Ligand sensitivity of type-1 inositol 1,4,5-trisphosphate receptor is enhanced by the D2594K mutation

Figures

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  Fig. 1. Closed IP3R1 channel structure. a Illustration of the 3D structure of IP3R1 (PDB ID: 3JAV; PyMOL) viewed from the cytosolic side down through the channel and along the membrane plane. The residue of interest D2594 is marked in red. Marked in blue are the conserved positively charged residues and in orange are the residues that correspond to the suggested IP3R1 channel’s pore gate. b Amino acid sequence alignment for the region containing IP3R1-D2594 and the corresponding site RYR2-E4872. Green line identifies the IP3R1 transmembrane segment 6 (aa 2564–2596) accordingly to PDB: 3JAV. Sequence alignment was done using the following IP3R1 sequences: Mus musculus (UniProt ID: P11881), Rattus norvegicus (UniProt ID: P29994), Homo sapiens (UniProt ID: Q14643). RYR2 sequences used in alignment include Mus musculus (UniProt ID: E9Q401), Rattus norvegicus (UniProt ID: B0LPN4), and Homo sapiens (UniProt ID: Q92736). Alignment was performed using Clustal Omega (Sievers & Higgins, 2018)

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  Fig. 2. Concentration-dependent Ca2+ release induced by ATP. a Traces show typical intracellular Ca2+ transients in HEK 293 cells expressing IP3R1-WT (left) or IP3R1-D2594K (right) evoked by the indicated ATP concentrations. b Maximal Ca.2+ transient amplitude plotted as a function of [ATP] from cells expressing IP3R1-WT (filled circles) and IP3R1-D2594K (open circles). Data were fitted by a Hill equation with an EC50 of 10.9 ± 0.75 and 3.3 ± 0.7 µM for WT and mutant IP3R1, respectively (n = 6 independent plates for each [ATP]). Data points are mean ± SEM. Significance was determined using unpair t test *p value 0.0002 and two-way ANOVA with Šídák test p < 0.0001

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  Fig. 3. Elementary Ca2+ release events induced by ATP. a Ca2+ puffs recorded from HEK293 cells expressing IP3R1-WT or IP3R1-D2594K during perfusion of 0 Ca2+ KRH external solution containing 20 nM ATP. Fluorescence kymograph images and traces show Ca2+ puffs time course and frequency. b Pool data comparison of Ca2+ puffs amplitude, kinetic attributes, and frequency of occurrence evaluated from averaged traces recorded from HEK293 cells expressing either IP3R1-D2594K (open circles; n = 64 cells) or IP3R1-WT (filled circles; n = 60 cells). Data points are mean ± SEM, unpair t test with Welch’s correction had a p < 0.0001

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  Fig. 4. Ion conductance of single IP3R1-WT and IP3R1-D2594K channels. Symmetrical solutions contained (in mM): 250 CsCH3SO3, 1 EGTA, 0.00014 free Ca2+, 10 HEPES, and pH 7.4. Solution in the cytosolic side of the channel also contained (in mM) 0.010 IP3 and 2 ATP. a Sample single WT and mutant (D2594K) IP3R1 recordings obtained at the indicated membrane potentials. Arrows labelled “c” mark the closed non-conducting state. b Pooled data (mean ± SEM; n = 3–8) of unitary current from WT (filled circles) and D2594K (open circles) IP3R1 plotted as a function of membrane potential and fitted by linear regression with a conductance of 248 ± 5 pS

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  Fig. 5. Cytosolic IP3 sensitivity of single IP3R1-WT and IP3R1-D2594K channels. a Cytosolic and luminal solutions contained symmetrical 250 mM CsCH3SO3, 1 mM EGTA, 140 nM free Ca2+, pH 7.4, with 2 mM ATP and the indicated IP3 concentration in the cytosolic side. Sample single IP3R1-WT and IP3R1-D2594K recordings made at + 30 mV are shown with 50 nM or 5 µM cytosolic IP3 present. Arrows labelled “c” mark the closed non-conducting state. Upward deflections represent channel openings. b Open probability expressed as nPo of IP3R1-WT and IP3R1-D2594K channels plotted as a function of log [IP3]. Pooled data (mean ± SEM; n = 3–9) were fitted by a Hill equation with an EC50 of 660 amf 406 nM and a Hill coefficient of 1.8 and 2 for IP3R1-WT (filled circles) and IP3R1-D2594K (open circles), respectively. Statistical significance between WT and mutant data points was evaluated with an unpaired t test with p < 0.05 and two-way ANOVA with Šídák test p < 0.0001

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  Fig. 6. Cytosolic Ca2+ dependency of IP3R1-WT, IP3R1-D2594K, and IP3R1-D2594A. The cytosolic and luminal solutions respectively contained (in mM): 500/50 CsCH3SO3, 1/1 EGTA, various/0.00007 [Ca2+]free, 2/0 ATP, 0.01/0 IP3 at pH 7.4. a Single-channel activity from IP3R1-WT (left), IP3R1-D2594K (center), and IP3R1-D2594A (right), recorded at 0 mV at the indicated cytosolic [Ca2+]. Downward deflections indicate channel openings. b Open probability as a function of Log10 cytosolic [Ca.2+] for IP3R1- WT (filled circles), IP3R1-D2594A (gray circles), and IP3R1-D2594K (open circles). Pooled data shown as mean ± SEM (n = 9–12) were fitted with a biphasic Hill equation. Significance was determined using ANOVA with Šídák test with p values 0.006 (WT vs. D2594K) and 0.962 (WT vs. D2594A)

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  Fig. 7. Luminal Ca2+ sensitivity of single IP3R1-WT and IP3R1-D2594K channels. The cytosolic and luminal solutions respectively contained (in mM) 250/250 CsCH3SO3, 0.5/0.5 EGTA, 0.5/0 BAPTA, 0.00007/various [Ca2+]free, 0.01/0 IP3, 2/0 ATP and pH 7.4. a Sample single IP3R1-WT and IP3R1-D2594K recordings made at + 30 mV with 10 µM luminal free Ca2+ present. Arrow labelled “c” mark the closed non-conducting state. Upward deflections represent channel openings. b Left, IP3R1-WT (filled triangles) and -D2594K (open triangles) nPo plotted as a function of luminal [Ca2+] at negative applied voltages. Data are mean ± SEM (n = 3–9) and were fitted by linear regression. b Right, nPo of IP3R1-WT (filled circles) and -D2594K (open circles) plotted as a function of luminal [Ca.2+] at positive applied voltages. Data are mean ± SEM (n = 4–9) fit by a biphasic Hill equation. Statistical significance at positive potentials by unpaired t test with Welch’s correction had a p value 0.0015

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  Fig. 8. Luminal Ca2+ dependency of single IP3R1-WT channels gating kinetics. The cytosolic and luminal solutions contained symmetrical 250 mM CsCH3SO3, 1 mM EGTA, 140 nM [Ca2+]free, pH 7.4. a Unitary Cs+ currents at + 40 mV with 2 mM ATP, 2 µM IP3 at the indicated 140 nM (upper), 60 µM (middle), or 5 mM (lower) free luminal Ca2+. Label “c” marks the closed non-conducting state. Upward deflections represent channel openings. b Normalized mean open time of IP3R1-WT channel plotted as a function of the electrochemical Ca2+ driving force (Em − ECa) in mV, where Em is the transmembrane potential applied to the bilayer (i.e., − 40, − 20, + 20, or + 40 mV) with the cis side held at ground and ECa is the Nernst potential of Ca2+ in mV, calculated at 22 ºC by the formula ECa = (RT/zCa2+F) ln([Ca2+]Cis/[Ca2+]Trans), where R is the universal gas constant, 8314 mJ/(K mol); F is Faraday’s constant, 96,485 C/mol; T is temperature, 295.15 K; and zCa2+ is the valence of Ca2+ ions, + 2. With these formulas, positive electrochemical driving forces indicate trans-to-cis Ca2+ fluxes. Data points were measured at − 40, − 20 mV (open circles), + 20, and + 40 mV (black circles). Data fitted with a double Hill…