Abstract
Auxiliary beta subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca(2+) channels in heterologous cells. In brain, all four beta isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of beta subunits and their role in membrane expression of Ca(V)1.2 alpha(1) subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous beta subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged beta subunits demonstrated that all beta subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca(V)1.2, thus indicating a great promiscuity in alpha(1)-beta interactions. In contrast, restricted axonal targeting of beta(1) and weak colocalization of beta(4b) with Ca(V)1.2 indicated isoform-specific differences in local channel complex formation. Membrane expression of external hemagglutinin epitope-tagged Ca(V)1.2 was strongly enhanced by all beta subunits in an isoform-specific manner. Conversely, mutating the alpha-interaction domain of Ca(V)1.2 (W440A) abolished membrane expression and targeting into dendritic spines. This demonstrates that in neurons the interaction of a beta subunit with the alpha-interaction domain is absolutely essential for membrane expression of alpha(1) subunits, as well as for the subcellular localization of beta subunits, which by themselves possess little or no targeting properties.
Highlights
Voltage-gated Ca2ϩ channels (CaV)3 provide key pathways for Ca2ϩ entry into neurons and translate membrane depolarization into neurotransmitter secretion and gene regulation
Cultured Hippocampal Neurons Express mRNA and Protein of All Four Ca2ϩ Channel  Subunits—It has previously been shown that the hippocampus expresses mRNA and protein of the CaV ␣1 subunits (CaV1.2, CaV1.3, CaV2.1, CaV2.2, and CaV2.3), ␣2␦ (␣2␦-1, ␣2␦-2, and ␣2␦-3), and of all four  subunits [12,13,14,15, 21, 23]
Little is known about the subcellular distribution of  subunits in neurons and about the specific subunit composition of CaV complexes in pre- and postsynaptic compartments
Summary
Low density cultures of hippocampal neurons were prepared from 16.5-day-old embryonic BALB/c mice as described previously [15, 37, 69]. Plasmids were introduced into neurons on day 6 using Lipofectamine 2000 transfection reagent (Invitrogen) as described previously [15]. For single transfection experiments (pA--V5 constructs), 0.5–2 g of DNA at a molar ratio of 1:1 were used, and for cotransfection experiments (pA-eGFP, pA-CaV1.2-HA, and pA-x-V5), 1–2.5 g of total DNA at a molar ratio of 1:1 were used. Cells were immunostained and analyzed 6 –19 days after transfection
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