Store-Operated and Receptor-Operated Calcium Channels (SOCCs & ROCEs)

Comprehensive review flashcards covering store-operated calcium channels (SOCCs/CRAC), STIM1 and Orai1 structure and gating, electrophysiology, pharmacology, and receptor-operated calcium entry (ROCE) through TRPC channels.

What is store-operated calcium entry (SOCE), and what primary cellular event triggers it?

Store-operated calcium entry (SOCE) is a sustained influx of extracellular $\text{Ca}^{2+}$ across the plasma membrane triggered by the depletion of intracellular $\text{Ca}^{2+}$ stores from the endoplasmic reticulum (ER). It typically initiates when GPCR or receptor tyrosine kinase activation stimulates phospholipase C (PLC) to produce $\text{IP}_3$, which binds to $\text{IP}_3\text{R}$ on the ER to release luminal $\text{Ca}^{2+}$.

What role does the SERCA pump play following store-operated calcium entry (SOCE)?

The Sarcoplasmic/Endoplasmic Reticulum $\text{Ca}^{2+}$-ATPase (SERCA) utilizes ATP to pump cytosolic $\text{Ca}^{2+}$ back into the ER lumen, refilling the intracellular stores after SOCE.

In what years and under what names were the ER $\text{Ca}^{2+}$ sensor and the pore-forming CRAC channel subunit identified?

STIM1 (stromal interaction molecule 1) was identified in 2005 as the ER $\text{Ca}^{2+}$ sensor. Orai1 (originally termed $\text{Ca}^{2+}$ release-activated $\text{Ca}^{2+}$ channel molecule 1, or CRACM1) was identified in 2006 as the pore-forming subunit of CRAC channels in the plasma membrane.

Identify this linear protein diagram and describe its primary molecular domains from N-terminus to C-terminus.

This diagram depicts the molecular domains of the $77\,\text{kDa}$ STIM1 protein: luminal EF-hand motifs (cEF and hEF) that bind $\text{Ca}^{2+}$, a sterile alpha motif (SAM) that senses $\text{Ca}^{2+}$ unbinding, a single transmembrane domain (TMD), coiled-coil 1 (CC1; comprising $C\alpha1\text{--}3$), the STIM1-Orai activating region (SOAR; comprising $S\alpha1$/CC2 and $S\alpha4$/CC3), an inhibitory domain (ID), and a lysine-rich (K-rich) polybasic domain at the C-terminus.

What post-translational modification can occur on the two luminal cysteine residues of STIM1?

They are subject to S-nitrosylation (the addition of a nitric oxide [NO] group), which affects STIM1 function.

How is STIM1 held in an inactive state under resting ER conditions?

Under resting conditions with high luminal $[\text{Ca}^{2+}]$ ($500\text{--}800\,\mu\text{M}$), $\text{Ca}^{2+}$ binds to the EF-hand, keeping SAM and TMD separated in a loose homodimer. The CC1 domain remains folded, and its $C\alpha3$ segment interacts with and sequesters the SOAR domain in an inactive state through residues including $\text{L251}$.

What conformational changes occur in STIM1 upon ER luminal $\text{Ca}^{2+}$ store depletion?

$\text{Ca}^{2+}$ dissociates from the luminal EF-hand, destabilizing the EF-SAM region and driving STIM1 oligomerization. This structural change propagates through the transmembrane domain, causing the CC1 domain ($C\alpha1\text{--}3$) to unfold and elongate, which releases SOAR from $C\alpha3$ and projects it toward the plasma membrane.

What is the function of the lysine-rich (K-rich) C-terminal domain of STIM1?

The polybasic K-rich domain interacts with and recruits negatively charged $\text{PIP}_2$ phospholipids in the inner leaflet of the plasma membrane, anchoring the ER to the plasma membrane to form ER-PM junctions and stabilizing STIM1-Orai1 interactions.

What membrane protein topology is depicted here, and which domain directly mediates physical activation by STIM1?

This depicts the transmembrane topology of Orai1, which features 4 transmembrane domains (TM1–TM4) with both N- and C-termini in the cytosol. Its negatively charged C-terminal coiled-coil domain (CT-CCD) directly interacts with the positively charged SOAR domain of STIM1.

What are the roles of the TM1 segment and the proline-rich N-terminal region in Orai1?

The TM1 segment forms the inner lining of the channel pore and dictates $\text{Ca}^{2+}$ selectivity and permeability. The intracellular N-terminal proline-rich region is critical for channel gating kinetics.

What is the stoichiometry of the fully assembled, active CRAC channel complex?

Six Orai1 subunits assemble into a hexameric pore, which is engaged by 6 STIM1 molecules (via the $\text{F394}^+$ residue in SOAR interacting with the CT-CCD of Orai1) to form the functional conducting channel.

What unique current-voltage (I-V) relationship is demonstrated in this recording of an activated CRAC channel ($I_{\text{CRAC}}$)?

Prominent inward rectification: the inward $\text{Ca}^{2+}$ current increases exponentially as membrane potential ($V_M$) becomes more negative, while current approaches zero at positive membrane potentials.

What is the unitary conductance of the CRAC channel under physiological conditions ($2\,\text{mM}\,\text{Ca}^{2+}$)?

The unitary single-channel conductance is extremely low, measured at approximately $9\,\text{fS}$.

How does ion selectivity of CRAC channels behave in physiological vs. divalent-free solutions?

In physiological solutions containing divalent cations, CRAC channels are exceptionally selective for $\text{Ca}^{2+}$ ($P_{\text{Ca}}/P_{\text{Na}} > 1000$). In divalent-free solutions, the channel readily conducts $\text{Na}^+$, but this $\text{Na}^+$ flux is completely blocked by adding micromolar ($\mu\text{M}$) concentrations of $\text{Ca}^{2+}$.

What phenomenon occurs in this fluorescence microscopy experiment when cells expressing mCh-STIM1 and eGFP-Orai1 are treated with thapsigargin (+TG)?

ER $\text{Ca}^{2+}$ store depletion by thapsigargin induces STIM1 and Orai1 to co-cluster into discrete puncta at ER-PM junctions, appearing as yellow overlapping puncta in the merged fluorescence image.

What is the mechanism of action of thapsigargin (TG) in experimental studies of CRAC channels?

Thapsigargin is a selective, irreversible inhibitor of the SERCA pump on the ER membrane. By blocking $\text{Ca}^{2+}$ reuptake into the ER, it passively depletes ER luminal $\text{Ca}^{2+}$ stores, thereby activating STIM1 and CRAC channels without receptor stimulation.

What pharmacological inhibitors are commonly used to block SOCCs/CRAC channels?

Trivalent lanthanide ions ($\text{La}^{3+}$ and $\text{Gd}^{3+}$) which completely block Orai channels in the sub-micromolar range; 2-APB at concentrations above $30\text{--}50\,\mu\text{M}$; and pyrazole compounds such as BTP1, BTP2 (YM-58483), and BTP3.

Which compounds can be included in the intracellular pipette solution during whole-cell voltage-clamp experiments to activate CRAC channels?

BAPTA (a high-affinity $\text{Ca}^{2+}$ chelator to buffer cytosolic $\text{Ca}^{2+}$ and drive store emptying), $\text{IP}_3$ (to open $\text{IP}_3\text{Rs}$), or thapsigargin (TG, to inhibit SERCA).

How does receptor-operated calcium entry (ROCE) differ fundamentally from store-operated calcium entry (SOCE)?

ROCE is triggered directly by cell surface receptor activation independently of whether internal ER stores are depleted. In contrast, SOCE strictly requires the emptying of ER luminal $\text{Ca}^{2+}$ stores sensed by STIM1.

How many members are in the human TRPC channel family, and what is the status of TRPC2?

The TRPC family consists of 7 members (TRPC1–TRPC7), but in humans there are only 6 functional channel proteins because TRPC2 is a non-functional pseudogene.

What channel subunit topology is depicted here, and where are the two diacylglycerol (DAG) binding sites located?

This depicts the 6-transmembrane topology of TRPC3. The two DAG binding sites are located at: 1) the cytosolic pre-S1 elbow region, and 2) the S5–S6 pore domain.

What direct physiological ligand gates TRPC3, TRPC6, and TRPC7 channels downstream of $\text{G}_{\text{q/11}}$-coupled GPCRs?

Diacylglycerol (DAG), generated alongside $\text{IP}_3$ through the cleavage of $\text{PIP}_2$ by $\text{PLC}\beta$, directly binds to and activates TRPC3, TRPC6, and TRPC7 channels.

What is the consequence of the 'Moonwalker' mutation (T561A) in TRPC3 channels?

The Moonwalker mutation (T561A) is a gain-of-function activating mutation that promotes constitutive channel pore opening via a structural rearrangement of the S6 transmembrane helix.

How does the current-voltage (I-V) relationship of TRPC3 differ from that of Orai1-formed CRAC channels?

TRPC3 displays double rectification with a reversal potential near $0\,\text{mV}$ (conducting inward $\text{Na}^+$ and $\text{Ca}^{2+}$ currents at negative potentials, and outward $\text{K}^+$ current at positive potentials). In contrast, Orai1 CRAC channels show prominent inward rectification with negligible outward current at positive potentials.

What is the mechanism of functional synergy between STIM1 and TRPC3 channels at ER-PM junctions?

Although TRPC3 is gated by DAG and not store depletion, STIM1 oligomers recruited upon store depletion concentrate $\text{PIP}_2$ at ER-PM junctions via their K-rich domains. This recruits TRPC3 and $\text{PLC}\beta$ into the same microdomain, enhancing localized DAG production and TRPC3 channel activation.

What synthetic compound selectively inhibits TRPC3-containing channels?

Pyr3.