High-voltage activated (HVA) calcium channels are heteromultimers composed of a main pore-forming Ca_Vα1 subunit associated with auxiliary subunits β and α2δ. The β subunits modulate biophysical properties of the channels and promote the voltage-dependence of modulation of N-type calcium channels by G proteins (Meir et al. 2000). They bind to an interaction site of 18 amino-acids (QQxExxLxGYxxWIxxxE), the alpha-interaction domain (AID), conserved in the I-II loop of the Ca_Vα1 subunit of all HVA calcium channels. By mutating W391 to A in the AID of Ca_V2.2, Biacore experiments confirmed that this amino-acid is essential for β1b subunit binding (Fig. 1), for plasma membrane expression and for modulation of the biophysical properties of Ca_V2.2 channels. Whereas robust currents with 10 mM barium as charge carrier were recorded using the patch-clamp technique on tsA-201 cells co-expressing Ca_V2.2 with β1b (−219.5±41 pA/pF at +20 mV ; mean±S.E.M., n=12), co-transfection of Ca_V2.2(W391A) with β1b induced small currents (−31.7±7.4 pA/pF at +20 mV ; n=11; p<0.01 using an unpaired Student's t test). Moreover, without ,β Ca_V2.2 produced small but recordable currents (−8.2±1.1 pA/pF at +20 mV ; n=13; p<0.01) while no currents were recorded when Ca2.2(W391A) was expressed alone (n=21), suggesting that some endogenous β may be present in tsA-201 cells and trafficking wild type Ca_V2.2. We then studied the G protein modulation of these channels by co-expressing a dopamine D2 receptor and activating it with the D2 receptor agonist quinpirole (100 nM). For the wild-type channel, quinpirole induced an inhibition of 53.5±3.8% (n=12; p<0.01) of the currents, a +7 mV shift of activation and prepulse potentiation was observed. The Ca_V2.2(W391A) mutant was still inhibited (46.4±8% n=11; p<0.01) by quinpirole and a positive shift of +6.5 mV for its activation still occurred but no prepulse potentiation was observed for this mutant. In contrast, only the expression of the channel at the plasma membrane was affected when Ca_Vβ2a was co-expressed with this mutant channel, while all the biophysical properties of the expressed Ca_V2.2W391A channels were still normally modulated by the palmitoylated Ca_Vβ2a including the voltage-dependence of G-protein modulation, suggesting another level of modulation by this subunit. These results confirm that the W391 is crucial for β subunit binding to the I-II linker of Ca_V2.2, and show that no Ca_V2.2 is functional at the plasma membrane in the complete absence of interaction with a β subunit. The results also provide evidence for the role of β subunits in the voltage-dependence of modulation of N-type calcium channels by G protein-coupled receptors.