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Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.

ABSTRACT: The subfamily of G-protein-linked inwardly rectifying potassium channels (GIRKs) is coupled to G-protein receptors throughout the CNS and in the heart. We used mutational analysis to address the role of a specific hydrophobic region of the GIRK1 subunit. Deletion of the GIRK1 C-terminal residues 330-384, as well as the point mutation I331R, resulted in a decrease in channel function when coexpressed with GIRK4 in oocytes and in COS-7 cells. Surface protein expression of GIRK1 I331R coexpressed with GIRK4 was comparable with wild type, indicating that subunits assemble and are correctly localized to the membrane. Subsequent mutation of homologous residues in both the GIRK4 subunit and Kir2.1 (Gbetagamma-independent inward rectifier) also resulted in a decrease in channel function. Intracellular domain associations resulted in the coimmunoprecipitation of the GIRK1 N and C termini and GIRK4 N and C termini. The point mutation I331R in the GIRK1 C terminus or L337R in the GIRK4 C terminus decreased the association between the N and C termini. Mutation of a GIRK1 N-terminal hydrophobic residue, predicted structurally to interact with the C-terminal domain, also resulted in a decrease in channel function and termini association. We hypothesize that the hydrophobic nature of this GIRK1 subunit region is critical for interaction between adjacent termini and is permissive for channel gating. In addition, the homologous mutation in cytoplasmic domains of Kir2.1 (L330R) did not disrupt association, suggesting that the overall structural integrity of this region is critical for inward rectifier function.

SUBMITTER: Sarac R

PROVIDER: S-EPMC6725930 | biostudies-literature | 2005 Feb

REPOSITORIES: biostudies-literature

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Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.

Sarac Radmila R Hou Ping P Hurley Karen M KM Hriciste David D Cohen Noam A NA Nelson Deborah J DJ

The Journal of neuroscience : the official journal of the Society for Neuroscience 20050201 7

The subfamily of G-protein-linked inwardly rectifying potassium channels (GIRKs) is coupled to G-protein receptors throughout the CNS and in the heart. We used mutational analysis to address the role of a specific hydrophobic region of the GIRK1 subunit. Deletion of the GIRK1 C-terminal residues 330-384, as well as the point mutation I331R, resulted in a decrease in channel function when coexpressed with GIRK4 in oocytes and in COS-7 cells. Surface protein expression of GIRK1 I331R coexpressed w ...[more]

PMID: 15716420

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Project description:Small fiber neuropathy (SFN) is a common condition affecting thinly myelinated Aδ and unmyelinated C fibers, often resulting in excruciating pain and dysautonomia. SFN has been associated with several conditions, but a significant number of cases have no discernible cause. Recent genetic studies have identified potentially pathogenic gain-of-function mutations in several pore-forming voltage-gated sodium channel α subunits (NaV) in a subset of patients with SFN, but the auxiliary sodium channel β subunits have been less implicated in the development of the disease. β subunits modulate NaV trafficking and gating, and several mutations have been linked to epilepsy and cardiac dysfunction. Recently, we provided the first evidence for the contribution of a mutation in the β2 subunit to pain in human painful diabetic neuropathy. Here, we provide the first evidence for the involvement of a sodium channel β subunit mutation in the pathogenesis of SFN with no other known causes. We show, through current-clamp analysis, that the newly identified Y69H variant of the β2 subunit induces neuronal hyperexcitability in dorsal root ganglion neurons, lowering the threshold for action potential firing and allowing for increased repetitive action potential spiking. Underlying the hyperexcitability induced by the β2-Y69H variant, we demonstrate an upregulation in tetrodotoxin-sensitive, but not tetrodotoxin-resistant sodium currents. This provides the first evidence for the involvement of β2 subunits in SFN and strengthens the link between sodium channel β subunits and the development of neuropathic pain in humans.NEW & NOTEWORTHY Small fiber neuropathy (SFN) often has no discernible cause, although mutations in the voltage-gated sodium channel α subunits have been implicated in some cases. We identify a patient suffering from SFN with a mutation in the auxiliary β2 subunit and no other discernible causes for SFN. Functional assessment confirms this mutation renders dorsal root ganglion neurons hyperexcitable and upregulates tetrodotoxin-sensitive sodium currents. This study strengthens a newly emerging link between sodium channel β2 subunit mutations and human pain disorders.

Dataset Information

Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.

Publications

Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function.

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