Trimeric Intracellular Cation Channels Research Articles

The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.

CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta

Ca2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K+, a counter ion for Ca2+flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-β reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-β treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone.

Mild early course of osteogenesis imperfecta type XIV - a case report

<p><strong>Introduction.</strong> Mutations in TMEM38B gene, which encodes the endoplasmatic reticulum membrane trimeric intracellular cation channel (TRIC) type B, cause osteogenesis imperfecta type XIV. So far there have been only four different pathogenic variants reported in TMEM38B. Clinical features of osteogenesis imperfecta type XIV described in scarce reports include osteopenia, femoral bowing, low trauma fractures, scoliosis, muscular hypotonia and cardiac pathology. </p><p><strong>Case report.</strong> A 2-month-old male infant was referred to a clinical geneticist office due to bone deformities. The shortening of the limbs was observed during the prenatal ultrasound examination in seventh month of pregnancy. Prenatal cytogenetic analysis was performed from a fetal blood sample and showed normal findings. Neither fetal fractures were observed prenatally, nor any occurred during vaginal labor. During the first clinical exam by the clinical geneticist, discrete rhizomelia and bluish sclerae were observed. Due to the suspicion of skeletal dysplasia, indication for genetic analysis was established. Next generation sequencing panel for skeletal dysplasia showed homozygous deletion of exons 1 and 2 in the TMEM38B gene, confirming osteogenesis imperfecta type XIV. At the follow-up visit performed at 12 months of age, no fractures were reported. Several skeletal deformities were observable: discrete frontal bossing, rhizomelic upper extremities, slightly bowed thighs and shins. The infant achieved normal psychomotor development. A radiographic examination showed bowing of long bones of the lower extremities, without significant osteopenia. </p><p><strong>Conclusion.</strong> Absence of early fractures is rare in osteogenesis imperfecta type XIV. Relatively mild clinical features of our patient therefore contribute to the understanding of the phenotype of osteogenesis imperfecta type XIV and its relation to the genotype.</p>

Channel Function of Polycystin-2 in the Endoplasmic Reticulum Protects against Autosomal Dominant Polycystic Kidney Disease.

Mutations of PKD2, which encodes polycystin-2, cause autosomal dominant polycystic kidney disease (ADPKD). The prevailing view is that defects in polycystin-2-mediated calcium ion influx in the primary cilia play a central role in the pathogenesis of cyst growth. However, polycystin-2 is predominantly expressed in the endoplasmic reticulum (ER) and more permeable to potassium ions than to calcium ions. The trimeric intracellular cation (TRIC) channel TRIC-B is an ER-resident potassium channel that mediates potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium ion release. Using TRIC-B as a tool, we examined the function of ER-localized polycystin-2 and its role in ADPKD pathogenesis in cultured cells, zebrafish, and mouse models. Agonist-induced ER calcium ion release was defective in cells lacking polycystin-2 and reversed by exogenous expression of TRIC-B. Vice versa, exogenous polycystin-2 reversed an ER calcium-release defect in cells lacking TRIC-B. In a zebrafish model, expression of wild-type but not nonfunctional TRIC-B suppressed polycystin-2-deficient phenotypes. Similarly, these phenotypes were suppressed by targeting the ROMK potassium channel (normally expressed on the cell surface) to the ER. In cultured cells and polycystin-2-deficient zebrafish phenotypes, polycystin-2 remained capable of reversing the ER calcium release defect even when it was not present in the cilia. Transgenic expression of Tric-b ameliorated cystogenesis in the kidneys of conditional Pkd2-inactivated mice, whereas Tric-b deletion enhanced cystogenesis in Pkd2-heterozygous kidneys. Polycystin-2 in the ER appears to be critical for anticystogenesis and likely functions as a potassium ion channel to facilitate potassium-calcium counterion exchange for inositol trisphosphate-mediated calcium release. The results advance the understanding of ADPKD pathogenesis and provides proof of principle for pharmacotherapy by TRIC-B activators.

Zebrafish tmem38b Knock out unveils the in vivo role of Trimeric Intracellular Cation (TRIC) channel B on cell homeostasis

Zebrafish tmem38b Knock out unveils the in vivo role of Trimeric Intracellular Cation (TRIC) channel B on cell homeostasis

Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of -13.67 ± 9.02 (n = 7), -2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl-. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Knocking out TMEM38B in human foetal osteoblasts hFOB 1.19 by CRISPR/Cas9: A model for recessive OI type XIV.

Osteogenesis imperfecta (OI) type XIV is a rare recessive bone disorder characterized by variable degree of severity associated to osteopenia. It is caused by mutations in TMEM38B encoding for the trimeric intracellular cation channel TRIC-B, specific for potassium and ubiquitously present in the endoplasmic reticulum (ER) membrane. OI type XIV molecular basis is largely unknown and, due to the rarity of the disease, the availability of patients' osteoblasts is challenging. Thus, CRISPR/Cas9 was used to knock out (KO) TMEM38B in the human Foetal Osteoblast hFOB 1.19 to obtain an OI type XIV model. CRISPR/Cas9 is a powerful technology to generate in vitro and in vivo models for heritable disorders. Its limited cost and ease of use make this technique widely applicable in most laboratories. Nevertheless, to fully take advantage of this approach, it is important to be aware of its strengths and limitations. Three gRNAs were used and several KO clones lacking the expression of TRIC-B were obtained. Few clones were validated as good models for the disease since they reproduce the altered ER calcium flux, collagen I structure and impaired secretion and osteoblastic markers expression detected in patients' cells. Impaired proliferation and mineralization in KO clones unveiled the relevance of TRIC-B in osteoblasts functionality.

Knock out of tmem38b by CRISPR/Cas9 in zebrafish unveils the in vivo role of Trimeric intracellular cation (TRIC) channel B on cell homeostasis

Knock out of tmem38b by CRISPR/Cas9 in zebrafish unveils the in vivo role of Trimeric intracellular cation (TRIC) channel B on cell homeostasis

Detection of a Recurrent TMEM38B Gene Deletion Associated with Recessive Osteogenesis Imperfecta.

Osteogenesis imperfecta is a clinically and genetically group of heterogeneous disorders associated with decreased bone density, brittle bones, bone deformity, recurrent fractures, and growth retardation. Osteogenesis imperfecta is commonly associated with mutations of the genes encoding for type I collagen (COL1A1/COL1A2). Mutations in other genes, some associated with type I collagen post-translational processing, have also been identified as the cause of osteogenesis imperfecta. Mutations in the transmembrane protein 38B (TMEM38B) gene have been reported in a rare autosomal recessive form of osteogenesis imperfecta. TMEM38B encodes TRIC-B - a trimeric intracellular cation channel type B which is essential to modulate intracellular calcium signaling. In this study, we are reporting a case of osteogenesis imperfecta type XIV from a Saudi consanguineous family. Our patient was an eight-month-old child with short limbs, club feet, and lower limb deformities with developmental delay. Radiological findings were consistent with the evidence of osteogenesis imperfecta. There was no evidence of impaired hearing or blue sclera and based on the clinical assessment, we classified our patient as a non-syndromic osteogenesis imperfecta. A pathogenic deletion in the chromosome 9q31.2 region, partially encompassing the TMEM38B gene, was detected using chromosomal microarray analysis. This study expands our knowledge about the rare type of osteogenesis imperfecta in our consanguineous population. Besides, it emphasizes the use of genomic medicine in clinical practices to formulate early interventions to clinically improve the patient’s condition.

Decreased cardiac pacemaking and attenuated β-adrenergic response in TRIC-A knockout mice.

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ± 15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ± 5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a β-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to β-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.

TRICking SOCE into altered oscillations

Defective ER/SR-cytosol Ca2+ cycling is associated with increased ER stress, pathological heart conditions and muscular defects. Within the SR, ryanodine receptor 2 (RyR2) is required for excitation/contraction coupling. Ca2+ release from the SR is counterbalanced by K+ influx through trimeric intracellular cation (TRIC) channels to maintain ER/SR polarity. New functions of TRIC channels have been discovered.

Correction to: Trimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium Homeostasis.

HomeCirculation ResearchVol. 126, No. 10Correction to: Trimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium Homeostasis Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: Trimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium Homeostasis Originally published7 May 2020https://doi.org/10.1161/RES.0000000000000350Circulation Research. 2020;126:e119This article corrects the followingTrimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium HomeostasisIn the article by Zhou et al, “Trimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium Homeostasis,” which published in the February 14, 2014 issue of the journal (Circ Res. 2014;114:706-716. doi: 10.1161/CIRCRESAHA.114.301816), a correction was needed.The following funding source has been added: “J. Ma acknowledges grant support from RO1-AR061385.”The authors apologize for the omission.The corrected version of this article is available at www.ahajournals.org/doi/10.1161/CIRCRESAHA.114.301816. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesTrimeric Intracellular Cation Channels and Sarcoplasmic/Endoplasmic Reticulum Calcium HomeostasisXinyu Zhou, et al. Circulation Research. 2014;114:706-716 May 8, 2020Vol 126, Issue 10 Advertisement Article InformationMetrics © 2020 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000350PMID: 32379571 Originally publishedMay 7, 2020 PDF download Advertisement

TRIC-A shapes oscillatory Ca2+ signals by interaction with STIM1/Orai1 complexes.

Trimeric intracellular cation (TRIC) channels have been proposed to modulate Ca2+ release from the endoplasmic reticulum (ER) and determine oscillatory Ca2+ signals. Here, we report that TRIC-A–mediated amplitude and frequency modulation of ryanodine receptor 2 (RyR2)-mediated Ca2+ oscillations and inositol 1,4,5-triphosphate receptor (IP3R)-induced cytosolic signals is based on attenuating store-operated Ca2+ entry (SOCE). Further, TRIC-A–dependent delay in ER Ca2+ store refilling contributes to shaping the pattern of Ca2+ oscillations. Upon ER Ca2+ depletion, TRIC-A clusters with stromal interaction molecule 1 (STIM1) and Ca2+-release–activated Ca2+ channel 1 (Orai1) within ER–plasma membrane (PM) junctions and impairs assembly of the STIM1/Orai1 complex, causing a decrease in Orai1-mediated Ca2+ current and SOCE. Together, our findings demonstrate that TRIC-A is a negative regulator of STIM1/Orai1 function. Thus, aberrant SOCE could contribute to muscle disorders associated with loss of TRIC-A.

Enhanced activity of multiple TRIC-B channels: an endoplasmic reticulum/sarcoplasmic reticulum mechanism to boost counterion currents.

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. 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.

Structural basis for activity of TRIC counter-ion channels in calcium release

Trimeric intracellular cation (TRIC) channels are thought to provide counter-ion currents that facilitate the active release of Ca2+ from intracellular stores. TRIC activity is controlled by voltage and Ca2+ modulation, but underlying mechanisms have remained unknown. Here we describe high-resolution crystal structures of vertebrate TRIC-A and TRIC-B channels, both in Ca2+-bound and Ca2+-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, Ca2+ binding at the luminal surface of TRIC-A, on its threefold axis, stabilizes lysine blockage of the pores. During active Ca2+ release, luminal Ca2+ 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.

Abstract 327: Attenuated Beta-Adrenergic Response in the Tric-a Knock Out Mice

Trimeric intracellular cation (TRIC) channels are expressed on the surface of sarcoplasmic reticulum (SR) and regulate calcium release from ryanodine receptors (RyRs). In a previous study, Tric-a knock out (KO) mice showed diminished calcium release from RyRs following increased calcium-influx via L-type calcium channels, which results in enhanced vascular resistance and non-dipper type hypertension. Decreased activation of RyR1 by PKA in skeletal myocytes in Tric-a KO mice is also known. However, physiological importance of TRIC channels on cardiac rhythm formation and its importance on the sympathetic nerve regulation are still obscure. Therefore, we aimed to clarify the effects of Tric-a ablation on cardiac pace making using Tric-a KO mice. We measured systolic blood pressure (SBP) with tail-cuff method, ECG and spontaneous action potential with microelectrode in the Tric-a KO and wild type (WT) mice. Isoproterenol or propranolol was used for sympathetic nerve manipulation. Furthermore, we evaluated heart rate variability (HRV). Tric-a KO mice tended to show limited responses to isoproterenol (0.3 mg/kg) than the WT mice (-27 ± 6 and -32 ± 6 mmHg, n = 10, p =0.70), and to propranolol (4 ± 6 and 13 ± 7 mmHg, n = 5~6, p =0.48). In ECG analysis, ablation of Tric-a gene resulted in significantly decreased heart rate changes to isoproterenol (23 ± 6 and 99 ± 15 bpm, Tric-a KO and WT mice, respectively, n = 9~10, p <0.001). Response to propranolol was also significantly decreased in the Tric-a KO mice (-28 ± 20 and -122 ± 14 bpm, Tric-a KO and WT mice, respectively, n = 9~10, p <0.001). In the action potential recordings, Tric-a KO mice showed significantly decreased sinus rate changes to 1 microM isoproterenol (35 ± 9 and 71 ± 10 bpm, Tric-a KO and WT mice, respectively, n = 6~8, p <0.05). In HRV analysis, low-frequency/high-frequency (LF/HF) ratio tended to be lower in the Tric-a KO mice than the WT mice under the administration of isoproterenol (0.22 ± 0.31 and 0.65 ± 0.16 bpm, Tric-a KO and WT mice, respectively, n = 9~11, p =0.16), suggesting lower sympathetic nerve tonus in the Tric-a KO mice. In conclusion, our data indicates that Tric-a KO mice showed attenuated responses to beta-adrenergic stimulus, which indicates involvement of TRIC-A channels in sympathetic nerve regulation.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

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. The 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.

Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel

BackgroundTrimeric intracellular cation (TRIC) channels are crucial for Ca2+ handling in eukaryotes and are involved in K+ uptake in prokaryotes. Recent studies on the representative members of eukaryotic and prokaryotic TRIC channels demonstrated that they form homotrimeric units with the ion-conducting pores contained within each individual monomer.ResultsHere we report detailed insights into the ion- and water-binding sites inside the pore of a TRIC channel from Sulfolobus solfataricus (SsTRIC). Like the mammalian TRIC channels, SsTRIC is permeable to both K+ and Na+ with a slight preference for K+, and is nearly impermeable to Ca2+, Mg2+, or Cl–. In the 2.2-Å resolution K+-bound structure of SsTRIC, ion/water densities have been well resolved inside the pore. At the central region, a filter-like structure is shaped by the kinks on the second and fifth transmembrane helices and two nearby phenylalanine residues. Below the filter, the cytoplasmic vestibule is occluded by a plug-like motif attached to an array of pore-lining charged residues.ConclusionsThe asymmetric filter-like structure at the pore center of SsTRIC might serve as the basis for the channel to bind and select monovalent cations. A Velcro-like plug-pore interacting model has been proposed and suggests a unified framework accounting for the gating mechanisms of prokaryotic and eukaryotic TRIC channels.

Phenotypic Spectrum in Osteogenesis Imperfecta Due to Mutations in TMEM38B: Unraveling a Complex Cellular Defect.

Recessive mutations in TMEM38B cause type XIV osteogenesis imperfecta (OI) by dysregulating intracellular calcium flux. Clinical and bone material phenotype description and osteoblast differentiation studies. Natural history study in pediatric research centers. Eight patients with type XIV OI. Clinical examinations included bone mineral density, radiographs, echocardiography, and muscle biopsy. Bone biopsy samples (n = 3) were analyzed using histomorphometry, quantitative backscattered electron microscopy, and Raman microspectroscopy. Cellular differentiation studies were performed on proband and control osteoblasts and normal murine osteoclasts. Type XIV OI clinical phenotype ranges from asymptomatic to severe. Previously unreported features include vertebral fractures, periosteal cloaking, coxa vara, and extraskeletal features (muscular hypotonia, cardiac abnormalities). Proband lumbar spine bone density z score was reduced [median -3.3 (range -4.77 to +0.1; n = 7)] and increased by +1.7 (1.17 to 3.0; n = 3) following bisphosphonate therapy. TMEM38B mutant bone has reduced trabecular bone volume, osteoblast, and particularly osteoclast numbers, with >80% reduction in bone resorption. Bone matrix mineralization is normal and nanoporosity low. We demonstrate a complex osteoblast differentiation defect with decreased expression of early markers and increased expression of late and mineralization-related markers. Predominance of trimeric intracellular cation channel type B over type A expression in murine osteoclasts supports an intrinsic osteoclast defect underlying low bone turnover. OI type XIV has a bone histology, matrix mineralization, and osteoblast differentiation pattern that is distinct from OI with collagen defects. Probands are responsive to bisphosphonates and some show muscular and cardiovascular features possibly related to intracellular calcium flux abnormalities.

Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.