A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G-protein 3 subunit (G3). Translation of the alternatively spliced mRNA results in a protein product, G3-s, in which 41 amino acids are deleted from G3. 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 G3-s might play in ion channel modulation. To examine this aspect, G3 or G3-s along with either G2 or G5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein. In contrast to G3, expression of G3-s did not modulate N-type Ca²⁺ or G-protein-gated inwardly rectifying K⁺ channels. In addition, G3-s did not appear to complex with a pertussis toxin-insensitive mutant of G_i2 or couple to natively expressed ₂-adrenergic receptors. Finally, fluorescence resonance energy transfer (FRET) measurements indicated that EYFP-labeled G3-s does not form a G heterodimer when coexpressed with ECFP-labeled G2. Therefore, when expressed in sympathetic neurons, G3-s appears to lack biological activity--hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of G3.