A splice variant of the G protein beta 3-subunit implicated in disease states does not modulate ion channels.
A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G protein beta3 subunit (Gbeta3). Translation of the alternatively spliced mRNA results in a protein product, Gbeta3-s, in which 41 amino acids are deleted from Gbeta3. Interestingly, previous studies indicate that the C825T allele occurs with a high frequency in patients with certain vascular disorders. However, little information is available regarding the functional role Gbeta3-s might play in ion channel modulation. To examine this aspect, Gbeta3 or Gbeta3-s, along with either Ggamma2 or Ggamma5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein. In contrast to Gbeta3, expression of Gbeta3-s did not modulate N-type Ca(2+) or G protein-gated inwardly rectifying K(+) channels. In addition, Gbeta3-s did not appear to complex with a pertussis toxin-insensitive mutant of Galpha(i2) or couple to natively expressed alpha(2)-adrenergic receptors. Finally, fluorescence resonance energy transfer (FRET) measurements indicated that enhanced yellow fluorescent protein (EYFP)-labeled Gbeta3-s does not form a Gbetagamma heterodimer when coexpressed with enhanced cyan fluorescent protein (ECFP)-labeled Ggamma2. Therefore, when expressed in sympathetic neurons, Gbeta3-s appears to lack biological activity--hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of Gbeta3.
Ruiz-Velasco
Victor
V
Laboratory of Molecular Physiology, Guthrie Research Institute, Sayre, Pennsylvania 18840, USA. vruizvel@inet.guthrie.org
Ikeda
Stephen R
SR
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000235:genetics / Q000378:metabolism*; D002469:Cell Separation; D019281:Dimerization; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D018014:Gene Transfer Techniques; D014644:Genetic Variation / Q000235:genetics*; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D037342:Pertussis Toxin / Q000494:pharmacology; D010957:Plasmids / Q000096:biosynthesis / Q000235:genetics; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017783:Superior Cervical Ganglion / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D014652:Vascular Diseases / Q000235:genetics
D001426:Bacterial Proteins; D020864:Calcium Channels, N-Type; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C454420:G-protein beta3 subunit; D008164:Luminescent Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D037342:Pertussis Toxin; D020962:Heterotrimeric GTP-Binding Proteins