Inneuroscience,ball-and-chain inactivation is a model to explain the fast inactivation mechanism ofvoltage-gated ion channels. The process is also calledhinged-lid inactivation orN-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" ofamino acids connected to the mainprotein by a string of residues on thecytoplasmic side of the membrane. The ball enters the open channel and binds to thehydrophobic inner vestibule within the channel. This blockage causes inactivation of the channel by stopping the flow ofions.[1][2] This phenomenon has mainly been studied inpotassium channels andsodium channels.[3]
The initial evidence for a ball-and-chain inactivation came in 1977 withClay Armstrong andFrancisco Bezanilla's work.[4] The suggestion of a physical basis for non-conductance came from experiments insquid giant axons, showing that internal treatment withpronase 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 byhyperpolarising the membrane, causing the channel to open, and observing a delay in inactivation. Inactivation was not observed when the membrane wasdepolarised (closed). Introducingtetraethylammonium (TEA) on theintracellular side of the channel was found to mimic inactivation in non-inactivating channels.[5] Blockage of the channel by TEA is mutually exclusive with peptide-mediate blockage, suggesting that TEA competes for an inactivationbinding site.[6]
Mutagenesis experiments have identified an intracellular string of amino acids as prime candidates for the pore blocker.[5] The precise sequence of amino acids that makes up the channel-blocking ball in potassium channels was identified through the creation asynthetic peptide. The peptide was built based on the sequence of a 20 amino acid residue from theDrosophila melanogaster'sShaker ShB protein and applied on the intracellular side of a non-inactivating channel inXenopusoocytes. 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 initialmethionine have been identified as essential for inactivation. The initial residues have a sequence motif ofphenylalanine,isoleucine andtryptophan without which inactivation does not occur. Modifying the subsequent residues alters the speed and efficacy of inactivation without abolishing it.[7]
More recently,nuclear magnetic resonance studies inXenopus oocyteBK channels have shed further light on the structural properties of the ball-and-chain domain.[8] The introduction of the KCNMB2β 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 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 aflexible linker region between the two functional regions. The ball is at theN-terminus 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 fromserine at position 11 toaspartate at position 16. The structure of the chain domain is 4-turnα-helix structure.
The ball and chain domains are on the cytoplasmic side of the channel. The most precise structural studies have been carried out inShaker potassium channels, in which the precise residues involved in the process have been identified. The first 19amino acids of theN-terminus constitute the ball domain. This is made up of 11hydrophobic amino acids, followed by 8hydrophilic ones, of which 4 are positively charged.[9] The following 60 amino acids constitute the chain domain. Modifying the amino acids of the ball while preserving theirchemical properties does not disrupt the inactivation mechanism. This suggests that the ball occludes the channel by bindingelectrostatically rather thancovalently.[10] Structural studies have shown that the inner pore of the potassium channel is accessible only through side slits between the cytoplasmic domains of the fourα-subunits, rather than from a central route as previously thought.[11] The ball domain enters the channel through the side slits and attaches to abinding site deep in thecentral cavity. This process involves aconformational change, which allows the ball-and-chain blocker to elongate and reach the inner center of the channel.[12]
A positively charged region between the III and IVdomains of sodium channels is thought to act in a similar way.[9] The essential region for inactivation in sodium channels is four amino acid sequence made up ofisoleucine,phenylalanine,methionine andthreonine (IFMT).[13] TheT andF interact directly with the docking site in the channel pore.[14] When voltage-gated sodium channelsopen, 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 thealanine in domain III's S4–S5 segments and theasparagine in domain IV's S4–S5 segments.[15] This explains why inactivation can only occur once the channel is open.
Lateral slits are also present in sodium channels,[16] 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 inShaker potassium channels results from the direct blockage of the channel by the ball protein, while two-step inactivation, thought to occur inBK channels, requires an intermediate binding step.[17]
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 insodium channels.[18] 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.[19]
Potassium channels 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.[20] The effect is thought bestoichiometric, as the gradual introduction of un-tethered synthetic balls to the cytoplasm eventually restores inactivation.[21]
The interplay between opening and inactivation controls thefiring pattern of aneuron by changing the rate and amount of ion flow through the channels. Voltage-gated ion channels open upondepolarization of thecell membrane. This creates a current caused by the flow ofions through the channel. Shortly after opening, the channel is blocked by the peptide ball. The β1 subunit aids recovery from inactivation,[22] while β2 accelerates inactivation.[23] 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.[13]
Differences in persistent and resurgent currents have been implicated in certain humanneurological andneuromuscular disorders. Inepilepsy, mutations in sodium channels genes delay inactivation. This leads to the channel staying open for longer and thus longer-lasting neuronal firing.[24] Higher levels of persistent current are observed in epilepsy. This constant, low-levelneuronal stimulation has been linked to theseizures typical of this disorder.[25]
Inactivation anomalies have also been linked toBrugada syndrome. Mutations in genes encoding theα subunit incardiac sodium channels affect inactivation. These increase persistent current by interfering with inactivation, though different mutations have opposite effects in inactivation speed.[26]
Mutations in theα subunit ofskeletal muscles are also associated withmyotonia. 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.[27]