The modulation of cytoplasmic free Ca(2+) concentration (Ca(2+)i) is a universal intracellular signaling pathway that regulates numerous cellular physiological processes. Ubiquitous intracellular ...Ca(2+)-release channels localized to the endoplasmic/sarcoplasmic reticulum-inositol 1,4,5-trisphosphate receptor (InsP3R) and ryanodine receptor (RyR) channels-play a central role in Ca(2+)i signaling in all animal cells. Despite their intracellular localization, electrophysiological studies of the single-channel permeation and gating properties of these Ca(2+)-release channels using the powerful patch-clamp approach have been possible by application of this technique to isolated nuclei because the channels are present in membranes of the nuclear envelope. Here we provide a concise description of how nuclear patch-clamp experiments have been used to study single-channel properties of different InsP3R channels in the outer nuclear membrane. We compare this with other methods for studying intracellular Ca(2+) release. We also briefly describe application of the technique to InsP3R channels in the inner nuclear membrane and to channels in the outer nuclear membrane of HEK293 cells expressing recombinant RyR.
Patch-clamping the outer or inner nuclear membrane of isolated nuclei is very similar to patch-clamping the plasma membrane of isolated cells. This protocol describes in detail all the steps required ...to successfully obtain nuclear membrane patches, in various configurations, from both the outer and inner nuclear membranes of isolated nuclei.
Ca
uptake by mitochondria regulates bioenergetics, apoptosis, and Ca
signaling. The primary pathway for mitochondrial Ca
uptake is the mitochondrial calcium uniporter (MCU), a Ca
-selective ion ...channel in the inner mitochondrial membrane. MCU-mediated Ca
uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca
uptake is tightly regulated to maintain low matrix Ca
and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca
regulation of intermembrane space-localized MICU1/2 is controlled by Ca
-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca
that permeates through the channel pore regulates Ca
affinities of coupled inhibitory and activating sensors in the matrix. Ca
binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca
binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca
regulation of channel activity. Our results demonstrate how Ca
influx into mitochondria is tuned by coupled Ca
-regulatory mechanisms on both sides of the inner mitochondrial membrane.
Mechanisms that regulate cellular metabolism are a fundamental requirement of all cells. Most eukaryotic cells rely on aerobic mitochondrial metabolism to generate ATP. Nevertheless, regulation of ...mitochondrial activity is incompletely understood. Here we identified an unexpected and essential role for constitutive InsP3R-mediated Ca2+ release in maintaining cellular bioenergetics. Macroautophagy provides eukaryotes with an adaptive response to nutrient deprivation that prolongs survival. Constitutive InsP3R Ca2+ signaling is required for macroautophagy suppression in cells in nutrient-replete media. In its absence, cells become metabolically compromised due to diminished mitochondrial Ca2+ uptake. Mitochondrial uptake of InsP3R-released Ca2+ is fundamentally required to provide optimal bioenergetics by providing sufficient reducing equivalents to support oxidative phosphorylation. Absence of this Ca2+ transfer results in enhanced phosphorylation of pyruvate dehydrogenase and activation of AMPK, which activates prosurvival macroautophagy. Thus, constitutive InsP3R Ca2+ release to mitochondria is an essential cellular process that is required for efficient mitochondrial respiration and maintenance of normal cell bioenergetics.
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▸ Constitutive InsP3R Ca2+ release suppresses autophagy in nutrient-rich conditions ▸ Absence of constitutive InsP3R Ca2+ release reduces cell ATP and activates AMP kinase ▸ Mitochondrial uptake of released Ca2+ is required for sufficient ATP production ▸ Constitutive ER-to-mitochondria Ca2+ transfer is essential for normal bioenergetics
Abstract Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at ...least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.
Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP...R) Ca... release channels in the endoplasmic reticulum to modulate Ca... signals that affect cell viability. ...However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-... activates single InsP...R channels with a biphasic concentration dependence. The Bcl-x... Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-x... activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP...R that play critical roles in Ca... signaling and cell viability. (ProQuest: ... denotes formulae/symbols omitted.)
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