Kir2.6 Regulates the Surface Expression of Kir2.x Inward Rectifier Potassium Channels

Abstract

Precise trafficking, localization, and activity of inward rectifier potassium Kir2 channels are important for shaping the electrical response of skeletal muscle. However, how coordinated trafficking occurs to target sites remains unclear. Kir2 channels are tetrameric assemblies of Kir2.x subunits. By immunocytochemistry we show that endogenous Kir2.1 and Kir2.2 are localized at the plasma membrane and T-tubules in rodent skeletal muscle. Recently, a new subunit, Kir2.6, present in human skeletal muscle, was identified as a gene in which mutations confer susceptibility to thyrotoxic hypokalemic periodic paralysis. Here we characterize the trafficking and interaction of wild type Kir2.6 with other Kir2.x in COS-1 cells and skeletal muscle in vivo. Immunocytochemical and electrophysiological data demonstrate that Kir2.6 is largely retained in the endoplasmic reticulum, despite high sequence identity with Kir2.2 and conserved endoplasmic reticulum and Golgi trafficking motifs shared with Kir2.1 and Kir2.2. We identify amino acids responsible for the trafficking differences of Kir2.6. Significantly, we show that Kir2.6 subunits can coassemble with Kir2.1 and Kir2.2 in vitro and in vivo. Notably, this interaction limits the surface expression of both Kir2.1 and Kir2.2. We provide evidence that Kir2.6 functions as a dominant negative, in which incorporation of Kir2.6 as a subunit in a Kir2 channel heterotetramer reduces the abundance of Kir2 channels on the plasma membrane.Inward rectifier potassium (Kir2) channels are key skeletal muscle components involved in determination of muscle resting potential, regulation of electrical excitability, repolarization of the action potential, and clearance of K ϩ from the T-tubule 2 system (1-4 Recently, a search for genes involved in thyrotoxic periodic paralysis (TPP) revealed KCNJ18, which encodes a novel subtype of inward rectifier potassium channel subunit, Kir2.6 (22). Kir2.6 is expressed primarily in skeletal muscle and shares Ͼ98% identity with Kir2.2 (22). Mutations in human Kir2.6 confer susceptibility to TPP, and it has been shown that these disease-associated mutations contribute to atypical current signatures and altered cell excitability (22). Expression studies in heterologous cells demonstrate that Kir2.6 subunits are able to form inwardly rectifying channels and that disease-associated mutations include truncated subunits that do not form functional channels, as well as gain-of-function mutations that increase electrical activity because of misregulation by phosphorylation (22).In addition to their electrophysiological properties, ion channels contribute to electrical events by their abundance on the plasma membrane. Targeting ion channels to subcellular sites and the plasma membrane is important for shaping the electrical response of muscle. Surface expression levels and channel activity are crucial for determining muscle excitability. However, trafficking of Kir2.6 to the plasma membrane has not yet been explored. Moreover, the role of wild type Kir2.6 in normal...

Identification and Functional Characterization of Kir2.6 Mutations Associated with Non-familial Hypokalemic Periodic Paralysis

“…The report by Ryan et al (12) and ours indicate an important role of Kir2.6 in the regulation of skeletal muscle membrane potentials. During the course of our present study, Dassau et al (14) reported that Kir2.6 co-assembles with Kir2.1 and Kir2.2. Based on immunofluorescent staining of recombinant Kir2.6 in transfected rodent tissues, the authors suggested that Kir2.6 is predominantly distributed to the endoplasmic reticulum and functions as a dominant negative regulator of cell surface abundance of Kir2.x channels by retention in the endoplasmic reticulum.…”

“…Kir2.1 channel is also expressed in skeletal muscle and reportedly forms heteromultimers with Kir2.6 (14). We thus compared Kir2.1 and Kir2.6 channel properties and explored potential interactions between disease mutant Kir2.6 and wild type Kir2.1.…”

Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction

“…S2E and S3F and Table S1). Whereas there are conflicting reports about the location of ClC-1 in muscle (37,40), it is established that Kir channels are expressed in the transverse tubular system (44,45). A definitive confirmation of a partial detubulation will require optical methods that include electron microscopy.…”

Parálisis períodica hipocalcémica tirotóxica

“…Además, Parálisis Periódica Hipocalémica Tirotóxica se ha demostrado en el músculo estriado y cardíaco que la actividad de la bomba calcio ATPasa presente en el retículo sarcoplásmico, esta disminuida, permitiendo un retardo en la eliminación del calcio presente en el citoplasma, contribuyendo a la parálisis (11). Los ataques de parálisis perió-dica hipocaliémica tirotóxica pueden ser desencadenados por periodos de ejercicio intenso, la ingestión de dieta rica en carbohidratos o alcohol, trauma, estrés, exposición al frío, infección y fármacos como: diuréticos perdedores de potasio, insulina, amiodarona, beta miméticos, adrenalina, corticoides, piridostigmina, cosintropina y pilocarpina (6,20). La parálisis periódica hipocaliémica tirotóxica afecta principalmente a varones entre la segunda y cuarta década de vida (8), puede estar precedida de pródromos con calambres, rigidez y mialgias en miembros inferiores (6).…”