title: Role of S3 and S4 transmembrane domain charged amino acids in channel biogenesis and gating of KCa2.3 and KCa3.1.
abstract: 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, Ca(2+)-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.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261,USA.
authors.forename: Yajuan
authors.initials: Y
authors.lastname: Gao
authors.forename: Cavita K
authors.initials: CK
authors.lastname: Chotoo
authors.forename: Corina M
authors.initials: CM
authors.lastname: Balut
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
authors.forename: Mark A
authors.initials: MA
authors.lastname: Bailey
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Devor
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology
chemical_list: D000596:Amino Acids; C487709:DERL1 protein, human; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C406328:KCNN3 protein, human; C496923:KCNN4 protein, human; D008565:Membrane Proteins; C101338:PSMD2 protein, human; D051640:Small-Conductance Calcium-Activated Potassium Channels; D047992:TNF Receptor-Associated Factor 2; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex