Lecture 03: Ca2+ Signalling and Disease

Why is Ca2+ Described as the Most Versatile Signal in the Body?

• Regulates a wide range of cellular and physiological processes, including:

  • Cell cycle control: division, survival, apoptosis

  • Secretion: proteins and fluids

  • Sensory function

  • Neuronal function: nerve transmission, neurotransmitter release

  • Brain function: synaptic transmission, synaptic plasticity (LTP/LTD), learning and memory

  • Cardiovascular control: vascular smooth muscle contraction, endothelial-mediated vasodilation, and blood pressure

  • Heart function: force and timing of contraction

  • Muscle contraction and coordinated movement

• Ca²⁺ can regulate many opposing functions, even within the same cell

• This versatility arises because Ca²⁺ signals vary in amplitude, duration, frequency, and spatial localisation

• Ca²⁺ signals can be localised, global, or oscillatory, allowing precise and context-specific responses

How do frequency, amplitude, and spatial modulation of Ca²⁺ signalling generate biological specificity?

• For a given stimulus, Ca²⁺ signals can vary in amplitude and frequency, producing low- or high-frequency oscillations

• Ca²⁺ oscillations can occur over seconds, minutes, or hours

• The temporal pattern of Ca²⁺ signalling is critical:

  • Frequency-modulated signalling provides high levels of signalling fidelity

  • Frequency modulation is more versatile than amplitude modulation, as Ca²⁺ concentration has a limited usable range, whereas frequencies can vary widely

• Ca²⁺ signals can also be spatially restricted, activating local effectors in a given region only

  • e.g. localised Ca²⁺ signals in cerebellar Purkinje neurons

• This combination of frequency, amplitude, and spatial restriction produces a wide repertoire of Ca²⁺ signals and enables stimulus–response coupling and biologically specific cellular responses

What methods are used to measure [Ca²⁺]ᵢ and Ca²⁺ signalling machinery?

• Methods assess the dynamic nature of Ca²⁺ signalling in cells (in vivo or intracellular)

  • 1. Fluorescent Ca²⁺ indicators and dyes

    • Fluo-4, Indo-1, Rhod-2, Fura-2

  • 2. Genetically encoded Ca²⁺ indicators → can be genetically targeted to specific cells, tissues, or organelles

    • Aequorin, Cameleons, Pericam, GCaMP

  • 3. Electrophysiology

    • Whole-cell patch clamp: Ca²⁺-dependent ion channel currents as a readout of [Ca²⁺]ᵢ

  • 4. Single-channel recording

    • Artificial lipid bilayers used to measure IP₃R and RyR single-channel activity

  • 5. Specialised preparations

    • Patch-clamped isolated nuclei: IP₃R activity

How Can Fura-2 Be Used to Measure [Ca2+]i?

• A widely used organic fluorescent indicator and dual excitation dye

• Loaded into cells by attaching the acetoxymethyl ester, which is membrane permeable → diffuses into the cell

• Once in the cell, endogenous esterases cleave the ester group

• Free charged acid form of the dye trapped within the cytosol → becomes membrane impermeable and the extracellular dye is washed away

• Has 2 absorption/excitation maxima → 340nm and 380nm

  • Emitted fluorescence (at 510nm) changes when Ca2+ binds, and differs when excited at 340nm vs 380mm

  • At 340nm: the fluorescence emitted increases as Ca2+ concentration increases

  • At 380nm: fluorescence emitted decreases as Ca2+ increases

• Ratio of 340/380 excitation signal is proportional to Ca2+ concentration

• Ratiometric measurement of C2+ controls for changes in cell volume, cell thickness and dye concentration → confounding factors of single wavelength dyes

How Can Recombinant Ca2+ Sensors (GAPs) Be Used To Study Localised Ca2+ Signalling in Cells?

• They are recombinant luminescent proteins that are genetically engineered to be expressed under specific promoters, allowing for cell- or tissue-specific expression

• Can be targeted to defined subcellular compartments, regions of tissues and cells, allowing measurements of local Ca²⁺ signals in live cells

• Targeting sequences used:

  • CR + KDEL → ER

  • Cox VIII → mitochondria

  • GT → Golgi

  • Cytosolic and nuclear versions also exist

• Genetically encoded Ca²⁺ sensors (GAPs) enable direct visualisation of organelle-specific Ca²⁺ signalling, revealing spatial and functional Ca²⁺ compartmentalisation

What Are Some Examples of GAP Sensors?

• CytGAP: detects cytosolic Ca²⁺ increases in response to ATP/carbachol

• NucGAP: reveals distinct nuclear Ca²⁺ dynamics under different extracellular Ca²⁺ conditions

• MitGAP: tracks mitochondrial Ca²⁺ uptake; uptake is disrupted by FCCP

• ErGAP1: monitors ER Ca²⁺ levels; co-localiSes with SERCA2b and can be used with Fura-2 for simultaneous ER and cytosolic Ca²⁺ imaging

  • In DRG neurons: erGAP1 reveals ER Ca²⁺ release with caffeine; tested under EGTA + SERCA inhibition.

How Can Recombinant Reporter Proteins Be Used in Vivo, e.g. Rodent Studies?

• Recombinant Ca²⁺ reporters (e.g. aequorin, cameleons, pericam, GCaMP) can be genetically encoded

• Transgenic mice can be engineered to express these reporters in specific cell types or organelles (e.g. mitochondria)

• Allows real-time, in vivo visualisation of Ca²⁺ signals

• Example: mitochondria-targeted reporters reveal mitochondrial Ca²⁺ uptake in hindlimb muscle following sciatic nerve stimulation

How Can Whole Cell Patch Clamp Be Used to Measure [Ca2+]i

• An electrophysiological technique that allows the monitoring of Ca2+ without fluorescent dyes

• Ca2+ is measured indirectly assessing Ca²⁺-dependent ion channel activity

• Changes in Ca²⁺-dependent K⁺ and Ca²⁺-dependent Cl^- currents are recorded

• These currents faithfully report the Ca concentration beneath the membrane

How Can the Planar Lipid Bilayer Be Used to Study Ca2+ Signalling

• Technique used to study single-channel acitivty of Ca2+ permeable channels e.g. IP3 receptors, Ryr

• Consists of two chambers representing the cytosolic and extracellular environment with a small hole in between

• A lipid solution is painted onto the hole, forming a single lipid bilayer → resembles an artificial membrane

• Membrane vesicles enriched with channel of interest, e.g. IP3Rs from Cerebellar Purkinje fibres, are isolated via differential centrifugation and added to one chamber

• The vesicles then fuse and coalesce with the planar lipid bilayer, and the single channels insert into the membrane

• Voltage applied and current recorded allowing single-channel recording

• Allows for single-channel activity measurements (detailed analysis of channel conductance, gating, and Ca²⁺ permeability)

What Are the Key Players of Ca2+ Signalling Machinery

• Ca2+ release and entry

  • Ga/q-coupled GPCRs → PLC → IP3

  • IP3 Receptors (ER Ca2+ Release)

  • Voltage Operated Ca2+ Channels (VOCCs)

  • Receptor-operated Ca channels (e.g. P2X, NMDA, TRPC)

• Ca2+ Clerance and Storage

  • Sarco/Endoplasmic Reticulum Ca2+ ATPase (SERCA): ER/SR Ca2+ Uptake

  • Plasma Membrane Ca²⁺-ATPase (PMCA): Plasma membrane Ca2+ efflux

  • Secretory Pathway Ca²⁺-ATPase (SPCA): Golgi Ca2+ uptake

  • Na⁺/Ca²⁺-EXchanger (NCX): Na+-dependent Ca2+ extrusion

• Mitocondrial Ca2+ handling

  • Mitochondrial Ca²⁺-Uniporter (MCU): Ca2+ Uptake into Mitochondria

  • mPTP (Ca2+ release during stress/apoptosis)

How Do Gaq Coupled GPCRs Trigger Intracellular Ca2+ Release?

• Agonist binds Gαq-coupled GPCR → dissociation of G-protein

• Gαq activates phospholipase C (PLC)

• PLC hydrolyses PIP₂ (membrane lipid)→ DAG + IP₃

  • DAG activates PKC

• IP₃ (water-soluble) diffuses into the cytosol

• IP₃ binds to and activates IP₃ receptors on the ER membrane, causing Ca²⁺ release

• Released Ca²⁺ amplifies signalling via Ca²⁺-induced Ca²⁺ release → Ca2+ released from IP3 receptor feedbacks back on the same and neighbouring IP3 receptors or neighbouring RyRs on the ER/SR

What Are the Main Pathways of Ca2+ Entry Into Cells

• Voltage-operated Ca²⁺ channels (VOCCs)

  • Activated by membrane depolarisation

  • Mainly located in excitable cells

• Receptor-operated Ca²⁺ channels

  • Ligand-gated Ca2+ permeable channels (e.g. P2X, NMDA)

  • DAG-gated TRPC channels

How is Cytosolic Ca2+ Cleared After Signalling?

• Sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA): ATP-dependent pump that pumps Ca²⁺ back into ER/SR (2 Ca²⁺ per ATP)

• Plasma Membrane Ca²⁺-ATPase (PMCA): ATP-dependent pump that pumps Ca²⁺ out of the cell, across the plasma membrane (1 Ca²⁺ per ATP)

• Secretory Pathway Ca²⁺-ATPase (SPCA): ATP-dependent pump that pumps Ca²⁺/Mg²⁺ into Golgi lumen → maintains folding and trafficking of newly synthesised proteins

• Na⁺/Ca²⁺-EXchanger (NCX): a co-transporter that exchanges Na⁺ influx for Ca²⁺ efflux

  • Main Ca²⁺ extrusion pathway in excitable cells, e.g. cardiac myocytes

  • Driven by (inward) Na+ electrochemical gradient and membrane potential

  • Regulated by regulated by Na+/K+ ATPase

  • Can reverse (Ca2+ influx) during depolarisation

How is the Mitochondria involved in Ca2+ Signalling?

• Mitochondrial Ca²⁺-Uniporter (MCU) transports Ca²⁺ into the mitochondrial matrix → drives metabolism

  • Links Ca²⁺ signalling to metabolism

• mPTP: protein complex gated by high [Ca²⁺] and oxidative stress in mitochondiral matrix

  • Releases Ca²⁺

  • Triggers cytochrome c release and apoptosis (via Ca2+)

What are the main Ca²⁺ signalling components in excitable cells and their roles?

• In neurons, cardiac, smooth, skeletal muscle, endocrine cells

  • Voltage-Operated Ca²⁺ Channels (VOCCs): act as trigger sources; activated by membrane depolarisation

  • Ryanodine Receptors (RyRs, if expressed) act as amplifiers via Ca²⁺-induced Ca²⁺ release (CICR)

What are the main Ca²⁺ signalling components in non-excitable cells and their roles?

• In epithelia, glial cells, immune cells, and fibroblast cells

  • Voltage-Operated Ca²⁺ Channels (VOCCs): not expressed

  • IP3 Receptors: Act as trigger sources and amplifiers

    • In some cells, the Ryanodine Receptors (RyRs) if expressed act as amplifiers instead

How are intracellular Ca²⁺ oscillations regulated in excitable cells?

• Intrinsic oscillators are components of Ca²⁺ signalling machinery that drive and facilitate the propagation of Ca2+oscillations

  • Voltage-Operated Ca²⁺ Channels (VOCCs) are crucial for cyclical Ca²⁺ entry due to oscillations in membrane potential

• It leads to coordinated Ca²⁺ oscillations that regulate functions such as neurotransmission, muscle contraction, and secretion

• In non-excitable cells: instrictic oscialltors include IP3 receptors on the ER → generation of osciallations without membrane depolarisation via Ca2+ release from ER

How are Pancreatic Beta Cells Generate Ca2+ Oscillatiosn

• Cells secrete insulin and express VOCCs and K_ATP Channels

• Glucose metabolism alters the ATP/ADP ratio (↑) → K_ATP channels close → depolarisation → VOOCs open

  • Anion channels (e.g., VRACs) and cell volume changes may also contribute to depolarisation

    • changes in cell volume regulated by metabolism → ↑ osmotic gradient → cell swelling and volume change

• This depolarisation allows Ca²⁺ entry via VOCCs → triggers the upstroke of the Ca²⁺ signal

  • increased Ca2+ linked to membrane depolarisation → rapid AP firing when membrane potential threshold reached → Ca signal transmitted across beta cells

• Rise in cytosolic Ca²⁺ activates Ca²⁺-dependent K⁺ channels → hyperpolarizes membrane → VOCCs close

• Cytosolic Ca²⁺ clearance via SERCA, PMCA, and NCX contributes to the downstroke of Ca²⁺ oscillation

• Cycle repeats → cyclical changes in membrane potential drive repeated Ca²⁺ oscillations

How does Ca²⁺ regulate IP₃ receptor activity and contribute to Ca²⁺ oscillations?

• IP₃ receptors (IP₃R) requires both IP₃ and Ca²⁺ for activation

• IP3 recepttors show a bell-shaped Ca²⁺ dependence (shown using fixed [IP₃], above threshold of dependence with varied [Ca²⁺]):

  • Low Ca²⁺: increases open probability of receptor → Ca²⁺ release

  • High Ca²⁺: threshold reached → feedback inhibition → receptor closes → inhibts Ca²⁺ release

• Mechanism of release process:

  1. IP₃ binding to the receptor results in a small Ca²⁺ release → acts as a trigger

  2. This is followed by Ca²⁺-induced Ca²⁺ release (CICR) via IP3; RyR may be recruited to amplify this.

  3. Feedback Generates Ca2+ Oscillations

     1. Feedback onto IP3 binding site (Ca2+ dependent binding site) where Ca²⁺ potentiates further release at one site,

     2. Ca2+ dependent inhibition of IP3 receptor at a different site → Ca causes IP3 receptor to close → turns off trigger source and results in Ca2+ clearance

• Measured using planar lipid bilayer for single-channel activity studies of IP₃Rs

What factors determine the shape and pattern of intracellular Ca²⁺ signals?

• Spatio-temporal patterning: both time and location influence Ca²⁺ signalling

• Localised signals: Ca²⁺ can be restricted to specific regions of the cell, oscillating locally

• Propagating signals: Ca²⁺ can also travel across the cell as waves, coordinating cellular responses

How do endothelial cells generate localised and global Ca²⁺ signals?

• Ca²⁺ puffs are localised releases of Ca2+ from clusters of IP₃ receptors

• Triggered by low concentrations of agonist (e.g., ATP) which activate P2Y purinergic receptors and generate IP₃ → results in localised Ca2+ release events

• Localised Ca²⁺ oscillations may remain restricted to one region, without propagating to the rest of the cell

• Summation of localised Ca2+ puffs can propagate as a global Ca²⁺ wave across the cell

What Are the Key Ca2+ Signals?

• Ca²⁺ blip: release from a single IP₃ receptor

• Ca²⁺ quark: release from a single RyR

• Ca²⁺ puff: release from clusters of IP₃ receptors

• Ca²⁺ spark: release from clusters of RyRs

• Ca²⁺ wave: coordinated/sequential propagation of Ca²⁺ across the cell

  • “Wave front” → rising Ca²⁺; “wave end” → falling Ca²⁺

  • Ca2+ oscillation that carries temporal and spatial information

  • Generated by individual Ca2+ release events that spread to neighbouring channels

How do pancreatic acinar cells generate apically confined Ca²⁺ signals?

• Perigranular mitochondria act as a buffer, restricting Ca²⁺ to the apical region near secretory granules → prevent propagation of Ca2+ signal to basal pole

• Localised (small) IP₃ release occurs following small UV strobe on caged IP₃ → apical Ca²⁺ events only

  • caged IP3 with nitrophenol, inactive until the addition of light

• Function: triggers exocytosis of digestive enzymes from secretory granules at the apical end of the cell → regulation allows for specific functional responses at one end of the cell

  • Mitochondria positioned around the secretory granule area, and facilitates Ca2+ uptake and prevents Ca2+-induced Ca2+ release spreading to the basal end → prevents global Ca2+ wave

• Mitochondria create a spatial barrier to restrict Ca²⁺ signals, allowing precise functional responses

How do pancreatic acinar cells generate global Ca²⁺ signals?

• Global Ca²⁺ signals generated with UV flash, which releases all IP₃ from the nitrophenol cage → stronger Ca²⁺ signals

  • Mitochondrial inhibition (e.g., with CCCP, antimycin) prevents Ca²⁺ uptake → Ca²⁺ spreads to the basal pole

• Mitochondria create a spatial barrier to restrict Ca²⁺ signals, allowing precise functional responses

How do mitochondria both shape and respond to Ca²⁺ signals?

• Mitochondria act as a buffer and take up cytosolic Ca²⁺, shaping Ca²⁺ signals

• Ca²⁺ uptake stimulates mitochondrial metabolism and ATP production

• Many enzymes in the Krebs cycle and ETC respond to Ca²⁺ → stimulus–metabolism coupling

• This results in reciprocal regulation whereby various metabolites produced (e.g., NADH, NAD⁺) can feed back on Ca²⁺ release channels, influencing further Ca²⁺ signalling and miochondrial function

How Does Store-Operated Ca2+ Entry (SOCE) Work and How Is It Measured?

• Ca²⁺ entry is initiated by ER Ca²⁺ store depletion

• Measured using fluorescent dye

  • No external Ca²⁺: application of high [agonist] → transient spike in cytosolic Ca²⁺ → rapidly returns to baseline via Ca²⁺ clearance via pumps (ER uptake + extrusion)

  • With external Ca²⁺: application of high [agonist] → peak + plateau response; removal of external Ca²⁺ → cytosolic Ca²⁺ returns to baseline

• SOCE allows sustained Ca²⁺ entry after store depletion to maintain cytosolic Ca²⁺ signals

How does SOCE generate cytosolic Ca²⁺ signals following ER store depletion?

• Zero external Ca²⁺ + agonist: transient spike-like increase in cytosolic Ca²⁺ from ER release → recovers and returns to baseline as Ca²⁺ is pumped out

• Agonist removed (washed off): cytosolic Ca²⁺ remains low; ER stores depleted and not replenished due to lack of extracellular Ca²⁺

• Re-addition of extracellular Ca²⁺: classic overshoot response in cytosolic Ca²⁺ → store-operated Ca²⁺ entry activated even without agonist bound → increase in cytosolic Ca2+ caused store depletion medaited Ca2+ entry

  • No agonist bound, but Ca2+ present and Ca channels open, allowing Ca2+ influx

• Key concept: Ca²⁺ channels open in response to store depletion, allowing a flood of Ca²⁺ into the cell

How does SERCA inhibition with thapsigargin lead to ER Ca²⁺ store depletion and SOCE activation?

• Normally, Ca2+ pumped into the ER is balanced by ER Ca²⁺ leak via SERCA pumps → maintains low cytosolic Ca²⁺; PMCA extrudes any excess Ca²⁺

  • equilibrium between Ca leak from the ER and Ca reuptake via the SERCA to maintain low resting cytosolic

• Thapsigargin inhibits SERCA → prevents Ca²⁺ reuptake into ER, resulting in a net leak of Ca2+ from ER → cytosolic Ca²⁺ pumped out by PMCA → ER Ca²⁺ store depleted

  • (Ca2+ leak from ER domaintes → must be cleared → pumped out via PMCA)

• Key point: ER store depletion triggers store-operated Ca²⁺ entry (SOCE) when extracellular Ca²⁺ is available

How Does Thapsigargin Demonstrate SOCE?

• Thapsigargin treatment inhibits SERCA causing a slow, small rise in cytosolic Ca²⁺ due to net Ca leak from the ER (e.g., via translocon or leaky IP₃R)

• PMCA activity pumps Ca²⁺ out of the cell → ER stores eventually depleted

• Addition of External Ca²⁺ triggers Ca²⁺ overshoot response→ shows SOCE is activated without IP₃ or agonist, proving it’s store depletion–dependent (as same Ca2+ entry and increase in [cytosolic] is seen)

How is SOCE Activated, According to the Capacitivate Model?

• GPCR activation → IP₃ production → binds IP₃ receptor → Ca²⁺ released from ER

• ER Ca²⁺ store depletion triggers Ca²⁺ entry into the ER from the extracellular space

• involves:

  • SOC channels (Store-Operated Ca²⁺ channels)

  • CRAC channels (Ca²⁺-Release Activated Ca²⁺ channels)

What are the key properties of CRAC channels?

• Activation specifically triggered by ER Ca²⁺ store depletion

• Electrophysiology (measured via IV curves)

  • Inwardly rectifying (current increases at negative voltages)

  • Reversal potential: very positive (> +60 mV)

• Blocked by La³⁺ and Gd³⁺

What is the Molecular Identify of SOC (Store-Operated Ca²⁺ channels)?

• Two different proteins that work together to regulate Ca2+ entry; discovered through a Genome-wide RNAi screen with a high-throughput FLIPR screen

  • STIM1 (Stromal Interacting Molecule 1):

    • ER Ca²⁺ sensor → detects ER Ca²⁺ store depletion

    • Has a 2D topology and contains an EF-hand motif that binds Ca²⁺ in ER lumen

  • CRACM1 / Orai1:

    • Pore-forming subunit of CRAC channel

    • Located on the plasma membrane → mediates Ca²⁺ entry

What experimental approach identified key components of store-operated Ca²⁺ entry?

• Primary screen: high-throughput RNAi in 384-well plates

  • In each well, cells treated with RNAi targeting a different gene in the drosophilas genome

  • Readout: Ca²⁺ overshoot measured (FLIPR assay)

  • Wells showing inhibited Ca²⁺ overshoot show candidate genes that have been knocked down

• Secondary screen: low-throughput patch-clamp (FLIPR Screen)

  • Identified key components of SOCE and measured current

  • Confirms reduced CRAC currents when the candidate gene knocked down

• Outcome: identification of key SOCE components (e.g., STIM1, Orai1)

How did knowing the molecular identity of SOCE components (STIM1 and Orai1) change experimental approaches?

• It allowed for genetic modifications and the overexpression of both STIM1 and Orai1, which massively enhanced Ca²⁺ overshoot, potentiated Ca²⁺ entry, which is characteristic of CRAC currents

• Sequential mutation of key acidic residues in Orai1 pore caused a dramatic reduction in Ca²⁺ overshoot response, confirming Orai1 as a bona fide Ca²⁺ channel

• Knowing molecular identity allowed functional validation of SOC via overexpression and mutagenesis

How Does TIRF (Total Internal Reflection fluorescence) Microscopy Work?

• An imaging technique useful for visualising local Ca²⁺ microdomains with low-affinity dyes (e.g., Fluo-5F), beneath the membrane

• An incident light hits the glass-water interface at a particular angle → an evanescent wave of excitation light excites the fluorophores within ~200 nm of the membrane producong a fluroescnet optical slice

  • It allows optical sectioning of the plasma membrane region

How does TIRF microscopy reveal STIM1/Orai1 dynamics during SOCE?

• It allows for the visualisation of localised Ca²⁺ entry that coincides with the formation of STIM1 puncta (and accumulation of STIM-1 cherry)

• At rest: Cherry-STIM1 and GFP-Orai1 do not co-localise

• After ER store depletion via thapsigargin, both molecules form puncta that coincide with each other, suggesting a molecular interaction that enables Ca²⁺ entry

  • Shows molecular interactions and local Ca²⁺ signalling at the plasma membrane

• Cherry STIM-1 and GFP-Orai1 are stimulated with Thapsigargin → activation of SOCE → molecules in close proximity and interact → facilitate Ca2+ entry

What is the role of STIM1 in store-operated Ca²⁺ entry?

• STIM1 is a transmembrane ER protein with an EF-hand domain inside the ER lumen → binds Ca²⁺

• Activation of the receptor causes IP3-mediated Ca²⁺ release, depleting ER stores → Ca²⁺ unbinds EF-hand

• STIM1 molecules oligomerise and form puncta, moving close to Orai1 at the plasma membrane

  • This STIM1-Orai1 interaction activates CRAC channels, allowing Ca²⁺ entry into the cytosol

What Are the Key Domains of STIM1 and Their Roles?

• Coiled-coil domains form the 3D structure of STIM1

• CRAC-activating domain (CAD): essential for activating Orai1/CRAC channels

• Polybasic cluster: binds directly to the CRAC channel at the plasma membrane

  • Mutating each domain reveals its specific contribution to SOCE

What Are the Key Structural Features of Orai1 and Their Roles?

• Single Orai1 subunit → building block of CRAC channel

  • Structure: homohexameric subunits forming a central pore in the membrane

    • Highly Ca²⁺ selective

• 4 transmembrane domains: form the channel pore

• Coiled-coil domain: mediates coupling with STIM1

• Allosteric gating domains: control channel opening/closing

• Positively charged arginine residues: important for gating and Ca²⁺ selectivity

• Ca²⁺-calmodulin binding sites: regulate Ca²⁺-dependent inactivation

How Do STIM-1 and Orai1 Interact in Store Depletion

• The two proteins interact during store depletion-induced activation

• The charged domains of STIM -1 interact with Orai-1 → STIM1 binding opens the Orai1 pore, allowing store-operated Ca²⁺ entry

• Important to understand the molecular mechanisms and amino acids resiudes invovled to understand their implications in disease

How Does a Mutation in Orai-1 Lead to Severe Compromised Immune Deficiency (SCID)?

• A complex genetic disease → single point mutation (R91W) in Orai-1 → Impaired SOCE

• Affects immune cells: Reduced/ Impaired Orai-1/STIM-1 expression in T-cells, B-cells and NK cytotoxic cells

• This leads to

  • Impaired T cell activation

  • Reduced cytokine production

  • Impaired T cell proliferation

  • Defective antigen-specific antibody response

Give 3 Examples of Diseases Associated With Defective SOCE:

• Congenital myopathy (global muscular hypotonia)

  • Impaired skeletal muscle function → reduced muscle strength and endurance, poor head control

• Chronic pulmonary disease

  • Defective pulmonary muscle → chronic infections and respiratory issues

• Anhidrotic ectodermal dysplasia

  • Impaired sweat gland function → decreased sweat production, dry skin, heat intolerance/persistent fever

    • Sweat glands require SOCE

What are the effects and examples of Loss-of-Function (LoF) mutations in ORAI1 and STIM1?

• Effect: Impaired SOCE → reduced Ca²⁺ entry → CRAC channelopathy

• Caused by:

  • Null mutations (no protein expression):

    • ORAI1: A88SfsX25, A103E, L194P, H165PfsX1

    • STIM1: E128RfsX9, 1538-1G>A

  • Functional LoF (protein expressed but defective):

    • ORAI1: R91W (Ca²⁺ permeation blocked)

    • STIM1: R429C, P165Q, R426C

What are the clinical features of LoF mutations in ORAI1/STIM1?

• Immune system: SCID-like immunodeficiency, recurrent infections, autoimmunity (AIHA, thrombocytopenia)

• Muscle: Muscular hypotonia (reduced muscle tone)

• Ectodermal: Anhydrosis, abnormal teeth/hair/nails, defective dental enamel

What are Gain-of-Function (GoF) mutations and their consequences?

• Effect: Constitutive Ca²⁺ influx → constant activation of SOCE

• Associated diseases:

  • Stormorken syndrome: STIM1 R304W

  • York Platelet Syndrome: STIM1 R304W

  • Tubular Aggregate Myopathy (TAM): STIM1 EF-hand, ORAI1 transmembrane mutations

• Symptoms: Muscle cramps/weakness, platelet dysfunction, asplenia, ichthyosis, myalgia, miosis

Which organs/systems are affected by LoF and GoF mutations in ORAI1/STIM1?

• Immune system (LoF → immunodeficiency; GoF → immune dysregulation)

• Muscle function (LoF → hypotonia; GoF → myopathy)

• Platelet biology (LoF → thrombocytopenia; GoF → platelet dysfunction)