Voltage-sensitivity has recently been demonstrated for agonist potency and affinity at certain G protein-coupled receptors. Using an electrophysiology assay in Xenopus oocytes, we have previously shown that the potency of dopamine in activating G protein-coupled potassium channels (GIRK) via the dopamine D2S receptor is reduced by depolarization from −80 to 0 mV. We recently investigated the voltage-sensitivities of a range of structurally related dopaminergic agonists at the D2S receptor.The findings of this study led us to propose that a conformationally constrained interaction of the agonist with transmembrane segment (TM) VI of D2 is required for voltage-sensitivity. The hypothesis assumes that for the flexible phenethylamines, two hydroxyls (such as in dopamine) interacting with the conserved serines in TM V are necessary for voltage-sensitivity. Conversely, N,N-dipropyl-2-aminotetralin (DPAT) agonists do not require hydroxyls for voltage-sensitivity due to their inherently more rigid structure. To test this hypothesis, we mutated three conserved serines in TM V (S193A, S194A, and S197A) which have been shown to mediate binding to agonist hydroxyls.The voltage-sensitivity of non-hydroxylated DPAT was similar to that observed with the wild-type receptor at all of the three mutants, suggesting that the mutations did not allosterically alter the voltage-sensing properties of the receptor.The S193A mutation drastically diminished voltage-sensitivity of dopamine, concomitantly with a marked reduction in potency. However, the S194A mutation which slightly decreased potency, did not appreciably affect the voltage-sensitivity of dopamine. At the S197A mutant, dopamine efficacy was decreased to such a degree that voltage-sensitivity could not be assessed.In the literature, S193 has consistently been assigned a major role in dopamine binding. Our results suggest that this residue might also be important for voltage-sensitive interactions between dopamine and the D2S receptor.