Ca V 1 and Ca V 2 voltage-gated calcium channels are associated with β and α 2 δ accessory subunits. However, examination of cell surface-associated Ca V 2 channels has been hampered by the lack of antibodies to cell surface-accessible epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the plasma membrane, in both a neuronal cell line and neurons. We first examined the effect of the auxiliary subunits. Although α 2 δ subunits copurify with Ca V 2 channels, it has recently been suggested that this interaction is easily disrupted and nonquantitative. We have now tested whether α 2 δ subunits are associated with these channels at the cell surface. We found that, whereas α 2 δ-1 is readily observed at the plasma membrane when expressed alone, it appears absent when coexpressed with Ca V 2.2/β1b, despite our finding that α 2 δ-1 increases plasmamembrane Ca V 2.2 expression. However, this was due to occlusion of the antigenic epitope by association with Ca V 2.2, as revealed by antigen retrieval; thus, our data provide evidence for a tight interaction between α 2 δ-1 and the α 1 subunit at the plasma membrane. We further show that, although Ca V 2.2 cell-surface expression is reduced by gabapentin in the presence of wild-type α 2 δ-1 (but not a gabapentin-insensitive α 2 δ-1 mutant), the interaction between Ca V 2.2 and α 2 δ-1 is not disrupted by gabapentin. Altogether, these results demonstrate that Ca V 2.2 and α 2 δ-1 are intimately associated at the plasma membrane and allow us to infer a region of interaction. P urification of L-type voltage-gated calcium (Ca V ) channels from skeletal muscle shows that they consist of a pore-forming α 1 subunit, Ca V 1.1, associated with three accessory subunits, β1, α 2 δ-1, and γ 1 (1, 2). Cardiac L-type channels have a similar subunit composition, although the α 1 subunit is α1C and the γ subunit is not present (3). However, the study of cell surface-associated N-type (Ca V 2.2) and P/Q-type (Ca V 2.1) calcium channels has been hampered by the lack both of antibodies to cell-surface epitopes and of functional exofacially tagged Ca V 2 channels. Here we report the development of fully functional Ca V 2.2 constructs containing inserted surface-accessible exofacial tags, which allow visualization of only those channels at the cell surface, in both cell lines and neurons. Using this methodological advance, we can now examine directly the effect of the auxiliary subunits on cellsurface expression of Ca V 2 channels.Although α 2 δ subunits have been shown to be associated with Ca V 2.1 and Ca V 2.2 following purification (4, 5), it has recently been suggested that the α 2 δ subunits are associated only very loosely and nonquantitatively with Ca V 2 channels (6), calling into question their role as calcium channel subunits. This study found that the α 2 δ proteins α 2 δ-1, α 2 δ-2, and α 2 δ-3 c...

Fragile X syndrome (FXS), the most common heritable form of mental retardation, is characterized by synaptic dysfunction. Synaptic transmission depends critically on presynaptic calcium entry via voltage-gated calcium (CaV) channels. Here we show that the functional expression of neuronal N-type CaV channels (CaV2.2) is regulated by fragile X mental retardation protein (FMRP). We find that FMRP knockdown in dorsal root ganglion neurons increases CaV channel density in somata and in presynaptic terminals. We then show that FMRP controls CaV2.2 surface expression by targeting the channels to the proteasome for degradation. The interaction between FMRP and CaV2.2 occurs between the carboxy-terminal domain of FMRP and domains of CaV2.2 known to interact with the neurotransmitter release machinery. Finally, we show that FMRP controls synaptic exocytosis via CaV2.2 channels. Our data indicate that FMRP is a potent regulator of presynaptic activity, and its loss is likely to contribute to synaptic dysfunction in FXS.

As auxiliary subunits of voltage-gated Ca 2ϩ channels, the ␣ 2 ␦ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the ␣ 2 ␦-1 subunit in sensory dorsal root ganglion neurons contributes to the generation of chronic pain states; however, very little is known about the underlying molecular mechanisms. Here we show that the increased expression of ␣ 2 ␦-1 in rat sensory neurons leads to prolonged Ca 2ϩ responses evoked by membrane depolarization. This mechanism is coupled to Ca V 2.2 channel-mediated responses, as it is blocked by a -conotoxin GVIA application. Once initiated, the prolonged Ca 2ϩ transients are not dependent on extracellular Ca 2ϩ and do not require Ca 2ϩ release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca 2ϩ uptake demonstrates that ␣ 2 ␦-1-mediated prolonged Ca 2ϩ signals are buffered by mitochondria, preferentially activated by Ca 2ϩ influx through Ca V 2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the ␣ 2 ␦-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca 2ϩ signaling in dorsal root ganglion neurons.

N type Calcium Channels are voltage gated channels composed of a pore forming a1B (Cav2.2) subunit and 2 auxiliary subunits a2d and b. N type calcium channels regulate calcium influx and exocytosis in presynaptic terminals of neurons. Tight control of Cav2.2 trafficking is important as small changes in channel density at the cell surface can result in large changes in calcium ion influx and therefore neurotransmitter release. The auxiliary subunits a2d and b modulate the biophysical properties and surface channel density of Cav2.2 through mechanisms that are not fully defined. To investigate the contribution of these subunits to Cav2.2 trafficking we have created a fully functional exofacially tagged Cav2.2. Using this tool we are able to quantify the contribution of each subunit on Cav2.2 trafficking to the surface of N2a cells (mouse neuroblastoma derived cell line). Our results support previous electrophysiological data indicating the absolute requirement for the b subunit and additional enhancement by the a2d subunit. Our data also suggest an intracellular site of interaction between Cav2.2 and a2d. Epitope obstruction of a2d bound to Cav2.2 may indicate the region of a2d involved in interaction with Cav2.2.