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After tyrosine kinases and G protein-coupled receptors, voltage-gated ion channels make up the third largest group of receptors encoded by the human genome. With 40 members, voltage-gated K+ channels (KV) have various structures and their functions include repolarization of neuronal and cardiac action potentials and regulation of Ca2+ signalling and cell volume.
One of the most important members of this group is the hERG (human ether-a-go-go-related gene) channel which is vital for the normal functioning of the electrical activity of the heart. Its significance was first made obvious when inherited mutations in HERG were linked to long QT syndrome (LQTS). A prolonged QT interval can result in ventricular tachyarrhythmia and sometimes in the life threatening form torsade de pointes (TdP).
Before this discovery was made, a similar disorder was known that was caused by high doses of the hERG-channel blockers quinidine and dofetilide. Ironically, these medications were at the time used to prevent arrhythmia. Arrhythmia and sudden death via block of the hERG channel can, however, be triggered by common non-cardiac drugs such as some antihistamines and antibiotics. These therapeutic compounds were all, thus, withdrawn and it is now compulsory in the pharmaceutical industry to test therapeutics for any hERG -channel activity in the primary stages of drug development.
Structure of the hERG channel
A major part of our current knowledge on the structure of hERG channels was gained through detailed biophysical studies of the Drosophila Shaker Kv channel (Kv1.1). Our understanding of the structure and the mechanism of channel gating has now been further enhanced by the X-ray crystal structures of some bacterial K+ channels (KcsA, MthK and KvAP) and a mammalian channel (Kv1.2) (MacKinnon et al).
Kv channels are made up of four identical α-subunits, consisting of six (S1-S6) α-helical transmembrane domains. Each subunit comprises two functionally distinct segments, one that forms the K+-selective pore and one that senses the transmembrane potential. The pore is asymmetric and its dimensions are altered when the channel changes from a closed to an open conformation. The extracellular end of the pore forms the K+-selectivity filter, a narrow cylinder that is optimally structured for the conduction of K+. This filter is the primary hallmark of all K+ channels and is defined by the K+ signature sequence, the highly conserved sequence Thr-Val-Gly-Tyr-Gly.
Under the selectivity filter, the pore widens into a water-filled region. This region is termed the central cavity and is lined by the S6 α-helices. In the closed conformation, the four S6 domains criss-cross near the cytoplasm to form a narrow hole that is too small to allow entry of ions from the cytoplasm. This restrain is assuaged in the open conformation, via a kink and swivel at a highly conserved glycine residue central to the S6 helix, which expands the opening and enables the passage of ions and small molecules.
Furthermore, hERG contains a PAS (Per-Arnt-Sim) domain that slows channel deactivation, a 'turret' which is unusually long and contains a helix that can potentially interact with the outer mouth of the selectivity filter and a cyclic-nucleotide-binding domain that may form a tetramer that hangs below the pore domain (Figure 1).
Drug-induced block of hERG channels
The cardiotoxicity of common drugs that prolong the QT interval and cause TdP is extensive. For example, drug-induced TdP by quinidine, an antiarrhythmic drug, is a relatively common side effect which affects 2-9% of treated patients. Induction of TdP by medications other than antiarrhythmic agents, however, is rare. Cisapride-induced TdP, for instance, only occurs in 1 out of 120,000 patients prescribed the medication. Nevertheless, this risk is unacceptable, especially for medications such as terfenadine and cisapride which are used in the treatment of non-life-threatening gastrointestinal disorders and allergies.
Figure 1. Structure of a single subunit of hERG. The main features of the hERG subunit are indicated, including the S4 domain which contains basic (+) amino acids, the acidic Asp residues (-) in S1-S3 that can form salt bridges with specific basic residues in S4 during gating, the voltage sensor domain and the pore domain. Also shown are the locations of the PAS domain and the C-terminal cyclic-nucleotide-binding domain (cNBD).
LQTS caused by direct block of hERG channels
The most common pharmacological explanation to the cardiotoxicity of drugs that act on the hERG channel is direct blockage of the specific delayed rectifier current, IKr, generated by expression of HERG gene. The vulnerability of hERG to block has been attributed to its unique structure. First, the channel's promiscuous high-affinity drug-binding site is found in the central cavity of the protein, which traps hydrated K+ on their passage to the selectivity filter once the cytoplasmic gate is opened. Therefore, most blockers can bind to the channel only after they reach the central cavity, which is larger than in other K+ channels and can thus trap molecules after closure of the gate.
Second, the promiscuity of hERG blockers depends on the interactions between a positively charged tertiary amine found in most blocking drugs and the π-electrons of Tyr652 of the S6 domain that project into the cavity. The interaction between the therapeutic compound and the channel is further stabilized by strong, non-polar interactions between rigid, planar aromatic residues present in drugs and the π-electrons of Phe656, again located in S6 (Sanguinetti and Mitcheson).
Drug-induced LQTS by inhibition of hERG channel trafficking
A novel mechanism for LQTS that usually goes undetected in most conventional safety studies has also been described and at least three therapeutic compounds have been shown to reduce hERG /IKr currents not by direct block but by inhibition of hERG /IKr trafficking to the cell membrane.
Mutations in inherited LQTS2 produce trafficking-deficient hERG channels that are retained in the endoplasmic reticulum. Many of these mutant channels adopt a conformation that reaches the cell surface membrane and form functional channels on incubation with pharmacological chaperones. It is necessary to gain a better understanding of the mechanisms controlling hERG trafficking to the cell surface membrane as this type of 'channel rescue' can provide a therapeutic window. However, only few proteins of the complete hERG trafficking pathway have been recognized.
Of these proteins, Hsp90 is probably best characterized. The chaperone Hsp90 is associated with the late folding steps and catalyses conformational transitions close to the protein's native conformation. Consequently, inhibition of Hsp90 prevents maturation of hERG channels in the endoplasmic reticulum and reduces hERG /IKr currents at the cell membrane. A novel inhibitor of Hsp90 that has been identified is celastrol, which is also known to reduce hERG maturation and cell surface expression via an interaction site on Hsp90 which is different from the ATP-binding pocket used by the classical inhibitors radicicol and geldanamycin.
In As2O3- induced cardiotoxicity, As2O3 produces its effects indirectly, by reducing surface expression of hERG /IKr channels. As2O3 interferes with hERG trafficking by inhibiting the formation of channel- Hsp90 complexes. Arsenic is a metalloid and is, therefore, an unusual drug with broad activity against many cellular targets due to its high affinity for thiol groups and its tendency to cause oxidative stress in various cell types.
A more typical small organic molecule is pentamidine. This hERG trafficking inhibitor is used as second line treatment of Pneumocystis carinii pneumonia, a common opportunistic infection in patients with weak immune systems. Pentamidine disrupts hERG trafficking and reduces hERG currents in cardiac myocytes, resulting in delayed cardiac repolarization. Furthermore, probucol, a cholesterol-lowering drug can also reduce hERG /IKr currents and prolong the cardiac action potential via inhibition of hERG trafficking.
Finally, fluoxetine has also been described as an hERG trafficking inhibitor. Fluoxetine, commonly known as Prozac is a generally prescribed antidepressant which inhibits serotonin re-uptake in the brain. This drug was believed to directly block hERG at overdosing concentrations. It is now clear that fluoxetine causes LQTS via a dual mechanism of both direct channel block as well as inhibition of hERG channel trafficking (January et al).
Although, the hERG channel has received vast attention since it was first discovered in 1994, many issues regarding its pharmacology in relation to cardiotoxicity remain unresolved. The central residues of the binding site seem to have been identified but undoubtedly not all pharmaceuticals interact with the channel in the same way. Further studies are necessary in order to explain why hERG in particular can be blocked by such a variety of drugs. Irrespective of the mechanisms responsible for this promiscuity, a combination of ligand-binding models and structure-activity relationship studies can truly assess the binding affinity of newly developed drugs.