the subunits; the functions of the smaller channel subunits (beta, gamma and delta) are less well understood but it is accepted that they appear to regulate some ion channels. Segments S5 & S6, together with their connecting peptide chain, line the channel pore through which the ions pass. S4 is believed to shift within the membrane in response to changing membrane potential. Membrane depolarisation causes a conformational change of the protein, which leads to opening of the pore. This allows movement of ions through the channel. Changes in the amino acid sequence of the protein surrounding the pore will alter the permeation properties of the channel.

2. Evolution of channel structure

The regulation of function in an organism can be investigated at three levels:

• gene expression
• cell biochemistry
• organ physiology

These mechanisms of regulation appeared sequentially during evolution. The most recent is organ physiology since it could only operate after the appearance of complex multicellular animals (about 600 million years ago.) More than 1400 million years ago regulatory mechanisms evolved that were effected by changing cell biochemistry. The eukaryotes developed elaborate membrane systems to control their intracellular composition. The oldest of the regulatory mechanisms involves the synthesis of altered gene products, something that probably appeared soon after the dawn of life (about 3500 million years ago.)

A concrete example of these three levels of regulation would be the electrical activation of the heart (organ physiology), which is brought about by membrane currents that are regulated by conformational changes in the channel proteins (cell biochemistry). The electrical properties of the membrane can be modified when altered gene products are incorporated into the protein structures that make up these channels (gene expression).

The primitive ancestor of ion channels as we know them today was probably a much simpler molecule similar to a single domain. Early on in the evolution of eukaryotes this gene is thought to have undergone duplication and divergence to give rise to two classes of ion channels. The one class includes noncovalently linked domains of the potassium channel and the other includes the tetrameric calcium channels in which the constituents remain covalently linked. The structure of the calcium channel later underwent further divergence, giving rise to sodium channels, which generate large, rapidly depolarizing action potentials that conduct faster than most calcium-dependent action potentials.

Potassium channels

Potassium channels are considered to be the most primitive of the voltage dependent ion channels since they resemble the ancestral channel more closely than do the other ion channels. In the heart, for example, there is a vast array of different types of potassium channel. Mutations are responsible for certain types of LQTS.

Sodium channel

-subunits

Humans have at least ten genes that encode a-subunits of voltage-gated sodium channels. Most of these genes are expressed in excitable tissues e.g. brain, peripheral nerve and skeletal muscle. Hyperkalaemic periodic paralysis, paramyotonia congenita, potassium aggravated myotonia and long QT syndrome (Type lll) are all associated with defective sodium channels.

Calcium channels

Eight different genes code for six calcium channels (Types T, L, B, N, P & R). L-type channels are sensitive to dihydropyridines (DHP) e.g. nifedipine, phenylalkylamines e.g. verapamil and benzothiazepines e.g. diltiazem which has led to the misnomer dihydropyridine receptor . The term suggests ligand activation, when, in fact, the channel is voltage activated. Another type of calcium channel of interest is the ryanodine receptor (RYR) so called because of its sensitivity to the plant alkaloid, ryanodine. This channel mediates calcium release from the sarcoplasmic reticulum (SR) or the endoplasmic reticulum (ER), an essential step in the contraction of skeletal (RYR1), cardiac and smooth (RYR2) muscle.

DHP receptor and RYR1 are closely associated during skeletal muscle contraction. DHP receptors are situated within the transverse tubular membrane in the muscle fibre. During depolarisation, activation of the L-channel leads to a conformational change of certain intracellular loops, which subsequently open RYR1, allowing calcium to pass into the cytoplasm from the SR. This signal transmission between the t-tubular and SR membranes is referred to as excitation-contraction coupling. RYR1 mutations cause susceptibility to malignant hyperthermia and central core disease.

Chloride channels

These channels are present in the plasma membrane of most cells involved in cell volume regulation, transepithelial transport, secretion of fluid from secretory glands and stabilization of membrane potential.

Chloride channels have been classified into 3 large superfamilies:

1. GABAA and glycine receptor channels mediate chloride flux within the nervous system.
2. The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the second superfamily.
3. Voltage-gated chloride channels in excitable and epithelial cells constitute the third family.

3. Heritable diseases associated with ion channel mutations

The following table lists some well-defined disorders associated with defects in ion channels.

Ion channel diseases
Syndrome	Inheritance R=recessive D=dominant XL = X-linked A=autosomal
Myotonia congenita (Thomsen's disease)	AD
Periodic paralyses (hyper- & hypokalaemic)	AD
Malignant hyperthermia	AD
Long QT syndrome	AD / AR
Cystic fibrosis	AR
Heritable hypertension (Liddle's syndrome)	AR
Familial persistent hyperinsulinaemic hypoglycaemia of infancy	AR
Generalised myotonia (Becker's disease)	AR
Hereditary nephrolithiasis (Dent's disease)	XL
Masseter muscle rigidity	?
Central core disease	?

Cystic fibrosis (CF)

Manifestations of CF stem from a defect in the chloride-channel protein (CFTR) that prevents chloride crossing the cell membrane. Chloride channels situated at the apex of epithelial cells hinder the egress of chloride ions into the lumen. Control of sodium channels is also lost, increasing the reabsorption of sodium from the lumen. The result is thick, desiccated mucus - a primary clinical characteristic of the disease.

1 in 2500 – 3000 white persons is born with CF, and more than 450 mutations have been identified in CFTR. A deletion of phenylalanine at position 508 (DeltaF508) accounts for more than 70% of cases and is associated with severe pancreatic insufficiency and pulmonary disease. DF508 CFTR channel conducts chloride well once it is incorporated into a cell membrane. However, the mutant protein becomes stuck in intracellular organelles due to improper folding and is not inserted into the cell membrane.

Potential molecular strategies to treat CF include replacing the mutant chloride channel by gene therapy or protein delivery; improving secretion from the mutant CFTR protein with CFTR-channel openers; "chaperonins" which allow for proper incorporation of CFTR into the cell membrane; bypassing CFTR defect by activating other chloride channels; and blocking increased sodium reabsorption through sodium channels using aerosolised amiloride. Only the last therapy is currently in common clinical use.

Long QT syndrome

Disorder of cardiac repolarisation characterised by QT interval prolongation & T wave abnormalities that results in ventricular arrhythmias with syncope and sudden death in children and young adults. The exact aetiology of the disease was unclear for a long time but in the early 1990's researchers demonstrated convincingly that the disease is inherited. It was subsequently determined to be genetically heterogeneous, with six different genes being implicated. Further work elucidated the responsible mutations and showed that defects in particular ion channels lead to the long QT syndrome.

LQTS1 is linked to chromosome 11. Chromosomes 7 and 3 are implicated in LQTS2 and LQTS3 respectively. LQTS1 and LQTS2 are due to a defect in the cardiac potassium channel gene whereas a defective sodium channel gene causes LQTS3.

The gene linked to chromosome 7 has been labelled as the human ether-a-go-go-related gene (HERG). This is a potassium channel gene related to the drosophila ether-a-go-go (eag) gene, from which it was cloned. The eag locus is involved in the control of potassium currents in the membranes of drosophila nerve & muscle. Mutants in this locus display ether-induced leg shaking, hence the name “ether-a-go-go”. Mutations in HERG are responsible for LQTS2. In normal cardiac physiology HERG suppresses depolarisations that lead to premature firing. Subjects with LQTS2 lack protection from arrhythmogenic afterbeats and may be prone to sudden cardiac death.

Drugs which cause LQTS do so by blocking the potassium channels that are involved in LQTS1 and LQTS2. Examples of these drugs are:

• Quinidine
• Sotalol
• Erythromycin
• Trimethoprim & sulfamethoxazole
• Pentamidine
• Adrenaline
• Terfenidine
• Astemizole
• Diphenhydramine
• Cisapride
• Ketoconazole
• Fluconazole
• Amitryptiline
• Haloperidol

Cardiac repolarisation (T wave) occurs as a result of ion currents controlled by potassium channels. In LQTS there is a delay in the opening of channels, which prolongs the action potential, causing a prolonged QT interval. These patients have a propensity to torsade de pointes and ventricular tachycardia.

The inherited LQTS presents in one of 2 forms:

1. Jervell, Lange-Nielsen variant (rare) associated with congenital deafness.
2. Romano-Ward variant (more common; AR) with normal hearing.

Myotonic diseases

These are a group of clinically similar diseases that share the feature of myotonia: delayed muscle relaxation after voluntary contraction or mechanical stimulation. e.g. hyperkalaemic periodic paralysis (HKPP), paramyotonia congenita, myotonia congenita and myotonic dystrophy.

HKPP

Transient episodes of paralysis; AD; attacks of paralysis are frequent, brief and precipitated by rest after exercise, stress and ingestion of certain foods and potassium. Due to defective sodium channels.

Paramyotonia congenita

Cold-induced, there is prolonged, localised myotonia and weakness. Muscle activity aggravates the myotonia associated with paramyotonia congenita (paradoxical myotonia), in contrast to the improvement of symptoms seen with exercise in most patients with myotonic disorders (classic myotonia). Due to defective sodium channels.

Myotonia congenita

In this disorder muscle stiffness resolves with exercise. The autosomal dominant variant is "Thomsen's disease". The continual 'isometric exercise' that these individuals undergo gives them the habitus of a 'circus strongman', at least in their earlier years. The defect is in a chloride channel, as is the case with the autosomal recessive form, termed Becker's disease.

Malignant hypermetabolic syndrome ('Malignant hyperthermia', MH)

This is not a disease in the strict sense of the word, but a genetic predisposition of clinically inconspicuous individuals to respond abnormally when exposed to volatile anaesthetics or depolarizing muscle relaxants. It is a drug-induced, potentially lethal event in carriers of calcium channel mutations. A pathologically large increase in myoplasmic Ca++ concentration following exposure to triggering agents is seen in these patients. This causes an increase muscle metabolism and heat production resulting in symptoms of muscle rigidity, hyperthermia, metabolic acidosis, hyperkalaemia & hypoxia. Dantrolene, an inhibitor of calcium release from the SR is used to abort the crisis.

MH susceptibility in humans is genetically heterogeneous. The gene encoding the skeletal muscle ryanodine receptor (a calcium channel) is the villain in some families. To date, more than 20 disease-causing point mutations have been identified in humans. 2 mutations in the dihydropyridine receptor have also been described.

The malignant hypermetabolic syndrome is discussed in detail elsewhere on this website .

Central core disease (CCD)

This is allelic to MH. It is a congenital AD proximal myopathy with structural alterations of certain muscle fibre types. These patients are hypotonic at birth but muscle strength usually improves later in life. In spite of the fact that events similar to MH may occur in numerous muscle disorders during general anaesthesia, a genetic relation exists with certainty only in CCD and possibly in King-Denborough syndrome.

4. Targeting ion channels

There is a vast array of drugs that influence ion channel function:

• Calcium channel blockers
• Potassium channel blockers (oral hypoglycaemics)
• Diuretics (amiloride) - sodium channels
• Anticonvulsants (clonazepam; phenobarbitone) - chloride channels
• Anticonvulsants (carbamazepine; phenytoin; valproate) - sodium channels
• Antiarrhythmic drugs (amiodarone) - potassium channels
• Class l antiarrhythmics - sodium channels
• Local anaesthetics (lignocaine; bupivacaine) - sodium channels
• Antihypertensive drugs (diazoxide) - potassium channels
• Adenosine - potassium channels
• Benzodiazepine - chloride channels

An Example - Insulin secretion

ATP-sensitive potassium channels have been identified in the heart, skeletal muscle, brain, smooth muscle and pancreas. In the pancreas these channels play a major role in glucose homeostasis and regulation of insulin secretion. Rising plasma glucose concentration will increase intracellular concentration of ATP in islet beta cells, which in turn inhibit ATP-sensitive potassium channels. As these potassium channels close there is depolarisation of the cell membrane to a level where voltage-dependent calcium channels are activated. The resulting influx of calcium triggers insulin secretion. The converse occurs as the blood glucose concentration drops in response to insulin secretion - ATP levels drop, the cell membrane hyperpolarizes, and insulin secretion is terminated. Oral hypoglycaemic agents such as sulphonylureas bind to a specific receptor on the beta cell membrane that inhibits the activity of ATP-sensitive potassium channels, depolarising the membrane and promoting insulin secretion. Similarly, diazoxide is able to open potassium channels in vascular smooth muscle. Cell membranes are hyperpolarized and calcium channel activity is diminished. The result is a decrease in vascular tone. Small wonder that diazoxide may interfere with insulin secretion!

References

1. Marban E et al. J Physiol 1998 508.3 647-57.
2. Merck Manual {edition not stated} Section 14 Chapter 184.
3. Lehman-Horn F, Jurkat-Rott K Physiol Rev 79.4 October 1999 1317-56
4. Vincent GM Annual Rev Med 1998 49 263-74
5. Benatar MG Q J Med 1999 92 133-41
6. Ackerman MJ, Clapham DE New Engl J Med 1997 336 1575-86
7. Towbin JA New Engl J Med 1995 333 384-5
8. Katz A New Engl J Med 1993 328 1244-51
9. Ptacek LJ et al. New Engl J Med 1993 328 482-9.