Most of the experiments in our study focused on the surface interaction of VGCC with -subunits. Their interaction is particularly important for the activation of VGCC20,47 during the development of chronic pain26,48 and synaptogenesis,16 where the channels are particularly the dominant calcium channel in immature hippocampal glutamatergic synapses.49 Probing the dynamics of the two subunits showed that channels and -subunits are not permanently associated but rather transiently associate in both HEK293 cells and neurons. Using extracellular and intracellular tagged channels and -subunits revealed the existence of three populations: free channels, free -subunits, and associated channels with -subunits. If there will indeed be a stoichiometry between the channel subunits,6,18 the weak affinity () might be compensated by different expression levels and confinement of subunits in the membrane, particular in synapses. Comparing the dynamics of both subunits [Figs. 2(e) and 2(g), 4(a)–4(d), 5(a)–5(d)] confirmed a substantial difference in the confinement of channels and -subunits [Figs. 3(b) and 3(c), 4(a)–4(d), 5(a)–5(d)]. This observation was further substantiated by determining the colocalization of calcium channel subunits in the plane of the cell membrane using STED microscopy [Figs. 3(d) and 3(e)]. The physiological consequences are proposed by the altered voltage-dependent inactivation of evoked barium currents from tagged channels where the interaction between channels and -subunits is altered [, Fig. 1(d)]. Whether the dynamic subunit associations will be a mechanism that influences channel activity in their natural environment needs to be further explored. Interestingly, the affinity of -subunits and -subunits seems to be different between different calcium channels as observed in the altered diffusion coefficient of -subunits when expressed with , , , or channels [Fig. 2(f)]. The strongest association seems to exist between and -subunits, whereas channels do not seem to interact with -subunits at the cell surface. With respect to the physiological channel function and subunit association, -subunits are identified to tune the voltage-dependent opening of channels.20,47,50 Recently, the first crystal structure of the channel complex has been described by the use of cryo-electron microscopy. Here, the authors demonstrate that the interface of binding between and -subunit is between the extracellular loops of the transmembrane repeats I–III and the von Willebrand A domain and cache1 domain of the -subunit.18 Using voltage clamp fluorometry, it has been shown that the channel -subunit interaction with the voltage sensor domains within the transmembrane repeats I–III is relevant for the activation of the channel.20 In light of these recent structure-function data, we cannot fully exclude that the tagging of the -subunit on the N-terminus will influence the association of the subunits. However, the functional tests with and without HA-tagged -subunits (Fig. 1) speak against a major impact of the chosen tag position. Whether the proposed dynamic association between -subunits and channels will be of functional relevance in neurons has to be tested. In specific compartments, the synapse channel densities can be tenfold higher5,51,52 as in the axon controlled by many molecular interactions.53 Our first data in neurons suggest that -subunits are more confined in synapses, which is rather due to other interactions than a specific affinity to channels (Fig. 4). The interaction with extracellular matrix proteins16,54 might be effective to confine -subunits in the synapse. Our simulations suggest that such local molecular crowding has an impact on the association of channel subunits. The simulation further indicated that the copy numbers for channels and -subunits should be very different to have a certain population of -subunits bound to the channels. The confinement and increased density of channels themselves can be assigned to many intracellular interaction partners, particularly in the synapse.55–58 Whether other molecules, e.g., synaptic adhesion proteins such as neurexins,59 participate in the confinement of channels or -subunits remains to be tested.