Palmitoylation of the S0-S1 linker regulates cell surface expression of voltage- and calcium-activated potassium (BK) channels.
S-palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.
Jeffries
Owen
O
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Geiger
Nina
N
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom; Pharmacology and Toxicology, Institute of Pharmacy, University Tuebingen, 72076 Tuebingen, Germany.
Rowe
Iain C M
ICM
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Tian
Lijun
L
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
McClafferty
Heather
H
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Chen
Lie
L
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Bi
Danlei
D
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom.
Knaus
Hans Guenther
HG
Division for Molecular and Cellular Pharmacology, Department of Medical Genetics, Molecular and Clinical Pharmacology, Medical University Innsbruck, Peter-Mayr Strasse 1, 6020 Innsbruck, Austria.
Ruth
Peter
P
Pharmacology and Toxicology, Institute of Pharmacy, University Tuebingen, 72076 Tuebingen, Germany.
Shipston
Michael J
MJ
From the Centre for Integrative Physiology, College of Medicine & Veterinary Medicine, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, United Kingdom. Electronic address: mike.shipston@ed.ac.uk.
D017398:Alternative Splicing / Q000502:physiology; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D020125:Mutation, Missense; D019308:Palmitic Acid / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary
D051036:Large-Conductance Calcium-Activated Potassium Channels; D019308:Palmitic Acid