The Voltage-dependent Calcium Channel β Subunit Contains Two Stable Interacting Domains

Abstract

Voltage-dependent calcium channels selectively enable Ca 2؉ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. ␣ 1 is the membrane pore-forming subunit, whereas ␤ is an intracellular subunit that binds to ␣ 1 , facilitating and modulating channel function. We have expressed, purified, and characterized recombinant ␤ 3 and ␤ 2a using both biochemical and biophysical methods, including electrophysiology, to better understand the ␤ family's protein structural and functional correlates. Our results indicate that the ␤ protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when ␤ is not in complex with the channel. In addition, the ␤ structural core, comprised of just these two domains without other sequences, binds tightly to the ␣ interaction domain (AID) motif, a sequence derived from the ␣ 1 subunit and the principal anchor site of ␤. Domain II is responsible for this binding, but domain I enhances it.Voltage-dependent calcium channels (VDCCs) 1 permit the flow of Ca 2ϩ ions through cellular membranes as a function of membrane potential. These protein complexes are central components in a variety of physiological systems of organisms, ranging from yeast to human. They play pivotal roles in signal transduction and homeostasis processes.Functional roles for these channels vary based on cell type. In muscle, both skeletal and cardiac, the predominant VDCCs (Ca V 1.1 and Ca V 1.2) cause release of Ca 2ϩ into the cytosol from intracellular stores, thereby initiating contraction (1, 2). In neurons and endocrine cells, neurotransmitter or hormone secretion requires VDCC activity. In addition, electrical activity, specifically the action potential in cardiac myocytes, is regulated by VDCCs. Finally, calcium influx and concentration controlled by VDCCs plays a significant role in neuronal gene expression (3). Pathways have been elucidated, where for one example, VDCC activity gives rise to phosphorylation of CREB, a transcription factor (4), thereby activating transcription of a myriad of target genes.The VDCC comprises four distinct polypeptides: ␣ 1 , ␣ 2 ␦, ␤, and ␥ (5). ␣ 1 is the membrane pore-forming subunit and numbers between 1800 and 2400 residues in length. Its sequence exhibits repeats comprising four transmembrane modules or domains, akin to the tetrameric architecture of potassium channels. Each module contains the canonical transmembrane arrangement for voltage-gated ion channels i.e. six transmembrane segments. Modules are connected by linkers that are located in the intracellular milieu, as are both the N and C termini. The high voltage-activated channel subunits, Ca V 1.x and Ca V 2.x (␣ 1 ), numbering seven in total, share a high degree of sequence similarity but nevertheless encode distinct electrophysiological activities.The ␤ subunit was firs...

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