Calcium regulates a wide spectrum of physiological processes such as heartbeat, muscle contraction, neuronal communication, hormone release, cell division, and gene transcription. Major entry-ways for Ca2+ in excitable cells are high-voltage activated (HVA) Ca2+channels. These are plasma membrane proteins composed of several subunits, including α1, α2δ, β, and γ. Although the principal α1 subunit (Cavα1) contains the channel pore, gating machinery and most drug binding sites, the cytosolic auxiliary β subunit (Cavβ) plays an essential role in regulating the surface expression and gating properties of HVA Ca2+ channels. Cavβ is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, and the Ras-related RGK GTPases. New proteins have emerged in recent years that modulate HVA Ca2+ channels by binding to Cavβ. There are also indications that Cavβ may carry out Ca2+ channel-independent functions, including directly regulating gene transcription. All four subtypes of Cavβ, encoded by different genes, have a modular organization, consisting of three variable regions, a conserved guanylate kinase (GK) domain, and a conserved Src-homology 3 (SH3) domain, placing them into the membrane-associated guanylate kinase (MAGUK) protein family. Crystal structures of Cavβs reveal how they interact with Cavα1, open new research avenues, and prompt new inquiries. In this article, we review the structure and various biological functions of Cavβ, with both a historical perspective as well as an emphasis on recent advances.

Mutations in PKD1 and TRPP2 account for nearly all cases of autosomal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the pathogenic mechanism of some ADPKD-producing mutations.autosomal dominant polycystic kidney disease ͉ single-molecule imaging ͉ stoichiometry ͉ transient receptor potential channel ͉ X-ray crystallography

The voltage-gated Ca2+ channel β subunit (Cavβ) is a cytosolic auxiliary subunit that plays an essential role in regulating the surface expression and gating properties of high-voltage activated (HVA) Ca2+ channels. It is also crucial for the modulation of HVA Ca2+ channels by G proteins, kinases, Ras-related RGK GTPases, and other proteins. There are indications that Cavβ may carry out Ca2+ channel-independent functions. Cavβ knockouts are either non-viable or result in a severe pathophysiology, and mutations in Cavβ have been implicated in disease. In this article, we review the structure and various biological functions of Cavβ, as well as recent advances.

The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC(50) approximately 53 microM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid ( approximately 2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC(50) approximately 300 microM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 microM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation.

Mutations in PKD1 and TRPP2 account for nearly all cases of autoso-mal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the patho-genic mechanism of some ADPKD-producing mutations. autosomal dominant polycystic kidney disease single-molecule imaging stoichiometry transient receptor potential channel X-ray crystallography