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

, 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 over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...

s connected to the main

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

by a string of residues on the

cytoplasmic The cytoplasm describes all the material within a eukaryotic or prokaryotic cell, enclosed by the cell membrane, including the organelles and excluding the nucleus in eukaryotic cells. The material inside the nucleus of a eukaryotic cell and ...

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

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

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 convent ...

. 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 ...

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. Classification They are classified into 2 types: Function In e ...

Discovery

Electrophysiological evidence

The initial evidence for a ball and chain inactivation came in 1977 with Clay Armstrong 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 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) is a quaternary ammonium cation with the chemical formula , consisting of four ethyl groups (, denoted Et) attached to a central nitrogen atom. It is a counterion used in the research laboratory to prepare lipophilic salt ...

(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 includ ...

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 lab ...

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 (an insect of the Order (biology), 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", "pomace fly" ...

''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 with ...

'' oocytes. 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 non-essential amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine play ...

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 chemical formula, formula . It can be viewed as a benzyl group substituent, substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of ...

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 depro ...

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 aromat ...

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 disturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...

studies in ''Xenopus'' oocyte BK channels 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 atomic nucleus, nuclei in a strong constant magnetic field are disturbed by a weak oscillating magnetic field (in the near and far field, near field) and respond by producing ...

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 amin ...

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 − ...

at position 11 to

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...

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

alpha helix An alpha helix (or α-helix) is a sequence of amino acids in a protein that are twisted into a coil (a helix). The alpha helix is the most common structural arrangement in the Protein secondary structure, secondary structure of proteins. It is al ...

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 over 500 amino acids exist in nature, by far the most important are the Proteinogenic amino acid, 22 α-amino acids incorporated into p ...

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 n ...

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 property, material's properties that becomes evident during, or after, a chemical reaction; that is, any attribute that can be established only by changing a substance's chemical substance, chemical identit ...

does not disrupt the inactivation mechanism. This suggests that the ball occludes the channel by binding electrostatically rather than covalently. 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 othe ...

, 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 when dissolved in water), 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'' (Gerd Dudek, Buschi Niebergall, and Edward Vesala album), 1979 * ''Open'' (Go ...

, 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 sid ...

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 BK channels, 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 ...

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 () is the relationships between the masses of reactants and products before, during, and following chemical reactions. Stoichiometry is based on the law of conservation of mass; the total mass of reactants must equal the total m ...

, 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 Neural oscillations, or brainwaves, are rhythmic or repetitive patterns of neural activity in the central nervous system. Neural tissue can generate oscillatory activity in many ways, driven either by mechanisms within individual neurons or by ...

of a

neuron A neuron (American English), neurone (British English), or nerve cell, is an membrane potential#Cell excitability, excitable cell (biology), cell that fires electric signals called action potentials across a neural network (biology), neural net ...

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 (biology), cell, during which the cell undergoes a shift in electric charge distribution, resulting in less negative charge inside the cell compared to the outside. Depolar ...

of the

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

. 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 , "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 nervous system, which comprises the brain, the s ...

and neuromuscular disorders. In

epilepsy Epilepsy is a group of Non-communicable disease, non-communicable Neurological disorder, neurological disorders characterized by a tendency for recurrent, unprovoked Seizure, seizures. A seizure is a sudden burst of abnormal electrical activit ...

, 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 A seizure is a sudden, brief disruption of brain activity caused by abnormal, excessive, or synchronous neuronal firing. Depending on the regions of the brain involved, seizures can lead to changes in movement, sensation, behavior, awareness, o ...

typical of this disorder. Inactivation anomalies have also been linked to Brugada syndrome. 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 muscle (commonly referred to as muscle) is one of the three types of vertebrate muscle tissue, the others being cardiac muscle and smooth muscle. They are part of the somatic nervous system, voluntary muscular system and typically are a ...

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, and the muscle shows an abnor ...

. 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