IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.
Abstract-Activation of protein kinase A (PKA) increases Na ϩ current derived from the human cardiac Na ϩ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na ϩ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na ϩ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na ϩ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both ␣-subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na
Strong voltage sensitivity of inward-rectifier K+ (Kir) channels has been hypothesized to arise primarily from an intracellular blocker displacing up to five K+ ions from the wide, intracellular part of the ion conduction pore outwardly across the narrow ion selectivity filter. The validity of this hypothesis depends on two assumptions: i) that five ion sites are located intracellular to the filter, and ii) that the blocker can force essentially unidirectional K+ movement in a pore region generally wider than the combined dimensions of the blocker plus a K+ ion. Here, we present a crystal structure of the cytoplasmic portion of a Kir channel with five ions bound, and demonstrate that a constriction near the intracellular end of the pore, acting as a gasket, prevents K+ ions from bypassing the blocker. This heretofore unrecognized “gasket” ensures that the blocker can effectively displace K+ ions across the selectivity filter to generate exceedingly strong voltage sensitivity.
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