RESUMEN
The two-pore-domain potassium channels TASK-1 (KCNK3) and TASK-3 (KCNK9) modulate the electrical activity of neurons and many other cell types. We expressed TASK-1, TASK-3 and related reporter constructs in Xenopus oocytes, mammalian cell lines and various yeast strains to study the mechanisms controlling their transport to the surface membrane and the role of 14-3-3 proteins. We measured potassium currents with the voltage-clamp technique and fused N- and C-terminal fragments of the channels to various reporter proteins to study changes in subcellular localisation and surface expression. Mutational analysis showed that binding of 14-3-3 proteins to the extreme C-terminus of TASK-1 and TASK-3 masks a tri-basic motif, KRR, which differs in several important aspects from canonical arginine-based (RxR) or lysine-based (KKxx) retention signals. Pulldown experiments with GST fusion proteins showed that the KRR motif in the C-terminus of TASK-3 channels was able to bind to COPI coatomer. Disabling the binding of 14-3-3, which exposes the KRR motif, caused localisation of the GFP-tagged channel protein mainly to the Golgi complex. TASK-1 and TASK-3 also possess a di-basic N-terminal retention signal, KR, whose function was found to be independent of the binding of 14-3-3. Suppression of channel surface expression with dominant-negative channel mutants revealed that interaction with 14-3-3 has no significant effect on the dimeric assembly of the channels. Our results give a comprehensive description of the mechanisms by which 14-3-3 proteins, together with N- and C-terminal sorting signals, control the intracellular traffic of TASK-1 and TASK-3.
Asunto(s)
Proteínas 14-3-3/fisiología , Espacio Intracelular/fisiología , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Señales de Clasificación de Proteína/fisiología , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Espacio Intracelular/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Oocitos/fisiología , Canales de Potasio de Dominio Poro en Tándem/genética , Señales de Clasificación de Proteína/genética , Transporte de Proteínas/genética , Xenopus laevisRESUMEN
Arginine (R)-based ER localization signals are sorting motifs that confer transient ER localization to unassembled subunits of multimeric membrane proteins. The COPI vesicle coat binds R-based signals but the molecular details remain unknown. Here, we use reporter membrane proteins based on the proteolipid Pmp2 fused to GFP and allele swapping of COPI subunits to map the recognition site for R-based signals. We show that two highly conserved stretches--in the beta- and delta-COPI subunits--are required to maintain Pmp2GFP reporters exposing R-based signals in the ER. Combining a deletion of 21 residues in delta-COP together with the mutation of three residues in beta-COP gave rise to a COPI coat that had lost its ability to recognize R-based signals, whilst the recognition of C-terminal di-lysine signals remained unimpaired. A homology model of the COPI trunk domain illustrates the recognition of R-based signals by COPI.
Asunto(s)
Proteína Coatómero/química , Proteína Coatómero/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejo 1 de Proteína Adaptadora/metabolismo , Secuencia de Aminoácidos , Arginina , Sitios de Unión , Secuencia Conservada , Retículo Endoplásmico/metabolismo , Genes Fúngicos , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Señales de Clasificación de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Homología Estructural de ProteínaRESUMEN
Arginine (Arg)-based endoplasmic reticulum (ER)-localization signals are involved in the quality control of different heteromultimeric membrane protein complexes. ATP-sensitive potassium (KATP) channels are unique because each subunit in the heterooctamer contains an Arg-based ER-localization signal. We have dissected the inactivation events that override the ER-localization activity of the eight peptide-sorting motifs. Employing a 14-3-3-scavenger construct to lower the availability of 14-3-3 proteins, we found that 14-3-3 proteins promote the cell-surface expression of heterologously expressed and native KATP channels. 14-3-3 proteins were detected in physical association with KATP channels in a pancreatic beta-cell line. Our results suggest that the Arg-based signal present in Kir6.2 is sterically masked by the SUR1 subunit. By contrast, 14-3-3 proteins functionally antagonized the Arg-based signal present in SUR1. The last ten amino acids were required for efficient 14-3-3 recruitment to multimeric forms of the Kir6.2 C-terminus. Channels containing a pore-forming subunit lacking these residues reached the cell surface inefficiently but were functionally indistinguishable from channels formed by the full-length subunits. In conclusion, 14-3-3 proteins promote the cell-surface transport of correctly assembled complexes but do not regulate the activity of KATP channels at the cell surface.
Asunto(s)
Proteínas 14-3-3/fisiología , Membrana Celular/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Western Blotting , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Femenino , Expresión Génica/genética , Potenciales de la Membrana/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Canales de Potasio de Rectificación Interna/genética , Unión Proteica , Transporte de Proteínas/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , XenopusRESUMEN
Recent progress in our understanding of the trafficking of potassium channels can be seen in particular when considering the Kv-type channels. To date, we have discovered that folding of the Kv1.3 T1 domain begins in the ribosomal exit tunnel, and that the cell surface expression of Kv4 channels is enhanced by the presence of two recently identified accessory subunits. Current advances are beginning to enable us to understand the Kv supermolecular complex containing these subunits in crystallographic detail. In addition, determinants that govern the dendritic or axonal targeting of Kv channels have also been identified. In terms of the bigger picture, the careful analysis of gene expression patterns in the brain paves the way for studying trafficking in a physiological context. Indeed, neuronal activity has recently been shown to fine-tune the localization of Kv2.1 channels in microdomains of the neuronal plasma membrane.