Dynamics of Pore Domain Affected by Single Mutations in S4 Segment of Shaker Potassium Channel

The four voltage sensors in voltage-gated potassium (Kv) channels activate upon membrane depolarization and open the pore. The location and motion of the voltage-sensing S4 helix during the early activation steps and the final opening transition are unresolved. We studied Zn2+ bridges between two introduced His residues in Shaker Kv channels: one in the R1 position at the outer end of the S4 helix (R362H), and another in the S5 helix of the pore domain (A419H or F416H). Zn2+ bridges readily form between R362H and A419H in open channels after the S4 helix has undergone its final motion. In contrast, a distinct bridge forms between R362H and F416H after early S4 activation, but before the final S4 motion. Both bridges form rapidly, providing constraints on the average position of S4 relative to the pore. These results demonstrate that the outer ends of S4 and S5 remain in close proximity during the final opening transition, with the S4 helix translating a significant distance normal to the membrane plane.

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Deletion of the S3–S4 Linker in theShaker Potassium Channel Reveals Two Quenching Groups near the outside of S4

The Journal of General Physiology ◽

10.1085/jgp.115.2.209 ◽

2000 ◽

Vol 115 (2) ◽

pp. 209-222 ◽

Cited By ~ 28

Author(s):

J.B. Sørensen ◽

A. Cha ◽

R. Latorre ◽

E. Rosenman ◽

F. Bezanilla

Keyword(s):

Potassium Channel ◽

Conformational Changes ◽

Deletion Mutant ◽

Transient State ◽

Voltage Sensor ◽

Charge Movement ◽

Shaker Potassium Channel ◽

Gating Charge ◽

In The Wild ◽

S4 Segment

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (ΔF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391–408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3–S4 linker. In the deletion mutant, the maximal ΔF/F seen was diminished 10-fold, and the ΔF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual ΔF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of −90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a ΔF at extreme hyperpolarizations (more negative than −90 mV) only. A signal from the interaction with this group in the wt S3–S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic ΔF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.

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Structural model of a voltage-gated potassium channel based on spectroscopic data

Biochemical Society Transactions ◽

10.1042/bst0290589 ◽

2001 ◽

Vol 29 (4) ◽

pp. 589-593 ◽

Cited By ~ 2

Author(s):

P. I. Haris

Keyword(s):

Potassium Channel ◽

Structural Model ◽

Structural Data ◽

Theoretical Models ◽

Shaker Potassium Channel ◽

Voltage Gated ◽

Folded Structure ◽

Membrane Spanning ◽

Voltage Gated Ion Channels ◽

Pore Domain

It is estimated that membrane proteins comprise as much as 30% of most genomes. Yet our knowledge of membrane-protein folding is still in its infancy. Consequently, there is a great need for developing approaches that can further advance our understanding of how peptides and proteins interact with membranes and thereby attain their folded structure. An approach that we have been exploring involves dissecting voltage-gated ion channels into simple peptide domains for the purpose of determining their structure in different media using physical techniques. We have synthesized peptides corresponding to the six membrane-spanning segments, as well as the pore domain, of the Shaker channel and characterized their secondary structures. From these studies we have developed a model for the transmembrane structure of the Shaker potassium channel that is constructed from α-helices. The hard structural data obtained from these studies lends support to the recent theoretical models of this channel protein that have been developed by others.

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