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 of the
N-terminus constitute the ball domain. This is made up of 11
hydrophobic amino acids, followed by 8
hydrophilic ones, of which 4 are positively charged. The following 60 amino acids constitute the chain domain. Modifying the amino acids of the ball while preserving their
chemical properties 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
binding site deep in the
central cavity. This process involves a
conformational change, which allows the ball-and-chain blocker to elongate and reach the inner center of the channel. , showing the important residues for inactivation in red. The domain structure (I–IV) is further subdivided into segments (S1–S6). The S4 segment is the voltage sensor, which moves out during
depolarisation of the
cell membrane. This frees up the
alanine and
asparagine residues with which the IFMT residues in the ball domain bind to. The
T and
F interact directly with the docking site in the channel pore. When voltage-gated sodium channels
open, 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 in domain III's S4–S5 segments and the
asparagine 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
sodium channels. 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 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, as the gradual introduction of un-tethered synthetic balls to the cytoplasm eventually restores inactivation. ==Effects on neuronal firing==