Dopamine receptor type 1 modulates CaV2.2 current density

MARTINEZ DAMONTE, V.; CHOU-FREED CAMBRIA; MC CARTHY CLARA INES; RAINGO JESICA; RODRIGUEZ SILVIA S.

San Diego

Congreso; Meeting of the Society for Neuroscience 2018; 2018

Society fro Neuroscience

D1R interacts with CaV2.2 and modulates its trafficking to the plasma membrane. This effect has been shown to regulate CaV2.2 density at postsynaptic sites in the prefrontal cortex (Kisilevsky et al. 2008). Here we studied the cellular mechanisms involved in CaV2.2 regulation by D1R using a heterologous expression system. We manipulated the concentration of transfected CaV2.2 and YFP-tagged D1R cDNA in HEK293t cells and measured D1R expression levels by fluorescence signal. There was a linear correlation between the amount of D1R transfected and YFP signal level, as expected. We recorded whole-cell calcium currents in cells expressing CaV2.2, its auxiliary subunits and increasing concentrations of D1R. We found a positive correlation between CaV2.2 current density and D1R expression levels at low D1R/CaV2.2 cDNA molar ratios (D1R/CaV2.2 MR) (CaV2.2 alone= 79.31 ± 11.04 pA/pF, 0.1 D1R/CaV2.2 MR= 114.3 ± 9.42 pA/pF; P=0.023). Moreover, this effect was occluded by Cholero Toxin, a Gs protein inhibitor. To evaluate if this effect was due to an increase in functional channels at the plasma membrane, we measured total charge movement during ON gating current evoked by steps from -100 mV to the reversal potential. We found that the charge movement increased from 7.6 ± 1.2 fC/pF (CaV2.2 alone) to 12.6 ± 0.22 fC/pF (0.1 D1R/CaV2.2 MR). At D1R cDNA concentrations above 0.1 D1R/CaV2.2 MR, the CaV2.2 current density was reduced, reaching values below the control condition without D1R (CaV2.2 alone= 79.31 ± 11.04, 1 D1R/CaV2.2 MR= 43.35 ± 4.88 pA/pF; P=0.0008). This current reduction was insensitive to Cholero Toxin. We hypothesized that high D1R expression levels could promote the formation of D1R homodimers. We therefore assayed the effect of adding equimolar cDNA concentrations of other receptors that are able to form heterodimers with D1R (ghrelin receptor and D2R) and found that this maneuver occluded the calcium current increase. Finally, we compared the acute inhibitory effect of dopamine mediated activation of D1R on CaV2.2 currents. We found a shift in the EC50 (3.28 ± 0.32 μM at 0.1 D1R/CaV2.2 MR versus 0.79 ± 1.5 μM at 1 D1R/CaV2.2 MR) without changes in the maximum inhibitory effect (~50 % of inhibition). This result suggests that D1R expression level impacts its activation mode. In summary, we found a biphasic effect of D1R co-expression on CaV2.2 current: at low D1R expression levels, a stimulation of traffic that implicates Gs protein activity, and in contrast, at high D1R expression levels, a reduction in current and increased sensitivity to dopamine-mediated inhibition, possibly due to the formation of D1R dimers and involving a different signaling pathway.