Dataset Information

A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

ABSTRACT: N-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation and inactivation curves. We find that the midpoint of the steady state inactivation curve depends in part on a secondary interaction between the channel core and a region of the N-terminus just proximal to the pore blocking peptide that we call the Inactivation Proximal (IP) region. The IP interaction with the channel core produces a negative shift in the activation and inactivation curves, without blocking the pore. A tripeptide motif in the IP region was identified in a large number of different N-type inactivation domains most likely reflecting convergent evolution in addition to direct descent. Point mutating a conserved hydrophobic residue in this motif eliminates the gating voltage shift, accelerates recovery from inactivation and decreases the amount of pore block produced during inactivation. The IP interaction we have identified likely stabilizes the open state and positions the pore blocking region of the N-terminus at the internal opening to the transmembrane pore by forming a Pre-Block (P state) interaction with residues lining the side window vestibule of the channel.

SUBMITTER: Pfaffinger PJ

PROVIDER: S-EPMC3827413 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

Pfaffinger Paul J PJ

PloS one 20131113 11

N-type inactivation occurs when the N-terminus of a potassium channel binds into the open pore of the channel. This study examined the relationship between activation and steady state inactivation for mutations affecting the N-type inactivation properties of the Aplysia potassium channel AKv1 expressed in Xenopus oocytes. The results show that the traditional single-step model for N-type inactivation fails to properly account for the observed relationship between steady state channel activation ...[more]

PMID: 24236164

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Project description:Blockade of large-conductance Ca(2+)-activated K(+) (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca(2+)-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca(2+) reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance-voltage curves at high Ca(2+); (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.

Project description:Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1-354), including the eag domain (amino acids 1-135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (?2-135) or both the eag domain and the adjacent proximal domain (?2-354) had less rectifying current-voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1-135) or the eag domain plus the proximal domain (N1-354) with N-deleted hERG ?2-135 or hERG ?2-354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain-containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain-containing fragments effectively converted N-deleted channels into WT-like channels. Neither the addition of the proximal domain to the eag domain in N1-354 fragments nor the presence of the proximal domain in hERG ?2-135 channels measurably affected inactivation properties; in contrast, the proximal region regulated steady-state activation in hERG ?2-135 channels. The results show that N-terminal region-dependent regulation of channel inactivation and resurgent current properties are caused by a direct interaction of the eag domain with the rest of the hERG channel.

Dataset Information

A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

Publications

A conserved pre-block interaction motif regulates potassium channel activation and N-type inactivation.

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