Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

BACKGROUND AND PURPOSEThe cysteine-rich head region, which is adjacent to the proposed ATP-binding pocket in the extracellular ligand-binding loop of P2X receptors for ATP, is absent in the antagonist-insensitive Dictyostelium receptors. In this study we have determined the contribution of the head region to the antagonist action of NF449 and suramin at the human P2X1 receptor.EXPERIMENTAL APPROACHChimeras and point mutations in the cysteine-rich head region were made between human P2X1 and P2X2 receptors. Mutant receptors were expressed in Xenopus oocytes and P2X receptor currents characterized using two-electrode voltage clamp.KEY RESULTSThe chimera replacing the region between the third and fourth conserved cysteine residues of the P2X1 receptor with the corresponding part of P2X2 reduced NF449 sensitivity a thousand fold from an IC50 of ∼1 nM at the P2X1 receptor to that of the P2X2 receptor (IC50∼1 µM). A similar decrease in sensitivity resulted from mutation of four positively charged P2X1 receptor residues in this region that are absent from the P2X2 receptor. These chimeras and mutations were also involved in determining sensitivity to the antagonist suramin. Reciprocal chimeras and mutations in the P2X2 receptor produced modest increases in antagonist sensitivity.CONCLUSIONS AND IMPLICATIONSThese data indicate that a cluster of positively charged residues at the base of the cysteine-rich head region can account for the highly selective antagonism of the P2X1 receptor by the suramin derivative NF449.

Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. AbstractThe type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.

Key points There are two subtypes of trimeric intracellular cation (TRIC) channels but their distinct single‐channel properties and physiological regulation have not been characterized. We examined the differences in function between native skeletal muscle sarcoplasmic reticulum (SR) K+‐channels from wild‐type (WT) mice (where TRIC‐A is the principal subtype) and from Tric‐a knockout (KO) mice that only express TRIC‐B. We find that lone SR K+‐channels from Tric‐a KO mice have a lower open probability and gate more frequently in subconducting states than channels from WT mice but, unlike channels from WT mice, multiple channels gate with high open probability with a more than six‐fold increase in activity when four channels are present in the bilayer. No evidence was found for a direct gating interaction between ryanodine receptor and SR K+‐channels in Tric‐a KO SR, suggesting that TRIC‐B–TRIC‐B interactions are highly specific and may be important for meeting counterion requirements during excitation–contraction coupling in tissues where TRIC‐A is sparse or absent. Abstract The trimeric intracellular cation channels, TRIC‐A and TRIC‐B, represent two subtypes of sarcoplasmic reticulum (SR) K+‐channel but their individual functional roles are unknown. We therefore compared the biophysical properties of SR K+‐channels derived from the skeletal muscle of wild‐type (WT) or Tric‐a knockout (KO) mice. Because TRIC‐A is the major TRIC‐subtype in skeletal muscle, WT SR will predominantly contain TRIC‐A channels, whereas Tric‐a KO SR will only contain TRIC‐B channels. When lone SR K+‐channels were incorporated into bilayers, the open probability (Po) of channels from Tric‐a KO mice was markedly lower than that of channels from WT mice; gating was characterized by shorter opening bursts and more frequent brief subconductance openings. However, unlike channels from WT mice, the Po of SR K+‐channels from Tric‐a KO mice increased as increasing channel numbers were present in the bilayer, driving the channels into long sojourns in the fully open state. When co‐incorporated into bilayers, ryanodine receptor channels did not directly affect the gating of SR K+‐channels, nor did the presence or absence of SR K+‐channels influence ryanodine receptor activity. We suggest that because of high expression levels in striated muscle, TRIC‐A produces most of the counterion flux required during excitation‐contraction coupling. TRIC‐B, in contrast, is sparsely expressed in most cells and, although lone TRIC‐B channels exhibit low Po, the high Po levels reached by multiple TRIC‐B channels may provide a compensatory mechanism to rapidly restore K+ gradients and charge differences across the SR of tissues containing few TRIC‐A channels.

were observed at 10-100 mM Ca 2þ . However, mutations on E3893 and E3967 impaired Ca 2þ -dependent activation of RyR1. E3893Q/E3967Q double mutant showed relatively high activity at submicromolar Ca 2þ and was inhibited by 10-100 mM Ca 2þ , while the mutant retained activation by ATP and caffeine. T5001A mutation decreased the affinity for the activation Ca 2þ . A mutation on Q3970 was reported to be associated with congenital skeletal myopathy, central core disease (CCD) [2] . We found that CCD-linked Q3970K mutation attenuated Ca 2þ activation of RyR1 as well as caffeine and ATP activation in single channel recordings. An interesting finding is that Q3970K mutation did not alter single channel conductance of RyR1, while previous studies showed decreased single channel conductance of other loss-of-function CCD mutants of RyR1. Combined with in silico analysis of the mutant structure, our functional studies reveal that the Ca 2þ binding site identified in the high resolution RyR1 structure serves as a Ca 2þ regulatory domain. Supported by NIH. Ryanodine receptors (RyRs), a group of homotetrameric intracellular calcium release channels, play a key role in muscular and neuronal activities. RyRsmediated calcium release can be activated and regulated by Ca(2þ) binding in skeletal muscle (to RyR1) and cardiac muscle (to RyR2). Despite recent progress in structural studies of RyRs, a detailed understanding of the activation mechanism of RyRs has been obscured by the lack of high-resolution structural and dynamical information for RyRs in different functional states. To elucidate how Ca(2þ) binding impacts the dynamics and energetics of RyR1, we performed molecular dynamics simulation of the C-terminal core domain of an RyR1 Cryo-EM structure in the presence and absence of Ca(2þ). Our simulation revealed elevated dynamics and specific interactions in the presence of Ca(2þ), which may prime RyR1 for activation. The structural and dynamic insights gained from this study will guide future functional studies of the RyR1 Ca(2þ) activation. The type 2 ryanodine receptor (RyR2) is the Ca 2þ release channel on the sarcoplasmic reticulum and plays a pivotal role in the excitation-contraction coupling in the heart. It is known that excessive activation of RyR2 by abnormal phosphorylation or amino acid mutation increases spontaneous Ca 2þ release in cardiomyocytes and results in arrhythmia. Therefore, drugs that suppress activity of RyR2 are expected to have anti-arrhythmic effects, but specific inhibitors of RyR2 have not been reported yet. Recently, we searched for novel RyR2 inhibitors by high-throughput screening using HEK293 cells expressing RyR2 and ER Ca 2þ indicator R-CEPIA1er, and found four candidates as RyR2 inhibitors. In this study, we assessed their effects on non-cardiac and cardiac cells. All four compounds suppressed spontaneous Ca 2þ release in HEK293 cells expressing RyR2. These compounds more or less suppressed Ca 2þ -dependent [ 3 H]ryanodine binding in microsomes obtained from RyR2 expressing cells. The effe...