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

Abstract

Inositol 1,4,5-trisphosphate receptor (IP₃R) and ryanodine receptor (RyR) are homologous cation channels that mediate release of Ca²⁺ from the endoplasmic/sarcoplasmic reticulum (ER/SR) and thereby are involved in many physiological processes. In previous studies, we determined that when the D2594 residue, located at or near the gate of the IP₃R type 1, was replaced by lysine (D2594K), a gain of function was obtained. This mutant phenotype was characterized by increased IP₃ sensitivity. We hypothesized the IP₃R1-D2594 determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states. To test this possibility, the relationship between the D2594 site and IP₃R1 regulation by IP₃, cytosolic, and luminal Ca²⁺ was determined at the cellular, subcellular, and single-channel levels using fluorescence Ca²⁺ imaging and single-channel reconstitution. We found that in cells, D2594K mutation enhances the IP₃ ligand sensitivity. Single-channel IP₃R1 studies revealed that the conductance of IP₃R1-WT and -D2594K channels is similar. However, IP₃R1-D2594K channels exhibit higher IP₃ sensitivity, with substantially greater efficacy. In addition, like its wild type (WT) counterpart, IP₃R1-D2594K showed a bell-shape cytosolic Ca²⁺-dependency, but D2594K had greater activity at each tested cytosolic free Ca²⁺ concentration. The IP₃R1-D2594K also had altered luminal Ca²⁺ sensitivity. Unlike IP₃R1-WT, D2594K channel activity did not decrease at low luminal Ca²⁺ levels. Taken together, our functional studies indicate that the substitution of a negatively charged residue by a positive one at the channels’ pore cytosolic exit affects the channel’s gating behavior thereby explaining the enhanced ligand-channel’s sensitivity.

Introduction

The endoplasmic/sarcoplasmic reticulum (ER/SR) have specialized Ca²⁺ release channels that mediate intracellular Ca²⁺ signals, which regulate many cellular processes as diverse as fertilization, apoptosis, muscle contraction, transcription, secretion, learning, and memory [1, 2, 29]. These Ca²⁺ release channels are the inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs), each having 3 different isoforms. Although in most cells, a mix of the 3 isoforms is present, there are some tissues where only one type predominates. For example, the type 1 IP₃R (IP₃R1) is the predominant intracellular Ca²⁺ release channel in cerebellum [23, 33], while the type 2 RyR (RyR2) predominates in the heart [32, 34]. The IP₃Rs and RyRs share a high degree of functional and structural homology [28, 30, 41, 42, 45] which can be exploited to advance our understanding of these channels.

Production of cytosolic IP₃ occurs when extracellular ligands (e.g., hormones, neurotransmitters) bind to Gq-protein–coupled receptors in the surface membrane to initiate an intracellular signaling cascade [1, 23, 36]. IP₃R channel activity is primarily regulated by the availability of IP₃ and Ca²⁺ [5, 27, 37, 38]. Specifically, single-IP₃R1 channels are activated when they bind IP₃, and the extent of IP₃R1 activation (i.e., opening duration/frequency) is a bell-shaped function of the cytosolic-free Ca²⁺ concentration [5, 7, 24, 37, 38]. The bell-shaped cytosolic Ca²⁺ dependency of the IP₃R1 is explained by the presence of a cytosolic Ca²⁺ activation site and a putative lower affinity cytosolic Ca²⁺ inhibition site [20, 31, 43]. An intense research topic of single IP₃R1 Ca²⁺ control has been the mechanism by which intra-ER Ca²⁺ modulates the channel activity [4, 18, 44, 47, 48]. Specifically, the controversial mechanisms that have been investigated are (a) intra-ER (luminal) Ca²⁺ interaction with a luminal regulatory site on the channel itself [8, 44, 48], or (b) on a closely associated regulatory protein [15, 47, 49], and/or (c) luminal Ca²⁺ flowing through open IP₃Rs (lumen-to-cytosol) acting on the IP₃R’s cytosolic Ca²⁺ activation and/or Ca²⁺ inhibitory sites [4, 17, 49, 50]. This latter process is commonly referred to as feedthrough (FT) Ca²⁺ regulation. Overall, the IP₃R1 ligand regulatory scenario involves allosterically interacting inputs [24,25,26, 53]. For example, Mak et al. (1998) proposed that cytosolic IP₃ allosterically “tunes” cytosolic IP₃R Ca²⁺ inhibition. Likewise, cytosolic ATP is a complementary allosteric regulator of IP₃R gating [26, 48].

Single RyR activity is also governed simultaneously by multiple synergistically acting ligands. For example, single RyR2 function is controlled by cytosolic ATP and Ca²⁺ (via its cytosolic Ca²⁺ activation and inhibition sites) and by luminal Ca²⁺ (via Ca²⁺ binding sites in the channel and accessory proteins) [10, 14, 22, 52]. In regard to the luminal [Ca²⁺] control, Chen et al., using RyR2 site-directed mutagenesis of negatively charged residues within the channel’s permeation pathway, identified the RyR2-E4872 residue as a critical site involved in RyR2 luminal Ca²⁺ regulation [9]. Furthermore, functional and structural analyses revealed that the negatively charged RyR2-E4872 residue in one subunit forms a salt-bridge with the positively charged RyR2-R4874 residue in the neighboring subunit in the closed state. This inter-subunit network of electrostatic interactions is believed to stabilize the closed state of RyR2. Thus, a model was put forward in which Ca²⁺ binding to E4872 disrupts the electrostatic interactions with neighboring amino acids favoring the transition from closed to open state [9, 35].

The corresponding IP₃R1 site to RyR2-E4872 residue is the IP₃R1-D2594 at the transmembrane region 6 (TMR6). Figure 1a shows the location of this residue in a 3D structure of the rat IP₃R1 channel in a non-conductive closed state (4.7 Å resolution; [12]). The IP₃R results from the assembly of four subunits arranged around the central channel axis, where two major regions can be defined, the bulky cytosolic region connected via “stalk” densities to the transmembrane regions (TMRs; [12]). The boxes show a close-up view of the ion conduction pathway, along the membrane plane (bottom) and from the cytosol (top). In orange, the F2586 residues are shown (F2585 mouse) which have been suggested to serve as the pore gate. Away from the gate, in the ion conduction pathway, the D2595 (D2594 mouse; red) and R2597 (R2596 mouse; blue) residues are marked. Similarly, to RyR2-R4874 residue, IP₃R1-R2596 have been suggested to participate in a network of TMR interactions that could play a role in transmitting signals to the gate, in part, through an interaction between neighboring TMR6 helices [13]. Figure 1b details the amino acid sequence alignment of the region containing the IP₃R1-D2594 and the corresponding site RYR2-E4872 in different species. Although Bhanumathy et al. did not find a substantial change in the IP₃R1 Ca²⁺ signaling when the D2594 residue (Fig. 1, residue in red) was replaced by alanine mutagenesis [6], we have recently shown that its substitution with a positive charge (IP₃R1-D2594K) results in a gain-of-function phenotype, while its alanine substitution (IP₃R1-D2594A) induces a reduction of its function. To gain mechanistic insights into the functional significance of IP₃R1-D2594K, a mouse model was created. The ITPR1‐D2594K^+/− mutant mice exhibited pathological ramifications that included male infertility, azoospermia, and acrosome loss [46]. These studies indicated that changing the electric nature of IP₃R1-D2594 residue allosterically influences the IP₃R1 channel’s activity. We speculated that changing the charge of this residue gives rise to electrostatic repulsion between side chains favoring the opening of the channel. Therefore, we hypothesized the IP₃R1-D2594 residue determines the ligand sensitivity of the channel by electrostatically affecting the stability of the closed and open states of the channel. Consequently, the aim of the present work was to define for the first time the molecular mechanism by which the IP₃R1-D2594K mutation induces a gain-of-function phenotype. To this end, we measured the influence of the D2594K mutation on the IP₃ activation, cytosolic Ca²⁺ sensitivity, and luminal Ca²⁺ dependency of IP₃R1 at the cellular, subcellular, and single-channel levels. Our results indicate that replacing this negative charge at the pore’s cytosolic exit with a positive charge (D2594K) dramatically potentiated the IP₃R1 channel’s IP₃, cytosolic, and luminal Ca²⁺ sensitivity. Our interpretation is that this residue influences the efficacy of cytosolic (IP₃ and Ca²⁺) and luminal (Ca²⁺) ligands perhaps by electrostatically influencing the stability of the closed and / or open state of the channel, and thus the operation of the IP₃R gating process.

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)

Materials and methods

Generation and culture of stable inducible IP₃R1 expressing cell lines

Briefly, as previously described [46], stable inducible WT- and D2594K-IP₃R1 expressing HEK293 cell lines were generated using full-length ITPR1 cDNA that was sub-cloned into an inducible expression vector, pcDNA5/FRT/TO. The recombinant expression of rat IP₃R1 was achieved by the Flp-In T-REx-293 system (Invitrogen). Cells were incubated at 37 °C under 5% CO₂ with DMEM supplemented with 0.1 mM nonessential amino acids, 2 mM L‐glutamine, 100 units of penicillin/ml, 1% streptomycin/ml, and 5% fetal bovine serum. Cells were selected for Flp positive in 200 µg/ml hygromycin selection medium. Induction of IP₃R1-WT or IP₃R1-D2594K expression was initiated upon incubation with DMEM containing 1 μg/ml tetracycline.

High throughput recording of global Ca²⁺ transients

To evaluate the changes in cytosolic Ca²⁺ concentration, stable inducible HEK293 cells were subcultured in poly-d-lysine pretreated black wall/clear bottom 96-well plates and incubated at 37 °C under 5% CO₂. Each 96-well plate was split to have IP₃R1-WT and -D2594K seeded cells. For the corresponding IP₃R1 expression, after 24 h, cells were induced with tetracycline (1 µg/ml). On the day of the experiment, the media was removed and replaced with Fluo-8 NW (ATT Bioquest) solution containing 10 × pluronic F127 Plus and KRH solution (2 mM Ca²⁺, 125 mM NaCl, 5 mM KCl, 6 mM glucose, 1.2 mM MgCl₂, 25 mM HEPES, pH 7.4). After 60-min incubation at room temperature, Fluo-8 NW solution was removed, and each well was rinsed twice with KRH 0 Ca²⁺ solution. KRH 0 Ca²⁺ solution was added back to each well, and the 96-well plate was placed into FLIPR® Tetra High Throughput Cellular Screening System (Molecular Devices). Each well on the plate was simultaneously challenged with KRH 0 Ca²⁺ solution plus different concentrations of ATP (Sigma-Aldrich) to induce Ca²⁺ release via IP₃R1s. LED excitation wavelength of 490 nm and a 520-nm bandpass emission filter were used to acquire images. The images were captured at every 2 or 4 s. Each determination was done by duplicate at 3 different days. The resulting Fluo-8 NW fluorescent signals were measured for each well using ScreenWorks software (Molecular Devices).

Linescan confocal microscopy imaging of Ca²⁺ puffs

Ca²⁺ puffs were recorded with linescan confocal microscopy in tetracycline-induced HEK293 cells expressing either IP₃R1-WT or IP₃R1-D2594K. Cells were plated on glass bottom (0.13–0.17 mm thick) plates and induced for 48 h. On the day of the experiment, cells were incubated for 30 min in normal KRH external solution containing 2 mM Ca²⁺ for ER loading, followed by 45-min incubation in the dark and at room temperature in 1 ml KRH solution containing the fluorophore Cal-520 AM (AAT Bioquest). Cal-520 AM 45 µM stock solution was prepared by dissolving 50 µg of Cal-520 AM in 20% pluronic acid in DMSO (Molecular Probes) in KRH solution. The incubation period was ended by rinsing the cells with dye-free normal external solution at room temperature for 15 min. To record Ca²⁺ puffs, Cal-520 was excited with the 488 nm line of an argon laser, and the emitted fluorescence was collected through a 515-nm long pass emission filter. Cal-520 images were recorded in bidirectional linescan mode with 256 × 512 pixels per frame at 4 Hz and x-time series images at 10 Hz. For consistency, all linescan confocal microscope settings were kept the same for all experiments (laser power: 5%, photomultiplier gain: 140, pin size: 0.9, pixel size: 0.63). All experiments started with perfusion with external KRH 0 Ca²⁺ solution for 1 min followed by 2-min perfusion of 20 nM ATP. Fluorescence images were analyzed with Elements v4.4 (Nikon) and ImageJ v1.52 (NIH) software. Briefly, Ca²⁺ puffs were interactively detected as areas of high fluorescence compared with the standard deviation (threshold arbitrarily set between 5 and 6.5 standard deviations) of the background fluorescence. Puff duration was measured as the dwell time at the 50% of the peak amplitude (full-duration-half-maximum; FDHM). Puffs frequency was expressed in Hz as number of Ca²⁺ puffs per cell, while their kinetic attributes were estimated by measuring time to peak (time from baseline to maximal), rise time (time from 20 to 80% maximal amplitude), and decaying time (from 80 to 20% maximal amplitude; Lock et al., 2015). All puff parameters were measured, pooled, and averaged for either IP₃R1-WT or IP₃R1-D2594K.

Isolation of microsomal vesicles

Crude microsomes were obtained from control (WT), ITPR1‐D2594K^+/− and -D2594A^+/− mutant knock-in (KI) mice models developed previously [46]. Mouse cerebellar microsomes were prepared as described previously [51] with some modifications. Briefly, cerebella were homogenized in a glass-Teflon Potter homogenizer in solution A (10 mM Tris-maleate, pH 6.8, 0.5 mM DTT, and protease inhibitor cocktail; Roche cOmplete EDTA free; Sigma-Aldrich). The homogenate was centrifuged for 10 min at 4000 × gmax. The supernatant was re-centrifuged for 5 min at 15,000 × gmax, and the pellet was resuspended in solution B (10 mM Tris-Maleate, 100 mM KCl, pH 6.8, and 0.5 mM DTT). Then, the supernatant was centrifuged for 90 min at 165,000 × gmax, and the resulting pellet was resuspended in an appropriate volume of solution B. All pellets were frozen in liquid nitrogen and stored at − 80 °C.

Single-channel recordings

Reconstitution of IP₃R1 channels into artificial planar lipid bilayers was performed as previously described [39]. Briefly, planar lipid bilayers were formed on ~ 100-µm-diameter hole in Teflon septa, separating two compartments: cis and trans. Bilayers were formed using a 5:3:2 mixture of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine (Avanti Polar Lipids) dissolved in decane to a final concentration of 50 mg/ml. Microsomes were added to the solution in cis that was held at ground potential. IP₃-gated channel activity was measured at room temperature in the presence of 2 mM ATP-Tris and various concentrations of IP₃ using Cs⁺ as current carrier. The trans side of the bilayer mimics the luminal (intra-ER) compartment, where the voltage was applied. Solutions (unless otherwise specified) contained symmetrical 250 mM CsCH₃SO₃, 20 mM HEPES pH 7.4, and 1 mM EGTA (70 nM or 140 nM free Ca²⁺). Channel sidedness was determined by IP₃ sensitivity. Stock solutions of IP₃ and Ca²⁺ (Sigma-Aldrich) were made to obtain the desired final concentrations. The cytosolic and luminal free Ca²⁺ concentrations were calculated by MaxChelator [3]. Calculations of total Ca²⁺ needed to obtain a specified free Ca²⁺ concentration were based on the presence of 1 mM EGTA for the determination of cytosolic Ca²⁺ and IP₃ dependency. For the luminal Ca²⁺ dependency, 0.5 mM EGTA, 0.5 mM BAPTA was used in the cis solution while trans solution contained 0.5 mM EGTA, 0.5 mM BAPTA, and 0.5 mM dibromo-BAPTA. Unitary current was processed by a patch-clamp amplifier (Axopatch 200B, Axon Instruments) that was connected, via Ag/AgCl electrodes, to the cis and trans solutions through agar bridges. Data were filtered at 2 kHz and digitized at 20 kHz for computer analysis using pClamp 10 (Axon Instruments). Opening events were detected from the filtered records by the half-amplitude threshold crossing technique [11]. Events with durations < 300 µs were not included in the analysis. Channel open probability was measured from the idealized records longer than 3 min. The number of channels in each experiment was estimated from the maximal number of stacked opening events observed in the bilayer and corroborated in the total amplitude histogram of the raw data by the presence of multiple peaks [16]. IP₃R1 channel currents depicted as positive (upward deflections of the current) reflect cation flux from the trans (luminal) to the cis (cytosolic) compartment. Likewise, the downward deflections of the current reflect the cation flux in opposite direction. Single-channel gating kinetics analysis was done by constructing dwell-time histograms for the open and closed times and fitted with the corresponding probability density functions. Mean open and closed times were calculated as the sum of rate constants exiting each gating state [11].

Statistics

All studies were designed so that only one parameter was changed at a time. To test for differences between groups, we used Student’s t test (two‐tailed with Welch correction), and the two-way ANOVA with Šídák’s post hoc correction using GraphPad Prism software. Data is reported as mean ± SEM. Differences were considered statistically significant at p < 0.05.

Results

Effects of IP₃R1-D2594K on intracellular Ca²⁺ dynamics

To evaluate the functional impact of IP₃R1 mutations on the cellular Ca²⁺ signaling, the IP₃R1-WT and IP₃R1-D2594K were expressed in HEK 293 cells. IP₃R-mediated response was induced through the production of endogenous IP₃ by stimulating Gq-protein-coupled purinergic receptors in the surface membrane with ATP. Intracellular Ca²⁺ signals were recorded with automated fluorescence plate reader in Fluo-8 NW-loaded cells. Figure 2a compares averaged fluorescence Ca²⁺ signals evoked by ATP with no added Ca²⁺, in cells expressing IP₃R1-WT or IP₃R1-D2594K. The evoked Ca²⁺ transients were larger in cells harboring the mutated IP₃R channel compared to the WT. Figure 2b shows pooled measurements of intracellular Ca²⁺ transient amplitude. Maximal change in fluorescence expressed as ΔF/F₀ is plotted as a function of the extracellular ATP stimulus (WT filled, mutant open circles). It can be observed that cells expressing IP₃R1-D2594K produced a significantly larger signal at each [ATP] tested. The reproducibility of this response was examined by performing agonist dose–response curves by duplicate in three separate experiments. ATP stimuli greater than 1 µM evoked a significantly larger Ca²⁺ release transients when the mutant IP₃R1 was expressed.

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

Effects of IP₃R1-D2594K on Ca²⁺ puffs

To determine if local Ca²⁺ release events are altered by the mutated IP₃R1, Ca²⁺ puffs were imaged in intact HEK cells expressing IP₃R1-WT or IP₃R1-D2594K. IP₃R-mediated Ca²⁺ puffs are analogous to RyR-mediated Ca²⁺ sparks in striated muscle. Compared to the global Ca²⁺ transients, the attributes of Ca²⁺ puffs reflect closely the molecular IP₃R functional properties, such as unitary conductance and single-channel gating (opening/closing) kinetics. Ca²⁺ puffs were promoted by addition of 20 nM of extracellular ATP. Our dose–response titration experiments ranging from 10 to 100 nM indicated that 20 nM ATP consistently promoted Ca²⁺ puffs and rarely induced global intracellular Ca²⁺ transients. Note these local Ca²⁺ signals are not the result of extracellular Ca²⁺ entry because the extracellular solution contained 0 mM Ca²⁺. Ca²⁺ puffs were measured during 2-min periods that were preceded and followed by control periods (no extracellular ATP present). Figure 3a shows example line scan images of Ca²⁺ puffs recorded in cells expressing IP₃R1-WT or IP₃R1-D2594K channels. Confocal images obtained under these conditions indicate that HEK cells expressing IP₃R1-D2594K channels exhibit substantially higher Ca²⁺ puff activity elicited by ATP than those expressing IP₃R1-WT. Subcellular areas with high Ca²⁺ puff activity were identified and selected as regions of interests (ROIs). In these selected regions, x-time images were generated with 10-µm kymograph lines to define Ca²⁺ puffs’ properties, which are better visualized by their fluorescence intensity profile plotted under each x-time image. From this type of fluorescent traces, Ca²⁺ puff kinetic attributes (i.e., rise time, time to peak, decay time, and half duration) were measured. Frequency of occurrence was directly quantified from selected ROIs in the x–y linescan images. Figure 3b reveals that IP₃R1-D2594K-expressing cells showed more than 2.5-fold higher Ca²⁺ puffs frequency (in Hz, 0.1 ± 0.002 vs 0.04 ± 0.004 puffs/cell), while the mutant puffs’ kinetics exhibited significantly longer decay (2.28 ± 0.09 vs 0.35 ± 0.01 s), longer half duration (2.39 ± 0.09 vs 0.68 ± 0.02 s), longer time to peak (1.43 ± 0.04 vs 0.57 ± 0.02 s), and longer rise time (0.76 ± 0.03 vs 0.35 ± 0.01 s) than their counterpart from WT. To evaluate the contribution of HEK293 cells endogenous IP₃Rs to Ca²⁺ release events, IP₃R1-D2594K and IP₃R1-WT cells were not induced with tetracycline, and linescan confocal images were recorded during application of external ATP. Under these conditions, infrequent or no intracellular Ca²⁺ signals were recorded in either cell type, indicating that the activity of endogenous IP₃R was not detectable. Overall, these cellular results (Figs. 2 and 3) indicate the IP₃R1-D2594K exhibits a gain of function phenotype.

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

Effects of IP₃R1-D2594K on unitary currents

Since these studies were conducted in a recombinant heterologous system, it was possible that the augmented responses observed with the IP₃R1-D2594K mutant could have resulted from an increased receptor expression. To address this possibility, IP₃R1-WT and -D2594K single-channel activity was evaluated from our WT and KI mice. The corresponding IP₃R1 microsomal fractions from cerebellum were reconstituted in planar lipid bilayers, and single-channel activity was studied under control conditions. As shown in Fig. 4a, the current amplitude as a function of voltage for the mutant channel was very similar to that observed in the WT. Using Cs⁺ as charge carrier, the unitary conductance of 248 ± 5 pS was not significantly different between the IP₃R1-WT and the IP₃R1-D2594K channels (Fig. 4b). Like IP₃R1-WT, the IP₃R1-D2594K Cs⁺ conductance was reduced when Ca²⁺ flux was favored by increased luminal [Ca²⁺] (see Fig. 8a and Online Resource 1), being consistent with the anomalous mole fraction effect previously described for IP₃R1 [4].

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

Effects of IP₃R1-D2594K on IP₃ sensitivity

To gain insight about the mechanism for the enhanced response to ATP by cells expressing IP₃R1-D2594K, its ligand sensitivity was determined at the single-channel level. Figure 5 compares the cytosolic IP₃ sensitivity of the WT and mutant IP₃R1 channels. Representative single IP₃R1 recordings with 50 nM and 5 µM IP₃ present are shown (Fig. 5a). The frequency of WT and mutant channel openings is higher at 5 µM IP₃ (compared to 50 nM). However, the mutant IP₃R1 is far more active (compared to WT) at 5 µM IP₃. Figure 5b plots pooled open probability normalized by the number of channels present in the bilayer (expressed as nPo) of WT and mutant channels as a function of cytosolic [IP₃]. These data were well fitted by a Hill equation with significantly different EC₅₀ values (660 nM for WT and 406 nM for mutant). In addition, IP₃ activated the mutant IP₃R to a significantly higher Po level. The maximal Po reached at 10 µM IP₃ was 3.6-fold higher for the IP₃R1-D2594K compared to the WT channel. Thus, the IP₃R1-D2594K mutation increased the sensitivity and efficacy of IP₃ activation.

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

Effects of IP₃R1-D2594K on cytosolic Ca²⁺ sensitivity

In a previous study, we found that when the IP₃R1-D2594 residue was replaced by alanine, the concentration-dependent response to IP₃ was reduced, while replacing it by a lysine or arginine, the response was substantially increased [46]. One possible mechanism to explain these effects is that the D2594 residue determines the channel sensitivity to cytosolic Ca²⁺. To evaluate this possibility, the experiments shown in Fig. 6 compare the cytosolic Ca²⁺ sensitivity of single IP₃R1-WT with that of D2594 mutated IP₃R1 channels. Representative single IP₃R1 channel activity obtained in 70 nM, 300 nM, and 1 µM cytosolic Ca²⁺ is shown (Fig. 6a). At 70 nM cytosolic Ca²⁺, openings of all three IP₃R1 channels are brief and relatively rare, but for IP₃R1-WT and IP₃R1-D2594K, they become longer and more frequent when the cytosolic Ca²⁺ is increased to 300 nM. Further increasing the cytosolic Ca²⁺ to 1 µM reduced the activity of these three types of IP₃R1 channels. Still, the activity of the IP₃R1-D2594K channel at all tested cytosolic Ca²⁺ levels was considerably greater than the WT channel. Although the IP₃R1-D2594A activity also exhibited a bell-shaped dependency, the opening events remained brief and scarce at the three cytosolic Ca²⁺ concentrations. Figure 6b plots nPo of IP₃R1-WT, IP₃R1-D2594K, and IP₃R1-D2594A channels as a function of cytosolic Ca²⁺. These experiments were conducted in the presence of 10 μM cytosolic IP₃ and 70 nM luminal Ca²⁺. The cytosolic Ca²⁺ dependency of all three channels exhibited a bell-shaped relationship, but the IP₃R1-D2594K activity was considerably larger at all tested [Ca²⁺]. These data were well fitted by a biphasic Hill equation with the following EC₅₀ and IC₅₀ values: 78 and 562 nM for IP₃R1-WT, 101 and 1380 nM for IP₃R1-D2594K, and 89 and 399 nM for IP₃R1-D2594A, respectively. Across the range of all Ca²⁺ levels tested, cytosolic Ca²⁺ activated the IP₃R-D2594K to a significantly larger Po level. The maximal Po reached near 300 nM Ca²⁺ was roughly threefold higher for the IP₃R1-D2594K than for the other two channel types. This shows that the D2594K mutation also increases the cytosolic Ca²⁺ activation efficacy without substantially altering the channel’s cytosolic Ca²⁺ sensitivity given the similarity of EC₅₀ values.

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)

Effects of IP₃R1-D2594K on luminal Ca²⁺ sensitivity

The mechanism of luminal Ca²⁺ regulation has been the focus of intense research [4, 8, 18, 24, 44, 48,49,50]; therefore, the effect of IP₃R1-D2594K on this property was investigated. Figure 7a shows sample single IP₃R1-WT and IP₃R1-D2594K channel recordings with 10 µM luminal Ca²⁺, 10 µM cytosolic IP₃, and 70 nM free cytosolic Ca²⁺. The mutant IP₃R1 is significantly more active than the WT channel. A possible mechanism of luminal Ca²⁺ regulation would involve luminal Ca²⁺ ions feeding through (FT) an open IP₃R1, as they move from lumen of the ER to cytosol, and binding to the channel’s own cytosolic Ca²⁺ activation and inhibition sites. Logically, the degree of Ca²⁺ regulation by FT will depend on the amplitude of Ca²⁺ flux through the open channel, which varies with both the trans-membrane Ca²⁺ gradient and the ER membrane potential. Figure 7b left compares the activity of IP₃R1-WT (filled triangles) with IP₃R1-D2594K (open triangles) normalized to the initial nPo, as a function of luminal [Ca²⁺] at − 40 mV of membrane potential, which favored cytosol-to-lumen Ca²⁺ flux. Under these conditions, the luminal Ca²⁺ dependency was essentially eliminated, as indicated by the straight lines fitted to the data points. In contrast, Fig. 7b right shows that at + 40 mV, which favored the lumen-to-cytosol Ca²⁺ flux, the luminal Ca²⁺ dependency of both types of channels becomes bell-shaped, resembling the cytosolic Ca²⁺ dependency. This bell-shaped behavior indicates the predominance of activation over inhibition at low luminal [Ca²⁺], and the predominant inhibitory effect as luminal [Ca²⁺] is increased. However, the activity of IP₃R1-D2594K (open circles) as a function of luminal [Ca²⁺] was more than threefold greater than that of the WT channel (filled circles) and peaked at about one order of magnitude lower luminal [Ca²⁺] (10 vs 100 μM, respectively). These dramatic effects observed across most of luminal Ca²⁺ levels tested were unlikely due to the mutant conducting a larger flux because as shown in Fig. 4, the mutation does not alter IP₃R1 unitary conductance.

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

Discussion

IP₃R and RyR share many structural and functional similarities. In RyR2, the negatively charged residue E4872 electrostatically interacts with the positively charged residue R4874 in the neighboring subunit to stabilize the closed state. When open, E4872 moves away from R4874, but closer to the negatively charged residue E4878. These residues (E4872 and E4878) are thought to be involved in electrostatic interaction with luminal Ca²⁺ [19, 35]. The corresponding E4872 residue in IP₃R1 is D2594, but its role in IP₃R function is unknown. We have previously found that when the D2594 residue’s charge was neutralized, the IP₃ sensitivity of IP₃R1 was reduced, but the IP₃ sensitivity increased if it was replaced by a positive residue like lysine or arginine [46]. In this work, we found that in intact cells, this substitution increased the efficacy of activation by IP₃ (shifted the EC₅₀), while at the subcellular level, it increased the frequency, amplitude, and duration of Ca²⁺ puffs. These actions could be explained by differences in cell densities, expression levels, and/or altered receptor regulation. To address these possibilities, we defined how the D2594K alters IP₃R1 function at the single-channel level. We showed (Fig. 5b) the mutation increased Po and shifted the IP₃ EC₅₀, indicative of an increase in the channel’s IP₃ sensitivity. However, this could also be explained in part by changes in either IP₃R1 cytosolic and/or luminal [Ca²⁺] regulation. We found that IP₃R1-D2594K, like its WT counterpart, exhibited a bell-shaped cytosolic-free [Ca²⁺] dependency, but the mutant exhibited higher Po even when the lumen-to-cytosol Ca²⁺ flux was minimized by reducing the Ca²⁺ driving force with 70 nM luminal [Ca²⁺]. As seen in Fig. 6, the channel activity at both 70 and 300 nM cytosolic [Ca²⁺] was considerably higher in the mutant channel than in the WT with low luminal [Ca²⁺]. Likewise, WT and D2594K channels were both sensitive to luminal [Ca²⁺], but the mutant’s Po was higher as well. Although potential effects of D2594K on ionic permeability/selectivity were not ruled out, our Ca²⁺/Cs⁺ selectivity experiments suggest this possibility is unlikely (see Fig. 4 and Online resource 1). Taken together, it appears that this mutation near the channel’s gate simultaneously alters the IP₃ and Ca²⁺ (cytosolic and luminal) sensitivities of the channel to produce its clear gain-of -function phenotype. This is consistent with the notion that the D2594 residue stabilizes the IP₃R1 closed state, as it has been proposed by Ogawa’s group for the corresponding residue in RyR2, where specific interactions within the pore stabilize and prevent hyperactivity [21]. Furthermore, this possibility has already been suggested for the IP₃R as point mutations in the channel permeation pathway alter intra/inter-domain interactions and have secondary regulatory actions [7, 40].

Specifically, we speculate that IP₃R1 D2594 residue electrostatically interacts with positively charged R2596 in the neighboring subunit. This forms a network of salt bridges that stabilizes the IP₃R1 closed state. This makes the R2594 residue key for IP₃R1 stabilization and prevention of potentially deleterious hyperactivity.

As mentioned, the IP₃R1-D2594 residue is homologous to RyR2-E4872 which (in RyR2) influences luminal Ca²⁺ regulation [9]. Replacing RyR2-E4872 for alanine abolished luminal Ca²⁺ dependency, but it was restored by introducing a glutamate residue nearby (G4871E). Thus, we sought to better detail the potential influence of IP₃R1-D2594 on luminal Ca²⁺ regulation. Figure 8 shows the actions of luminal [Ca²⁺] and membrane potential on the duration of IP₃R1-WT opening and closing events. Single IP₃R1-WT channel activity obtained at three different luminal [Ca²⁺] at + 40 mV (Fig. 8a) illustrates the clear luminal [Ca²⁺] dependency of IP₃R1 Po. Note the unitary Cs⁺ current is small at 5 mM luminal (as Ca²⁺ competes with Cs⁺ for occupancy of the pore). The duration of IP₃R1 opening and closing events varied with luminal [Ca²⁺]. We analyzed mean open time (MOT) and mean closed time (MCT) separately because lumen-to-cytosol Ca²⁺ flux carried by an open channel may act on cytosolic Ca²⁺ regulatory sites and extend mean open time (MOT; [50]). Intuitively, lumen-to-cytosol Ca²⁺ flux should not influence closed channel function. In Fig. 8b, MOT was normalized (to its initial value) and plotted as a function of the electrochemical Ca²⁺ driving force (EDF, measured as the difference between the membrane potential and the Ca²⁺ Nernst potential; E_m − E_Ca). Interestingly, single IP₃R1 MOT displayed a bell-shaped dependency on EDF. As indicated by the vertical arrows, MOT decreased as the ER [Ca²⁺] is reduced. Arrows represent the expected EDFs at room temperature, for an ER that is full of Ca²⁺ (solid arrow), a depleted ER (dash arrow), and an extremely depleted ER (square dot arrow). Note that the activation phase lies within the physiological range of electrochemical driving forces for Ca²⁺. The MOT behavior is reminiscent of the IP₃R1’s bell-shaped cytosolic Ca²⁺ sensitivity (see Fig. 6) and strongly suggests luminal Ca²⁺ feeds through an open IP₃R1 and acts on its cytosolic Ca²⁺ activation and inactivation sites. Interestingly, the mean closed time (MCT) also exhibited a profound dependency on EDF as illustrated in Fig. 8c. In fact, the experimental data points were well described by the bell-shaped curve fitted to the MOT (double-Hill equation scaled and inverted; Fig. 8c; dashed curve). Although our results cannot definitively rule out the presence of a luminal Ca²⁺ binding site (in the channel itself and/or in an accessory protein), our analysis indicates that the IP₃R1 dependence on luminal [Ca²⁺] is strongly influenced by the Ca²⁺ ions passing through the IP₃R1. Unfortunately, the same type of kinetic analysis could not be performed on IP₃R-D2594K channels because multiple (not single) mutant channels were typically present. However, based on our cytosolic and luminal [Ca²⁺] studies, we believe mutant channel dwell times would likely exhibit similar dependencies on EDF.

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 (E_m − E_Ca) in mV, where E_m is the transmembrane potential applied to the bilayer (i.e., − 40, − 20, + 20, or + 40 mV) with the cis side held at ground and E_Ca is the Nernst potential of Ca²⁺ in mV, calculated at 22 ºC by the formula E_Ca = (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 equation (MOT = 1 + Range∙(1 + (Va₅₀/EDF)^Ha) − 1 (1 + (EDF/Vi₅₀)^Hi) − 1) with Va₅₀ = 111.5 mV, Ha = 7.8, Vi₅₀ = 170.7 mV, Hi = 3.7. Arrows represent the electrochemical driving force for Ca²⁺ at E_m = 0 mV (the physiological transmembrane potential at the ER membrane) at full (1 mM), depleted (100 µM) or an extremely depleted (10 µM) ER, with 140 nM Ca²⁺ at the cytosol. c Normalized closed time of IP₃R1-WT channel plotted as a function of the electrochemical Ca²⁺ driving force. Data points were measured at + 20 (dashed circles) and + 40 mV (gray circles). For comparison, the curve fitted to MOT (flipped and scaled) is included as a dashed line

In summary, a ligand gated channel’s gate interacts (likely allosterically) with ligand binding sites. Our data show the intra-pore near-the-gate IP₃R1-D2594 residue critically contributes to ligand interactions (likely allosterically) of multiple IP₃R1 binding sites. Thus, the IP₃R1-D2594 residue is located in an ideal position to influence gating kinetics by establishing specific interactions within the pore to stabilize and prevent hyperactivity.