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J. Biol. Chem., Vol. 283, Issue 14, 9049-9059, April 4, 2008

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Role of S3 and S4 Transmembrane Domain Charged Amino Acids in Channel Biogenesis and Gating of KCa2.3 and KCa3.1^*

From the Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

The role of positively charged arginines in the fourth transmembrane domain (S4) and a single negatively charged amino acid in the third transmembrane domain (S3) on channel biogenesis and gating of voltage-gated K⁺ channels (Kv) has been well established. Both intermediate (KCa3.1) and small (KCa2.x) conductance, Ca²⁺-activated K⁺ channels have two conserved arginines in S4 and a single conserved glutamic acid in S3, although these channels are voltage-independent. We demonstrate that mutation of any of these charged amino acids in KCa3.1 or KCa2.3 to alanine, glutamine, or charge reversal mutations results in a rapid degradation (<30 min) of total protein, confirming the critical role of these amino acids in channel biogenesis. Mutation of the S4 arginine closest to the cytosolic side of KCa3.1 to histidine resulted in expression at the cell surface. Excised patch clamp experiments revealed that this Arg/His mutation had a dramatically reduced open probability (P_o), relative to wild type channels. Additionally, we demonstrate, using a combination of short hairpin RNA, dominant negative, and co-immunoprecipitation studies, that both KCa3.1 and KCa2.3 are translocated out of the endoplasmic reticulum associated with Derlin-1. These misfolded channels are poly-ubiquitylated, recognized by p97, and targeted for proteasomal degradation. Our results suggest that S3 and S4 charged amino acids play an evolutionarily conserved role in the biogenesis and gating of KCa channels. Furthermore, these improperly folded K⁺ channels are translocated out of the endoplasmic reticulum in a Derlin-1- and p97-dependent fashion, poly-ubiquitylated, and targeted for proteasomal degradation.

Received for publication, September 25, 2007 , and in revised form, January 14, 2008.

^* This work was supported by a National Institutes of Health Grant HL083060 (to D. C. D.). 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 To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S331 BST, 3500 Terrace St., Pittsburgh, PA 15261. E-mail: dd2{at}pitt.edu.

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