Reciprocal Interactions Regulate Targeting of Calcium Channel β Subunits and Membrane Expression of α1 Subunits in Cultured Hippocampal Neurons

Abstract

Auxiliary ␤ subunits modulate current properties and mediate the functional membrane expression of voltage-gated Ca 2؉ channels in heterologous cells. In brain, all four ␤ isoforms are widely expressed, yet little is known about their specific roles in neuronal functions. Here, we investigated the expression and targeting properties of ␤ subunits and their role in membrane expression of Ca V 1.2 ␣ 1 subunits in cultured hippocampal neurons. Quantitative reverse transcription-PCR showed equal expression, and immunofluorescence showed a similar distribution of all endogenous ␤ subunits throughout dendrites and axons. High resolution microscopy of hippocampal neurons transfected with six different V5 epitope-tagged ␤ subunits demonstrated that all ␤ subunits were able to accumulate in synaptic terminals and to colocalize with postsynaptic Ca V 1.2, thus indicating a great promiscuity in ␣ 1 -␤ interactions. In contrast, restricted axonal targeting of ␤ 1 and weak colocalization of ␤ 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 ␤ subunits in an isoform-specific manner. Conversely, mutating the ␣-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 ␤ subunit with the ␣-interaction domain is absolutely essential for membrane expression of ␣ 1 subunits, as well as for the subcellular localization of ␤ subunits, which by themselves possess little or no targeting properties.Voltage-gated Ca 2ϩ channels (Ca V ) 3 provide key pathways for Ca 2ϩ entry into neurons and translate membrane depolarization into neurotransmitter secretion and gene regulation.Ca V s are composed of a pore-forming ␣ 1 subunit and the auxiliary ␣ 2 ␦ and ␤ subunits (1). Whereas the ␣ 1 subunits are responsible for voltage sensing and ion conduction, the auxiliary subunits have been implicated in membrane targeting and modulation of channel properties (for review see Ref.2). Presynaptic Ca V s regulate neurotransmitter release (3), and postsynaptic Ca V s activate the transcriptional regulators cAMP-response element-binding protein (CREB) and nuclear factor of activated T-cells (NFAT) (4, 5) and thus modulate long term potentiation (6). These functions reflect both the diversity of Ca V isoforms expressed in brain (7-11) and their differential subcellular localization in neurons (12-15).Four distinct ␤ isoforms have been identified (16 -19), all of which are expressed in brain (20 -23). They contain an Src homology 3 domain and a guanylate kinase domain (24 -27). However, the guanylate kinase fold is modified so that it can bind with high affinity to the so-called ␣-interaction domain (AID) in the intracellular I-II linker of Ca V ␣ 1 subunits (28, 29). The Src homology 3 and the guanylate kinase-like domains are highly conserved among the four genes encoding ␤ subunits (Cacnb1-b4; Fig. 1C), whereas t...

Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

“…S5). Membrane expression of Ca V 1.2 in cultured hippocampal neurons was also disrupted after mutation of the key tryptophan residue to alanine (58). Furthermore, double mutations in the same region completely eradicated the Ca V ␤3 stimulation of Fig.…”

The Cavβ subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels

“…4). We also examined the ubiquitination of a mutant Cav1.2 calcium channel construct that cannot associate with the Cavβ subunit (HACav1.2 W440A ) 24,25 . The mutant channel showed robust ubiquitination irrespective of the coexpression of Cavβ 1b (Fig.…”

The Ca2+channel β2subunit is selectively targeted to the axon terminals of supraoptic neurons

“…It has been proposed by Obermair et al 14 that the selective targeting of Ca V β subunits to neuronal terminals depends on their association with specific Ca V α 1 subunits that are themselves selectively targeted and this could form an alternative hypothesis for the targeting of the Ca V β 2 subunit to MNC terminals. These authors suggested that Ca V β 2 may be slightly more likely to associate with somatodendritic Ca V 1.2 channels whereas Ca V β 4 may be more likely to interact with presynaptic Ca V channels.…”

“…In contrast, a study of the distribution of fluorescently labeled Ca V β subunits expressed in cultured hippocampal neurons found that all Ca V β subunits accumulate in synaptic terminals, 14 which led the authors to conclude that Ca V β subunits do not possess targeting properties.…”