FARAZA JAVEDMPHIL PHARMACOLOGY

Ion Channels

Ion Channels

Ion channels are pore-forming membrane proteins whose function is establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane, controlling the flow of ions across membranes, and regulating cell volume.

They are often described as narrow, water-filled tunnels that accept only specific type of ions. This characteristic is called selective permeability.

Ion channels are integral membrane proteins, formed as assemblies of several proteins. Such "multi-subunit" assemblies usually make a circular arrangement of identical or homologous proteins closely packed around a water-filled pore through the plane of the lipid bilayer membrane.

Ion channels are different from other transporter proteins:

The rate of ion transport through the channel is very high (often 106 ions per second or above).

Ions pass through channels down their electrochemical gradient, which is a function of ion concentration and membrane potential, "downhill", without the input of metabolic energy (e.g. Adenosine triphosphate, active transport mechanisms, co-transport mechanisms).

History

By the late 1800s,  the chemical mechanism underlying  nerve and muscle tissue messaging was a mystery. 

Ludimar Hermann was able to conclude that nerve and muscle cells were capable of exhibiting a "self-propagating wave of negative charge which advances in steps along the tissue ". 

Julius Bernstein made the first real theoretical contribution, for he  postulated the ionic theory, the Nernst equation, and the assumption of a semi-permeable membrane surrounding nerve and muscle cells could all help explain the mounting electrophysiological evidence of the previous statement.

Sidney Ringer used a solution of water and ran it through the vessels of an isolated heart from a frog in the 1880s and discovered that in order for the heart to continue beating salts needed to be present in the water.  

Specifically, sodium, calcium, and potassium salts were needed and they had to be in special concentrations relative to each other.   

In 1937, John Z. Young use squid neuron to study ion current.

He made important experiments possible for the first time, including the first intracellular recordings of the nerve cell action potential.

The next improvement in instrumentation took place in the late 1940s by Kenneth Cole.   This involved placing a glass electrode inside the cell in order to "voltage clamp" the interior of the cell.

Voltage clamping made it possible to distinguish the voltage effects caused by influx of sodium or efflux of potassium.

Later in the 1960s and 70s, many other small molecules and peptides would be discovered to help gate these channels, including glutamate, GABA, glycine, serotonin, dopamine, and norepinephrine.

In 1970s, the existence of ion channels was confirmed by the invention of ‘patch clamp’ technique by Erwin Neher and Bert Sakmann who won a Nobel Prize for it.

In 2003, the Nobel Prize was awarded to American scientists, Roderick MacKinon and Peter Agre for their x-ray crystallographic structure studies on ion channels.

Since the discovery of ion channels, the etiology of many diseases has been traced back to channelopathies. Many toxins produced by snakes, fish, spiders and other insects paralyze the ion channels. Many physiological mechanisms have been studied at molecular level and have confirmed the involvement of ion channels. The ion channels are the recent target sites for pharmaceutical biosynthesis of new drugs.

Types of Ion Channels

2 major types:

Voltage gated ion channelsLigand gated ion channels

Voltage Gated Ion Channels

Structure and Function

Voltage-dependent channels are made of three basic parts:Voltage sensorThe pore or conducting pathway andSelectivity filter

Voltage gated ion channels consist of a highly processed

α subunit, associated with auxiliary β subunits.

The pore-forming α subunit is sufficient for functional expression, but the kinetics and voltage dependence of channel gating are modified by the β subunits.

The α subunits are organized in four homologous domains (I-IV)  each with six transmembrane segments (S1-S6) - 24 transmembrane segments in total. The pore forming segments are formed by S5 and S6.

Each of these segments coils is called a transmembrane domain, and within a transmembrane domain the side chains necessarily face outward where they readily interact with the lipids of the membrane are known as

Polypeptide chain.

For clarity in the figure, the alpha-helices are shown spread out and in a row.

In an actual membrane, thealpha-helices are not in a line, but clustered. This is shown in top view in the figure. At the center of the four domains is the channel through which the ions movement take place.

Voltage Gated

Channels

Domains

TM Segmen

ts

Sub-Units

Pore Forming Regions

Voltage Sensor

V.G Na+ 4 6 1 S5-S6 S4

V.G Ca2+ 4 6 4 S5-S6 S4

V.G Cl- 4 6 4 S5-S6 S4

V.G K+ 4 6 1 S5-S6 S4

Channel Structure

In this diagram, a single transmembrane domain is shown as the voltage sensor that operates the gate.

The S4 segment of voltage gated channel is the voltage sensor that is responsible for changing conformation as the voltage changes. All voltage gated channels have this S4 segment.

Changes in the membrane potential modulate the channel's opening or closing, as changing the membrane potential changes the relative amounts of positive and negative charges on the inside and

outside of the membrane. Like charges repel, so thepositively charged S4 segment will be pushed away from a positive intracellular fluid towards the negative extracellular fluid, changing the protein's conformation and opening the channel. 

At a typical resting membrane potential (for example, -70 mV) the channel is closed. Then should any factor depolarize the

membrane potential sufficiently (for example, to -50 mV), the voltage sensor moves outward and the gate opens. The channel can also close (deactivate) by negative voltages that

restore the down position of S4 and close the gate.

Types of V.G Ion Channels

4 major types:

V.G Sodium ChannelsV.G Calcium ChannelsV.G Potassium ChannelsV.G Chloride Channels

Voltage Gated Sodium Channels

The founding member of the ion channel superfamily in terms of its discovery as a protein is the voltage gated sodium channel. These channels are

responsible for the rapid influx of sodium ions that underlies the rising phase of the action potential in nerve, muscle, and endocrine cells.

Sodium channel composed of one principal alpha subunit and one or two auxiliary beta subunits.

The a subunits of sodium channels are composed of four homologous domains that each contain six transmembrane segments.

Different distinct neurotoxin binding sites have been identified within the Na channel protein, with different effects on ion permeation and gating resulting in either inhibition or enhancement of Na currents.

Channelopathies

Extensive research has been done and is continued on ion channels. Ion channels are a favorite site for invention of new drugs.

Moreover many genetic disorders are found to be caused by defective channel proteins. In addition to that, many toxins and venoms produced by spiders, snakes, scorpion, bees, fish, snails and others act by incapacitating ion channels.

Paramyotonia Congenita (PMC)

Paramyotonia Congenita (PMC) is one of the periodic paralyses caused by mutations in (alpha-1 subunit) the sodium channel. PMC causes muscle stiffness (myotonia) which is made worse by chilling or activity. 

Mechanism: In Paramyotonia the sodium channels fail to regulate the flow of ions properly.  At first this imbalance causes the muscle fiber to contract uncontrollably, but as the imbalance worsens the muscle stops responding to nerve signals and becomes weak or paralyzed.

This can clearly be seen in an EMG measurement of muscle from a PMC patient.

Treatment:  Tocainide is a new antiarrhythmic agentwhich seems to reduce effectively sodium conductance.

Brugada syndrome

The Brugada syndrome is a genetic disease that is characterized by abnormal electrocardiogram (ECG) findings and an increased risk of sudden cardiac death. It is the major cause of sudden death in young’s and termed as sudden unexplained/ adult death syndrome (SUDS) or (SADS).

Genetics: The cases of Brugada syndrome have been

shown to be associated with mutations (loss of function mutation) in the gene (named SCN5A of alpha subunit) that encodes for the sodium ion channel in the cell membranes of the muscle cells of the heart (the myocytes).

Treatment: The cause of death in Brugada syndrome is ventricular fibrillation. These arrhythmias appear with no warning. While there is no exact treatment modality, treatment lies in termination of this lethal arrhythmia before it causes death.

This is done via implantation of an implantable cardioverter-defibrillator(ICD), which continuously monitors the heart rhythm and will defibrillate an individual if ventricular fibrillation is noted.

Voltage Gated Calcium Channels

Voltage-gated calcium channels mediate calcium influx in response to membrane depolarization and regulate intracellular processes such as contraction, secretion, neurotransmission.

Like sodium channels, the α1 subunit of voltage gated calcium channels is organized in four homologous domains (I-IV), with six transmembrane segments (S1-S6) in each.

An intracellular β subunit and a transmembrane, disulfide-linked α2β subunit complex are components of most types of calcium channels.

A γ subunit has also been found in skeletal muscle calcium channels, and related subunits are expressed in heart and brain.

There are several different kinds of high-voltage-gated calcium channels (VGCCs).

N-type channel (Most often found throughout the brain  and peripheral nervous system).

P/Q-type channel (Purkinje neurons in the cerebellum)

L-type (Skeletal muscle, smooth muscle, bone (osteoblasts), ventricular myocytes (responsible for prolonged action potential in cardiac cell), dendrites and dendritic spines of cortical neurones).

L-type channels are responsible for excitation-contraction coupling of skeletal, smooth, and cardiac muscle and for hormone secretion in endocrine cells.

Malignant Hyperthermia

MH is a life-threatening clinical syndrome of hypermetabolism involving the skeletal muscle. It is triggered in susceptible individuals primarily by the volatile inhalational anesthetic agents (Halothane) and the muscle relaxant succinylcholine.

Genetics: The defect is typically located on chromosome

19 (involving the ryanodine receptor) located in the N-terminus of the protein, which interacts with L-type calcium channels. This region is important for allowing Ca2+ passage through the protein following opening.

Treatment: During an episode of malignant hyperthermia, wrapping the patient in a cooling blanket can help reduce fever and the risk of serious complications.

The current treatment of choice is the intravenous administration of Dantrolene, the only known antidote, and supportive therapy directed at correcting hyperthermia.

Migraine

Calcium channel blockers are effective second-line agents. They are a viable alternative in patients who cannot tolerate β-blockers.

Mechanism: Results of recent studies suggest that cerebral blood flow during the initial phase of migraine is decreased and this decrease probably leads to ischemia and hypoxia. Cellular hypoxia, in turn, can cause an increase in the flow of calcium, resulting in calcium overload and cellular dysfunction.

Nimodipine, a calcium-channel blocker that exhibits selective effects on cerebral vessels, seems to offer protection against the cerebral ischemia and hypoxia presumed to be operative during migraine attacks.

Analgesic Activity of CCB

Ziconotide (Prialt) is the synthetic form of the N-type Ca2+ channel blocker in the final stages of clinical development, a peptide toxin derived from a marine cone snail.

In humans, spinal infusion of Prialt produces significant pain relief in patients with intractable pain associated with cancer, AIDS and in some neuropathic pain conditions.

Voltage Gated Potassium Channels

Potassium channels are the most widely distributed type of ion channel and are found in virtually all living organisms.

The α1 subunit of voltage gated potassium channels is organized in four homologous domains (I-IV), with six transmembrane segments (S1-S6) in each.

The core of the channel consists of helices 5 & 6 & the intervening H5 segment of each of the 4 copies of the protein.

Mutation studies showed that the H5 segment is essential for K+ selectivity.

Potassium channels act to set or reset the resting potential in many cells. In excitable cells, such as neurons, the delayed counter flow of potassium ions shapes the action potential.

They also regulate cellular processes such as the secretion of hormones (e.g., insulin release from beta-cells in the pancreas) so their malfunction can lead to diseases.

Congenital Hyperinsulinism

Congenital hyperinsulinism is a condition that causes individuals to have abnormally high levels of insulin.

People with this condition have frequent episodes of low blood sugar (hypoglycemia). These conditions are present at birth but milder forms may not be detected until adult years.

Mechanism: In approximately half of people with congenital

hyperinsulinism, the cause is unknown. Mutations in genes that regulate the release (secretion) of insulin, which is produced by beta cells in the pancreas is the proposed cause.

Treatment: Diazoxide and octreotide are the primary

medications used in long-term treatment of CHI.

Multiple sclerosis

Multiple sclerosis (MS) is an inflammatory disease of CNS characterized by demyelination of axons. Uregualtion of V.G Potassium channels increase the autoimmune disease process of MS.

The idea that neurologic function might be improved if conduction could be restored in CNS demyelinated axons led to the testing of potassium channel blockers as a symptomatic treatment.

To date, only 2 broad-spectrum K+ channel blockers, 4-aminopyridine (4-AP) and 3,4-diaminopyridine (3,4-DAP), have been tested in MS patients. Although both 4-AP and 3,4-DAP produce clear neurologic benefits, their use has been limited due to toxicity.

Epilepsy

Instead of blocking excitatory ion channels, another potentially antiexcitable strategy is to enhance the activity of Kv channels. The past decade has seen increased interest in Kv channel opening as antiepileptic mechanism with focus on Kv channels. The leading compound in the pipeline is Retigabine which activates neuronal Kv channels.

Retigabine has successfully completed Phase III trial and is presently awaiting approval as an antiepileptic.

Voltage Gated Chloride Channels

Chloride channels are a superfamily of poorly understood ion channels.

CLC is involved in setting and restoring the resting membrane potential of skeletal muscle, while other channels play important parts in solute concentration mechanisms in the kidney.

A number of human disease-causing mutations have been identified in the genes encoding CLCs.   These mutations have been demonstrated to reduce or abolish CLC function.  

Cystic Fibrosis

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is encoded by the CFTR gene. Mutations of the CFTR gene affecting chloride ion channel function lead to dysregulation of epithelial fluid transport in the lung, pancreas and other organs, resulting in cystic fibrosis.

Treatment:Respiratory therapy is any treatment that

slows down lung damage and improves breathing.

FDA has just approved a new drug called Ivacaftor that will almost certainly be a godsend for 4% of cystic fibrosis (CF) sufferers.

References

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Francisco Bezanilla. Voltage-Gated Ion Channels. Nanobioscience, Vol. 4, No. 1, March 2005.

Susan I.V. Judge, Christopher T. Bever Jr. Potassium channel blockers in multiple sclerosis: Neuronal Kv channels and effects of symptomatic treatment. Pharmacology & Therapeutics 111 (2006) 224 – 259.

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Felix Luessi, MD; Volker Siffrin; Frauke Zipp. Neurodegeneration in Multiple Sclerosis: Novel Treatment Strategies: Therapeutic Approaches to Neuronal Degeneration in MS. Medscape. 2013.

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