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J. Biol. Chem., Vol. 280, Issue 44, 37257-37265, November 4, 2005

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Role of an S4-S5 Linker Lysine in the Trafficking of the Ca²⁺-activated K⁺ Channels IK1 and SK3^*

Heather M. Jones, Kirk L. Hamilton1, and Daniel C. Devor2

From the Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the Department of Physiology, University of Otago, Dunedin, New Zealand

We have investigated the role of the S4-S5 linker in the trafficking of the intermediate (human (h) IK1) and small (rat SK3) conductance K⁺ channels using a combination of patch-clamp, protein biochemical, and immunofluorescence-based techniques. We demonstrate that a lysine residue (Lys¹⁹⁷) located on the intracellular loop between the S4 and S5 domains is necessary for the correct trafficking of hIK1 to the plasma membrane. Mutation of this residue to either alanine or methionine precluded trafficking of the channel to the membrane, whereas the charge-conserving arginine mutation had no effect on channel localization or function. Immunofluorescence localization demonstrated that the K197A mutation resulted in a channel that was primarily retained in the endoplasmic reticulum, and this could not be rescued by incubation at 27 °C. Furthermore, immunoblot analysis revealed that the K197A mutation was overexpressed compared with wild-type hIK1 and that this was due to a greatly diminished rate of channel degradation. Co-immunoprecipitation studies demonstrated that the K197A mutation did not preclude multimer formation. Indeed, the K197A mutation dramatically suppressed expression of wild-type hIK1 at the cell surface. Finally, mutation of this conserved lysine in rat SK3 similarly resulted in a channel that failed to correctly traffic to the plasma membrane. These results are the first to demonstrate a critical role for the S4-S5 linker in the trafficking and/or function of IK and SK channels.

Received for publication, August 4, 2005 , and in revised form, August 30, 2005.

^* This work was supported in part by National Institutes of Health Grant DK54941 (to D. C. D.) and Training Grant T32-DK061296 (to H. M. J.) and by the University of Otago Dean's Fund (to K. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Received sabbatical support from the Dept. of Physiology, University of Otago.

² To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S312 BST, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax: 412-648-8330; E-mail: dd2{at}pitt.edu.

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