The beta-subunit of voltage-gated Ca(2+) channels is essential for trafficking the channels to the plasma membrane and regulating their gating. It contains a Src homology 3 (SH3) domain and a guanylate kinase (GK) domain, which interact intramolecularly. We investigated the structural underpinnings of this intramolecular coupling and found that in addition to a previously described SH3 domain beta strand, two structural elements are crucial for maintaining a strong and yet potentially modifiable SH3-GK intramolecular coupling: an intrinsically weak SH3-GK interface and a direct connection of the SH3 and GK domains. Alterations of these elements uncouple the two functions of the beta-subunit, degrading its ability to regulate gating while leaving its chaperone effect intact.
G-protein (Gβγ)-mediated voltage-dependent inhibition of N- and P/Q-type Ca 2+ channels contributes to presynaptic inhibition and short-term synaptic plasticity. The voltage dependence derives from the dissociation of Gβγ from the inhibited channels, but the underlying molecular and biophysical mechanisms remain largely unclear. In this study we investigated the role in this process of Ca 2+ channel β subunit (Ca v β) and a rigid α-helical structure between the α-interacting domain (AID), the primary Ca v β docking site on the channel α 1 subunit, and the pore-lining IS6 segment. Gβγ inhibition of P/Q-type channels was reconstituted in giant inside-out membrane patches from Xenopus oocytes. Large populations of channels devoid of Ca v β were produced by washing out a mutant Ca v β with a reduced affinity for the AID. These β-less channels were still inhibited by Gβγ, but without any voltage dependence, indicating that Ca v β is indispensable for voltage-dependent Gβγ inhibition. A truncated Ca v β containing only the AID-binding guanylate kinase (GK) domain could fully confer voltage dependence to Gβγ inhibition. Gβγ did not alter inactivation properties, and channels recovered from Gβγ inhibition exhibited the same activation property as un-inhibited channels, indicating that Gβγ does not dislodge Ca v β from the inhibited channel. Furthermore, voltage-dependent Gβγ inhibition was abolished when the rigid α-helix between the AID and IS6 was disrupted by insertion of multiple glycines, which also eliminated Ca v β regulation of channel gating, revealing a pivotal role of this rigid α-helix in both processes. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gβγ-binding pocket through a rigid α-helix induced partly by the Ca v β GK domain, causes the dissociation of Gβγ and is fundamental to voltage-dependent Gβγ inhibition.
Trimeric intracellular cation (TRIC) channels are thought to provide counter-ion currents that facilitate the active release of Ca 2+ from intracellular stores. TRIC activity is controlled by voltage and Ca 2+ modulation, but underlying mechanisms have remained unknown. Here we describe high-resolution crystal structures of vertebrate TRIC-A and TRIC-B channels, both in Ca 2+ -bound and Ca 2+ -free states, and we analyze conductance properties in structure-inspired mutagenesis experiments. The TRIC channels are symmetric trimers, wherein we find a pore in each protomer that is gated by a highly conserved lysine residue. In the resting state, Ca 2+ binding at the luminal surface of TRIC-A, on its threefold axis, stabilizes lysine blockage of the pores. During active Ca 2+ release, luminal Ca 2+ depletion removes inhibition to permit the lysine-bearing and voltage-sensing helix to move in response to consequent membrane hyperpolarization. Diacylglycerol is found at interprotomer interfaces, suggesting a role in metabolic control.
Centromere is a specialized chromatin domain that plays a vital role in chromosome segregation. In most eukaryotes, centromere is surrounded by the epigenetically distinct heterochromatin domain. Heterochromatin has been shown to contribute to centromere function, but the precise role of heterochromatin in centromere specification remains elusive. Centromeres in most eukaryotes, including fission yeast (Schizosaccharomyces pombe), are defined epigenetically by the histone H3 (H3) variant CENP-A. In contrast, the budding yeast Saccharomyces cerevisiae has genetically-defined point centromeres. The transition between regional centromeres and point centromeres is considered as one of the most dramatic evolutionary events in centromere evolution. Here we demonstrated that Cse4, the budding yeast CENP-A homolog, can localize to centromeres in fission yeast and partially substitute fission yeast CENP-ACnp1. But overexpression of Cse4 results in its localization to heterochromatic regions. Cse4 is subject to efficient ubiquitin-dependent degradation in S. pombe, and its N-terminal domain dictates its centromere distribution via ubiquitination. Notably, without heterochromatin and RNA interference (RNAi), Cse4 fails to associate with centromeres. We showed that RNAi-dependent heterochromatin mediates centromeric localization of Cse4 by protecting Cse4 from ubiquitin-dependent degradation. Heterochromatin also contributes to the association of native CENP-ACnp1 with centromeres via the same mechanism. These findings suggest that protection of CENP-A from degradation by heterochromatin is a general mechanism used for centromere assembly, and also provide novel insights into centromere evolution.
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