neuroscience Neuroscience is the scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions and disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, development ...

, ball and chain inactivation is a model to explain the fast inactivation mechanism of voltage-gated ion channels. The process is also called hinged-lid inactivation or N-type inactivation. A voltage-gated ion channel can be in three states: open, closed, or inactivated. The inactivated state is mainly achieved through fast inactivation, by which a channel transitions rapidly from an open to an inactivated state. The model proposes that the inactivated state, which is stable and non-conducting, is caused by the physical blockage of the pore. The blockage is caused by a "ball" of

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...

s connected to the main

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...

by a string of residues on the

cytoplasmic In cell biology, the cytoplasm is all of the material within a eukaryotic cell, enclosed by the cell membrane, except for the cell nucleus. The material inside the nucleus and contained within the nuclear membrane is termed the nucleoplasm. Th ...

side of the membrane. The ball enters the open channel and binds to the

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, th ...

inner vestibule within the channel. This blockage causes inactivation of the channel by stopping the flow of

ions An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by conven ...

. This phenomenon has mainly been studied in

potassium channels Potassium channels are the most widely distributed type of ion channel found in virtually all organisms. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cell types and control a wide variety of cel ...

and

sodium channels Sodium channels are integral membrane proteins that form ion channels, conducting sodium ions (Na+) through a cell's membrane. They belong to the superfamily of cation channels and can be classified according to the trigger that opens the chann ...

Discovery

Electrophysiological evidence

The initial evidence for a ball and chain inactivation came in 1977 with

Clay Armstrong Clay Margrave Armstrong (born 1934) is an American physiologist and a former student of Andrew Fielding Huxley. Armstrong received his MD from Washington University School of Medicine in 1960. He is currently emeritus professor of Physiology at t ...

and Francisco Bezanilla's work. The suggestion of a physical basis for non-conductance came from experiments in

squid giant axon The squid giant axon is the very large (up to 1.5 mm in diameter; typically around 0.5 mm) axon that controls part of the water jet propulsion system in squid. It was first described by L. W. Williams in 1909, but this discovery was fo ...

s, showing that internal treatment with

pronase Pronase is a commercially available mixture of proteases isolated from the extracellular fluid of ''Streptomyces griseus''. Activity extends to both denatured and native proteins leading to complete or nearly complete digestion into individual amino ...

disrupted the inactivation phenomenon. This suggested a physical, tethered mechanism for inactivation as the pronase was inferred to degrade the channel blocker and abolish the inactivation process. These experiments also showed that inactivation can only occur after the opening of the channel. This was done by hyperpolarising the membrane, causing the channel to open, and observing a delay in inactivation. Inactivation was not observed when the membrane was depolarised (closed). Introducing

tetraethylammonium Tetraethylammonium (TEA), () or (Et4N+) is a quaternary ammonium cation consisting of four ethyl groups attached to a central nitrogen atom, and is positively charged. It is a counterion used in the research laboratory to prepare lipophilic salts ...

(TEA) on the

intracellular This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...

side of the channel was found to mimic inactivation in non-inactivating channels. Blockage of the channel by TEA is mutually exclusive with peptide-mediate blockage, suggesting that TEA competes for an inactivation

binding site In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may inclu ...

Molecular evidence

Mutagenesis Mutagenesis () is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using la ...

experiments have identified an intracellular string of amino acids as prime candidates for the pore blocker. The precise sequence of amino acids that makes up the channel-blocking ball in potassium channels was identified through the creation a

synthetic peptide In organic chemistry, peptide synthesis is the production of peptides, compounds where multiple amino acids are linked via amide bonds, also known as peptide bonds. Peptides are chemically synthesized by the condensation reaction of the carboxyl ...

. The peptide was built based on the sequence of a 20 amino acid residue from the ''

Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...

''s Shaker ShB protein and applied on the intracellular side of a non-inactivating channel in ''

Xenopus ''Xenopus'' () (Gk., ξενος, ''xenos''=strange, πους, ''pous''=foot, commonly known as the clawed frog) is a genus of highly aquatic frogs native to sub-Saharan Africa. Twenty species are currently described within it. The two best-known ...

oocytes An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female g ...

. The peptide restored inactivation to the channel, giving further support to the ball and chain model. In β₂ proteins, the first three residues after the initial

methionine Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical ro ...

have been identified as essential for inactivation. The initial residues have a sequence motif of

phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amino a ...

isoleucine Isoleucine (symbol Ile or I) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the deprot ...

and

tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α- carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromatic ...

without which inactivation does not occur. Modifying the subsequent residues alters the speed and efficacy of inactivation without abolishing it.

Structural evidence

More recently,

nuclear magnetic resonance Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...

studies in ''Xenopus'' oocyte

BK channel BK channels (big potassium), are large conductance calcium-activated potassium channels, also known as Maxi-K, slo1, or Kca1.1. BK channels are voltage-gated potassium channels that conduct large amounts of potassium ions (K+) across the ce ...

s have shed further light on the structural properties of the ball and chain domain. The introduction of the KCNMB2 β subunit to the cytoplasmic side of a non-inactivating channel restored inactivation, conforming to the expected behaviour of a ball and chain-type protein.

NMR Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with ...

analysis showed that the ball domain is composed of residues 1–17 and the chain region of residues 20–45. The three amino acids in the middle constitute a flexible linker region between the two functional regions. The ball is at the

N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the ami ...

of the β subunit and consists of a disordered part (residues 1–10) and a loop-helix motif formed by a block of amino acids spanning from

serine Serine (symbol Ser or S) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated − form under biological conditions), a carboxyl group (which is in the deprotonated − form un ...

at position 11 to aspartate at position 16. The structure of the chain domain is 4-turn

alpha helix The alpha helix (α-helix) is a common motif in the secondary structure of proteins and is a right hand-helix conformation in which every backbone N−H group hydrogen bonds to the backbone C=O group of the amino acid located four residues e ...

structure.

Structure

The ball and chain domains are on the cytoplasmic side of the channel. The most precise structural studies have been carried out in Shaker potassium channels, in which the precise residues involved in the process have been identified. The first 19

amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...

of the

constitute the ball domain. This is made up of 11

amino acids, 8

hydrophilic A hydrophile is a molecule or other molecular entity that is attracted to water molecules and tends to be dissolved by water.Liddell, H.G. & Scott, R. (1940). ''A Greek-English Lexicon'' Oxford: Clarendon Press. In contrast, hydrophobes are no ...

ones and 4 positively charged ones. The following 60 amino acids constitute the chain domain. Modifying the amino acids of the ball while preserving their

chemical properties A chemical property is any of a material's properties that becomes evident during, or after, a chemical reaction; that is, any quality that can be established only by changing a substance's chemical identity.William L. Masterton, Cecile N. Hurley, ...

does not disrupt the inactivation mechanism. This suggests that the ball occludes the channel by binding

electrostatically Electrostatics is a branch of physics that studies electric charges at rest (static electricity). Since classical times, it has been known that some materials, such as amber, attract lightweight particles after rubbing. The Greek word for amber ...

rather than

covalently A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atom ...

. Structural studies have shown that the inner pore of the potassium channel is accessible only through side slits between the cytoplasmic domains of the four α-subunits, rather than from a central route as previously thought. The ball domain enters the channel through the side slits and attaches to a

deep in the central cavity. This process involves a

conformational change In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or oth ...

, which allows the ball and chain blocker to elongate and reach the inner center of the channel. A positively charged region between the III and IV domains of sodium channels is thought to act in a similar way. The essential region for inactivation in sodium channels is four amino acid sequence made up of

and

threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), a carboxyl group (which is in the deprotonated −COO� ...

(IFMT). The T and F interact directly with the docking site in the channel pore. When voltage-gated sodium channels

open Open or OPEN may refer to: Music * Open (band), Australian pop/rock band * The Open (band), English indie rock band * ''Open'' (Blues Image album), 1969 * ''Open'' (Gotthard album), 1999 * ''Open'' (Cowboy Junkies album), 2001 * ''Open'' (YF ...

, the S4 segment moves outwards from the channel and into the extracellular side. This exposes hydrophobic residues in the S4 and S5 segments which interact with the inactivation ball. The phenylalanine of the ball interacts with the

alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side c ...

in domain III's S4-S5 segments and the

asparagine Asparagine (symbol Asn or N) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depro ...

in domain IV's S4-S5 segments. This explains why inactivation can only occur once the channel is open. Lateral slits are also present in sodium channels, suggesting that the access route for the ball domain may be similar. There is a distinction between direct inactivation and two-step inactivation. Direct inactivation, which occurs in Shaker potassium channels results from the direct blockage of the channel by the ball protein, while two-step inactivation, thought to occur in

s, requires an intermediate binding step. The mechanism of ball-and-chain inactivation is also distinct from that of voltage-dependent blockade by intracellular molecules or peptide regions of beta4 subunits in

. When these blocks contribute to sodium channel inactivation after channel opening, repolarization of the membrane reverses the block and can causes a resurgent current: a flow of ions between unblocking and closure of the channel.

Inactivation prevention domain

Potassium channels Potassium channels are the most widely distributed type of ion channel found in virtually all organisms. They form potassium-selective pores that span cell membranes. Potassium channels are found in most cell types and control a wide variety of cel ...

have an additional feature in the N-terminus which makes the channels unable to inactivate. The N-type inactivation-prevention (NIP) domain counteracts the effect of the peptide ball. Channels containing the NIP domain behave as mutated non-inactivating channels, as they have no inactivation activity. The effect is thought be

stoichiometric Stoichiometry refers to the relationship between the quantities of reactants and products before, during, and following chemical reactions. Stoichiometry is founded on the law of conservation of mass where the total mass of the reactants equ ...

, as the gradual introduction of un-tethered synthetic balls to the cytoplasm eventually restores inactivation.

Effects on neuronal firing

The interplay between opening and inactivation controls the firing pattern of a

neuron A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...

by changing the rate and amount of ion flow through the channels. Voltage-gated ion channels open upon

depolarization In biology, depolarization or hypopolarization is a change within a cell, during which the cell undergoes a shift in electric charge distribution, resulting in less negative charge inside the cell compared to the outside. Depolarization is esse ...

of the

cell membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...

. This creates a current caused by the flow of

through the channel. Shortly after opening, the channel is blocked by the peptide ball. The β1 subunit aids recovery from inactivation, while β2 accelerates inactivation. The β subunits can also interfere with ball and chain domains by blocking their entry into the channel. This leads to persistent currents, caused by the continued influx of ions. The β3 subunit can increase persistent current in certain sodium channels.

Implications for disease

Differences in persistent and resurgent currents have been implicated in certain human

neurological Neurology (from el, νεῦρον (neûron), "string, nerve" and the suffix -logia, "study of") is the branch of medicine dealing with the diagnosis and treatment of all categories of conditions and disease involving the brain, the spinal ...

and

neuromuscular A neuromuscular junction (or myoneural junction) is a chemical synapse between a motor neuron and a muscle fiber. It allows the motor neuron to transmit a signal to the muscle fiber, causing muscle contraction. Muscles require innervation t ...

disorders. In

epilepsy Epilepsy is a group of non-communicable neurological disorders characterized by recurrent epileptic seizures. Epileptic seizures can vary from brief and nearly undetectable periods to long periods of vigorous shaking due to abnormal electrical ...

, mutations in sodium channels genes delay inactivation. This leads to the channel staying open for longer and thus longer-lasting neuronal firing. Higher levels of persistent current are observed in epilepsy. This constant, low-level neuronal stimulation has been linked to the

seizures An epileptic seizure, informally known as a seizure, is a period of symptoms due to abnormally excessive or neural oscillation, synchronous neuronal activity in the brain. Outward effects vary from uncontrolled shaking movements involving much o ...

typical of this disorder. Inactivation anomalies have also been linked to

Brugada syndrome Brugada syndrome (BrS) is a genetic disorder in which the electrical activity of the heart is abnormal due to channelopathy. It increases the risk of abnormal heart rhythms and sudden cardiac death. Those affected may have episodes of syncope ...

. Mutations in genes encoding the α subunit in cardiac sodium channels affect inactivation. These increase persistent current by interfering with inactivation, though different mutations have opposite effects in inactivation speed. Mutations in the α subunit of

skeletal muscles Skeletal muscles (commonly referred to as muscles) are organs of the vertebrate muscular system and typically are attached by tendons to bones of a skeleton. The muscle cells of skeletal muscles are much longer than in the other types of muscle ...

are also associated with

myotonia Myotonia is a symptom of a small handful of certain neuromuscular disorders characterized by delayed relaxation (prolonged contraction) of the skeletal muscles after voluntary contraction or electrical stimulation. Myotonia is the defining sympt ...

. The characteristic muscular hyperexcitation of myotonia is mainly caused by the presence sodium channels which do not inactivate, causing high levels of persistent current in the muscles.

References

{{Reflist, 30em Cell anatomy Cell biology Cellular neuroscience Electrophysiology