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© The Rockefeller University Press, 0022-1295/1998//391/ $5.00
Journal of General Physiology, Volume 112, Number 4, 1998

Article

Structural Implications of Fluorescence Quenching in the Shaker K+ Channel

Albert Cha*,{ddagger} and Francisco Bezanilla*,{ddagger}

From the * Department of Physiology and {ddagger} Department of Anesthesiology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095

When attached to specific sites near the S4 segment of the nonconducting (W434F) Shaker potassium channel, the fluorescent probe tetramethylrhodamine maleimide undergoes voltage-dependent changes in intensity that correlate with the movement of the voltage sensor (Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Science. 271:213–216; Cha, A., and F. Bezanilla. 1997. Neuron. 19:1127–1140). The characteristics of this voltage-dependent fluorescence quenching are different in a conducting version of the channel with a different pore substitution (T449Y). Blocking the pore of the T449Y construct with either tetraethylammonium or agitoxin removes a fluorescence component that correlates with the voltage dependence but not the kinetics of ionic activation. This pore-mediated modulation of the fluorescence quenching near the S4 segment suggests that the fluorophore is affected by the state of the external pore. In addition, this modulation may reflect conformational changes associated with channel opening that are prevented by tetraethylammonium or agitoxin. Studies of pH titration, collisional quenchers, and anisotropy indicate that fluorophores attached to residues near the S4 segment are constrained by a nearby region of protein. The mechanism of fluorescence quenching near the S4 segment does not involve either reorientation of the fluorophore or a voltage-dependent excitation shift and is different from the quenching mechanism observed at a site near the S2 segment. Taken together, these results suggest that the extracellular portion of the S4 segment resides in an aqueous protein vestibule and is influenced by the state of the external pore.

Key Words: Shaker potassium channel • fluorescence quenching • pore conformational changes

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{at}ucla.edu

Abbreviations: dF, fluorescence difference; R, intensity ratio; TEA, tetraethylammonium; TMRM, tetramethylrhodamine maleimide


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