Deletion of the S 3 – S 4 Linker in the Shaker Potassium Channel Reveals Two Quenching Groups Near the Outside of S 4

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes ( D F) 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 D F/F seen was diminished 10-fold, and the D F at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3–S4 linker. The residual D F 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 2 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 D F at extreme hyperpolarizations (more negative than 2 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 D F 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. key words: Shaker K 1 channel • S3–S4 linker • S4 displacement • fluorescence quenching • gating currents I N T R O D U C T I O N The development of the technique of site-directed fluorescent labeling has made it possible to track movements in specific regions of the protein whether or not these rearrangements carry gating charge (Mannuzzu et al., 1996; Cha and Bezanilla, 1997), providing a timeresolved measurement of conformational changes of the channel. Using this technique to place fluorophores outside the S4 (in the S3–S4 linker), it was directly demonstrated that structural rearrangements around the S4 are associated with gating charge movement (Mannuzzu et al., 1996; Cha and Bezanilla, 1997). Placing probes in the pore of the channel, or P-region, elucidated rearrangements associated with slow inactivation of the ionic current (Cha and Bezanilla, 1997; Loots and Isacoff, 1998). Initially, the mechanism of fluorescence quenching near the S4 segment was hypothesized to result from changes in the hydrophobicity of the probe’s environment in response to voltage (Mannuzzu et al., 1996). However, spectral analysis determined that the fluorophores remained in an aqueous environment at all potentials (Cha and Bezanilla, 1997). Other studies pointed to the existence of a protein vestibule surrounding the extracellular portion of the S4 segment (Yang et al., 1997; Cha and Bezanilla, 1998). Using a combination of spectroscopic techniques, Cha and Bezanilla (1998) elucidated some properties of this aqueous vestibule, which was likely responsible for the voltage-dependent quenching of fluorophores attached near the S4 segment. This vestibule constrained the anisotropy and accessibility of fluorophores in the S4 segment in a voltage-dependent manner, as determined by anisotropy and quenching studies. The S4 fluorescence was also titrated by changes in external pH, but not by extracellular calcium, suggesting that the quenching groups in the vestibule contained pHtitratable residues. From the sequence of Shaker channel protein, a likely candidate for the putative protein vestibule is the z 31 Portions of this work were previously published in abstract form (Sørensen, J.B., A. Cha, R. Latorre, E. Rosenmann, and F. Bezanilla. 1999. Biophys. J . 76:A411). Address correspondence to Dr. F. Bezanilla, Dept. of Physiology, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095. Fax: 310-794-9612; E-mail: fbezanil@ucla.edu on A uust 6, 2017 jgp.rress. org D ow nladed fom