Effects of divalent cations on single-channel conduction properties of Xenopus IP3 receptor

Don-On Daniel Mak and J. Kevin Foskett

Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6100

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The effects of Mg2+ and Ba2+ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopus oocytes. In 140 mM K+ the IP3R channel kinetics and presence of conductance substates were similar over a range (0-9.5 mM) of free Mg2+. In 0 mM Mg2+ the channel current-voltage (I-V) relation was linear with conductance of ~320 pS. Conductance varied slowly and continuously over a wide range (SD approx  60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on either or both sides of the channel reduced the current (blocking constant ~0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ~120 pS near 0 mV and ~360 pS at ±70 mV in symmetrical 2.5 mM Mg2+). Ba2+ exhibited similar effects on channel conductance. Mg2+ and Ba2+ permeated the channel with a ratio of permeability of Ba2+ to Mg2+ to K+ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.

calcium signaling; inositol phosphates; calcium release channel; patch clamp; signal transduction

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MODULATION OF FREE CYTOPLASMIC Ca2+ concentration ([Ca2+]i) is a ubiquitous cellular signaling system. In many cell types, Ca2+ signals involve the generation of the intracellular second messenger inositol 1,4,5-trisphosphate (IP3) in response to the binding of ligands, including neurotransmitters and hormones, to plasma membrane receptors. IP3 causes the release of Ca2+ from intracellular stores, including the endoplasmic reticulum (ER), by binding to its receptor (IP3R) (2, 34, 44), which is a Ca2+ channel (42). Several types of IP3Rs as products of different genes with alternatively spliced isoforms (26, 27, 45, 53) have been identified and sequenced. The IP3Rs have ~2,700 amino acid residues in IP3 binding, regulatory (modulatory), and transmembrane channel domains (26, 27, 45). Some putative transmembrane helices of the receptors have sequence homology with some of those in the ryanodine receptor (26), a muscle sarcoplasmic reticulum Ca2+ channel (52). [Ca2+]i signals in nonexcitable cells exhibit complex spatial and temporal features that are believed to provide highly regulated global as well as localized control of Ca2+-dependent processes. Because Ca2+ release from intracellular stores is the central component in all models that account for [Ca2+]i signals, a detailed understanding of the mechanisms that generate such complexity requires knowledge of the ion channel properties of the IP3R. The single-channel properties of the IP3Rs have been studied by their reconstitution into synthetic bilayer membranes (3, 4), because their intracellular location has precluded patch-clamp recordings in the native membrane environment. More recently, however, the observed continuity of the ER with the outer membrane of the nuclear envelope (10) and the successful application of the patch-clamp technique to the nuclear envelope, despite the presence of nuclear pores (7, 25, 32, 43), have enabled application of the patch-clamp technique to study ER-localized ion channels in their native membrane. We (22, 23) and others (40) recently employed this approach to study the Xenopus laevis type 1 IP3R in the outer membrane of nuclei isolated from oocytes, where it has been localized (8, 19, 30, 31).

We established the identity of the IP3R channel in outer nuclear membrane patches by its activation by IP3 and inhibition by heparin. The Xenopus IP3R was found to be a weakly Ca2+-selective cation channel, with PCa/PK/PCl of 8:1:0.05 (where PCa, PK, and PCl are permeabilities of Ca2+, K+, and Cl-, respectively), and it exhibited multiple conductance states. With K+ as the charge carrier, the current-voltage (I-V) relation of the IP3R was rectified, with the conductance of the most frequently observed state (occurring ~90% of channel open time) being 113 pS at ~0 mV and increasing to ~300 pS at ±60 mV (22, 23). Under our experimental conditions the IP3R frequently showed robust kinetic behavior. The channel usually exhibited bursting-type kinetics with a high open probability (>0.8) during bursts that typically last over several seconds. A novel "flicker" kinetic mode of the IP3R was also observed in which the channel alternated rapidly between two conductance states F1 and F2, with values nearly one-fourth and three-fourths that of the main open state conductance (23). In addition, IP3-dependent inactivation or rundown of channel activity (time constant ~30 s) was observed for every channel, despite the continuous presence of IP3 and the absence of net Ca2+ flux or change in Ca2+ concentration. Some (~10%) of these channel disappearances could be reversed by an increase in voltage before they irreversibly inactivated (23). Mapping of the IP3R channel distribution by repeated patching of individual nuclei revealed that the channels have a high propensity to cluster. Interestingly, clustering significantly slowed the rate of channel inactivation. IP3R clustering and inactivation may contribute to the generation of complex spatial and temporal [Ca2+]i signals in cells.

Mg2+ is the second most abundant intracellular cation in cells (11), and it plays an important role in a large number of cellular processes, including regulation of ion transport. Intracellular Mg2+ modulates single-channel properties of K+ and Na+ channels (1), and binding of IP3 to the IP3R (49) and IP3-mediated Ca2+ release (50) may be affected by Mg2+. However, the effects of Mg2+ on IP3R channel properties have not been directly examined. Previous studies of the reconstituted IP3R (3, 41) determined its divalent cation conductance sequence, but the ionic conditions employed, including tens of millimoles of divalent ions on one side and absence of K+, leave unresolved the specific effects of Mg2+ on single-channel permeation or gating properties under more physiological ionic conditions. In the present study we have investigated the effects of cytoplasmic and luminal Mg2+ concentration on the single-channel properties of the type 1 IP3R in isolated Xenopus oocyte nuclei by the patch-clamp technique. To determine the specificity of the interactions between the channel and Mg2+, we also compared the effects on the IP3R of another divalent ion, Ba2+, with the effects of Mg2+.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Maintenance of Xenopus laevis, surgical extraction of ovaries, and storage of the extracted ovaries have been described previously (22, 23). Stage VI oocytes were isolated from the ovary just before the experiments and opened mechanically. Isolation of oocyte nuclei, patch clamping of the outer nuclear membrane, and acquisition and analysis of data were done as described previously. All experiments were performed in the "on-nucleus" configuration without excision of the patched membrane, with the solution in the perinuclear lumen between the outer and inner nuclear membranes in apparent equilibrium with the bath solution (22). The cytoplasmic aspect of the IP3R channel faced into the patch pipette. Following standard conventions, the applied potential (Vapp) is the potential of the pipette electrode relative to the reference bath electrode. Accordingly, positive current flowed from the pipette outward. The majority of the current records were obtained under positive Vapp, because the gigaohm seals were more stable under that polarity. However, all channel properties were observed under both positive and negative Vapp, unless explicitly stated otherwise. All experiments were performed at room temperature with the pipette solution containing 10 µM IP3, a saturating concentration to ensure that observed effects of experimental manipulations could not be attributable to effects on IP3 binding.

All pipette and bath solutions used in our experiments contained 140 mM KCl, 10 mM HEPES, and 0.5 mM Na2ATP, with pH adjusted to 7.1 with KOH. The Maxchelator software (C. Patton, Stanford University, Stanford, CA) was used to calculate the concentrations of free Ca2+ ([Ca2+]), Mg2+ ([Mg2+]), and Ba2+ ([Ba2+]) in those solutions. Solutions used in the Mg2+ experiments contained 0-10 mM MgCl2, and 0.1 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid was used with appropriate amounts of CaCl2 added (40-56 µM) to maintain [Ca2+] at ~200 nM in the presence of different [Mg2+]. Solutions used in the Ba2+ experiments contained 0 or 3 mM BaCl2, yielding calculated [Ba2+] of 0 or 2.5 mM, respectively, because of chelation by ATP. Ca2+ chelators were not used, because their high affinity for Ba2+ relative to Ca2+ makes them inappropriate for buffering [Ca2+] in the presence of millimolar [Ba2+]. Total Ca2+ content in these experimental solutions was determined by induction-coupled plasma mass spectrometry (Mayo Medical Laboratory, Rochester, MN) to be 6.5 ± 0.7 µM. Calculated [Ca2+] was 0.76 ± 0.08 and 5.9 ± 0.7 µM in the presence of 0 and 3 mM BaCl2, respectively, after chelation by ATP. Under our experimental conditions the open probability of the IP3R under all experimental [Ca2+] was >0.3 (24).

IP3 and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid were obtained from Molecular Probes (Eugene, OR), high-purity BaCl2 anhydrous salt (99.999%) from Aldrich (Milwaukee, WI), and other inorganic salts and ATP (sodium salt) from Sigma Chemical (St. Louis, MO).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Behavior of the IP3R in symmetrical Mg2+-containing solutions. In symmetrical solutions containing 2.5 mM free Mg2+, similar in composition to those used in our previous studies (22, 23), the IP3R was regularly observed in outer nuclear membrane patches, with the predominant conductance state M accounting for ~90% of channel open time. The channel was observed with a high open probability (Fig. 1A) and had a nonlinear I-V relation (Fig. 1, A and B) with a slope conductance (dI/dV) of 120 pS between ±20 mV and 360 pS at ±70 mV. These features, as well as the occurrence of long quiescent periods (open probability = 0) under high Vapp (50-60 mV) on the cytoplasmic side of the channel (Fig. 1A), inactivation of the channel (time constant ~ 30 s), and an I-V relation that was symmetrical with respect to the origin between -70 and 80 mV (Fig. 1B), are characteristics of the Xenopus IP3R (22, 23).


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Fig. 1.   A: typical current traces of inositol 1,4,5-trisphosphate receptor (IP3R) in symmetrical solutions with 2.5 mM free Mg2+ at various applied potentials (Vapp). In all current traces, dashed lines with letter C mark closed-channel current level. All current traces show data digitized at 2 kHz and filtered at 1 kHz and are plotted with same scale. B: current-voltage (I-V) curves of main state (M) of IP3R in symmetrical solutions. Current amplitudes were averaged from all channel opening and closing events (n > 100 in each record) detected automatically in data records. SE bars are smaller than symbols. bullet , Data obtained in 2.5 mM Mg2+ solution from 7 different patches. From patches obtained in 0 mM Mg2+ solution with stable channel conductances throughout current records, 3 records showing similar conductance at 40 mV were selected for I-V analysis (open circle , square , and triangle ). Solid curve, 5th-order odd polynomial [I = a1Vapp + a3(Vapp)3 + a5(Vapp)5] fitted to 2.5 mM Mg2+ I-V curve. Dotted lines, slope conductances (dI/dV) of 2.5 mM Mg2+ I-V curve: 120 and 360 pS at 0 and 70 mV, respectively. Dashed line, conductance of 370 pS fitted to 0 mM Mg2+ data. All curves fitted to data in this and subsequent figures were obtained using a least-squares iterative algorithm in Igor Pro 3.0 software (Wavemetrics, Lake Oswego, OR). C: typical current traces of IP3R in symmetrical 0 mM Mg2+ solutions under various Vapp.

In symmetrical solutions without Mg2+, the IP3R (n = 41 patches from 18 nuclei) conducted a significantly higher current (Fig. 1C) than in symmetrical 2.5 mM Mg2+ solutions under the same Vapp. This large conductance (~320 pS) was observed in single-channel and multichannel current records (containing up to 8 evenly spaced current levels; Fig. 1C). Unlike the highly nonlinear I-V curve characteristic of the IP3R in symmetrical 2.5 mM Mg2+ solution, the I-V relation in symmetrical 0 mM Mg2+ solution was linear between -40 and 60 mV (Fig. 1B). Although removal of Mg2+ altered the conduction properties of the IP3R, the kinetic properties of the channels were similar to those observed in symmetrical solutions containing 2.5 mM Mg2+. Channels in 0 mM Mg2+ solution had a high open probability and displayed quiescent periods whose frequency and duration increased at higher cytoplasmic potentials. The channels inactivated with a time course (~90% of channels inactivated within 2 min) similar to that observed for the IP3R in symmetrical solutions containing 2.5 mM Mg2+. Inactivated channels could be reactivated by a jump in Vapp in ~10% of the experiments, and in one experiment the channels were repeatedly reactivated. All these properties are characteristic of the IP3R in Mg2+-containing solutions (23).

To further confirm that the channels observed in symmetrical 0 mM Mg2+ solutions were indeed the IP3R, regions on the outer membrane of four different nuclei were identified where the probability of obtaining patches with IP3R channel activity was ~1 (23). A series of patches was obtained from these regions with the pipette solution alternately containing no IP3 or 10 µM IP3. Whereas 14 of 15 patches with IP3 exhibited channel activities similar to those shown in Fig. 1C, none of the 5 patches without IP3 exhibited similar activities. Thus the high-conductance channels observed in symmetrical 0 mM Mg2+ solutions were gated by IP3. Together with the characteristic IP3R kinetic properties, these results established the identity of the channels as the IP3R.

In addition to its higher conductance, a novel feature of the IP3R observed in symmetrical 0 mM Mg2+ solutions was that its conductance could achieve a wide range of values. In most of the current records of IP3R channels in symmetrical 0 mM Mg2+ solutions, the conductance of the open state (M) remained constant over extended periods lasting several to tens of seconds in the same experiment (Fig. 1C), which enabled steady-state I-V relations of the channel to be determined. However, even under the same Vapp, the conductance of IP3R channels was observed to modulate in three different temporal domains. First, the stable IP3R conductance varied from patch to patch (open amplitude histograms in Fig. 2A) over a wide range [SD of ~60 pS around a mean of 320 pS (Fig. 2B) vs. SD of 15 pS around a mean of 123 pS in symmetrical 2.5 mM Mg2+ solution (Fig. 2D)], even among patches obtained from the same nucleus with use of the same experimental solutions. Second, the conductance of channels changed during the course of a recording (in 8 of 41 patches; gray amplitude histogram in Fig. 2A), which usually lasted <2 min because of channel inactivation. Third, the channel conductance fluctuated during single-channel openings lasting tens of milliseconds (in 3 patches; Fig. 2, G and H). Such a wide range of conductances of channels among patches and fluctuations of conductance during single-channel openings has never been observed in the presence of 2.5 mM Mg2+ in hundreds of patches, in which the channels always gated between different well-defined conductance states (22, 23).


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Fig. 2.   A-F: transition event amplitude histograms of IP3R at 20 mV. Channel opening and closing events were detected automatically by MacTac 2.5.1 software (SKALAR Instruments, Seattle, WA) by use of 50% threshold technique (12). Event amplitude is change in measured current caused by a channel opening or closing. Data were digitized at 2 kHz and filtered at 1 kHz. Only events separated by >2 ms were used, thereby excluding events with amplitudes that had been reduced by filtering. Means ± SD are shown above each histogram (B-F). Current axes of graphs have same scale. A: event amplitude histograms for 3 different experiments (thick line, thin line, and stippled histograms) in symmetrical 0 mM Mg2+ solutions. B: total event amplitude histogram for 8 experiments in symmetrical 0 mM Mg2+ solutions. C: event amplitude histogram for 3 experiments in symmetrical 0.5 mM Mg2+ solutions. D: event amplitude histogram for 9 experiments in symmetrical 2.5 mM Mg2+ solutions. E: event amplitude histogram for 6 experiments in 0 mM [Mg2+]pip/2.5 mM [Mg2+]bath (where [Mg2+]pip is free Mg2+ concentration in pipette solution and [Mg2+]bath is free Mg2+ concentration in bath solution). F: event amplitude histogram for 2 experiments in 9.5 mM [Mg2+]pip/0 mM [Mg2+]bath. Smooth lines in D and F, Gaussian curves fitted to histograms. G and H: current traces of IP3R in a single-channel patch (G) and a multichannel patch (H) from a different experiment; both show spontaneous continuous fluctuations of channel conductance under constant Vapp. Experiments were in symmetrical 0 mM Mg2+ solutions, with data digitized at 2 kHz and filtered at 1 kHz.

Of interest were recordings that contained multiple channels. Among 84 patches containing IP3R channel activities in symmetrical 0 mM Mg2+ solution, 40 had two or more channels. In the majority of these multichannel patches the conductances of the individual channels detected in the same patch were similar, within 10% (experimental error limit) of one another (Fig. 3), despite the wide range of conductances observed among patches (185-412 pS). Even among those patches (8 of 40; filled circles in Fig. 3) in which the conductance changed during the recording, most (7 of 8) exhibited conductances that either changed in concert, so that they remained similar before and after the change, or became similar, although they were dissimilar at the beginning of the current recording (filled squares in Fig. 3). In only three experiments (open squares in Fig. 3) did the individual conductances remain dissimilar throughout the recording.


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Fig. 3.   IP3R channel conductances (G) in multichannel patches plotted in order in which they were obtained. Patches obtained from same oocyte nuclei are separated by dashed lines; patches obtained from different nuclei are separated by solid lines. Vertical bars, value (±10%) of each conductance level in a multichannel patch. In recordings during which conductance changed (bullet  at bottom of graph), vertical bars denote conductance values before change and bullet  with error bars denote conductance values after change. Squares at bottom of graph designate recordings in which dissimilar conductance values (differ from one another by >10%) occurred: black-square, recordings with dissimilar conductances that became similar over course of recordings; square , recordings with conductances that remained dissimilar throughout recordings.

Increasing the [Mg2+] from 0 to 2.5 mM in symmetrical solutions decreased the range of IP3R channel conductances observed among patches (SD decreased from 1.2 to 0.3 pA at Vapp = 20 mV; Fig. 2, B-D). The current amplitude distributions of channel closing and opening events were converted by Mg2+ from broad distributions with multiple peaks (Fig. 2B) in 0 mM Mg2+ solution to essentially single peaks resembling the Gaussian distributions in symmetrical 2.5 mM Mg2+ solution (Fig. 2D). Thus [Mg2+] in the millimolar range stabilized the IP3R channel conductance.

In addition, the stable conductance of the IP3R channel decreased continuously as [Mg2+] in the solutions was increased symmetrically from 0 to 9.5 mM (Fig. 4A). At 20 mV the IP3R channel conductances were 123.2 ± 0.2 and 84.4 ± 0.7 (SE) pS in symmetrical 2.5 and 9.5 mM Mg2+ solutions, respectively.


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Fig. 4.   IP3R channel current (mean ± SD) at 20 mV in solutions with symmetrical (A) and asymmetrical (B) Mg2+ concentrations. Tabulated numbers adjacent to each current point are number of transition events detected and number of experiments used (in parentheses) for corresponding solution. In A, data were fitted by a simple saturation kinetics curve {I = Iinfinity  + gamma /([Mg2+] + Ki)}, where blocking constant by Mg2+ (Ki) = 0.56 mM, with curve-fitting constants Iinfinity  = 1.4 pA and gamma  = 2.86 mM · pA.

Behavior of the IP3R in asymmetrical Mg2+-containing solutions. To determine from which side of the channel Mg2+ exerted its effects on conductance and conductance stability, asymmetrical solutions were used. With 2.5 mM free Mg2+ in the pipette solution ([Mg2+]pip = 2.5 mM) and 0 mM free Mg2+ in the bath solution ([Mg2+]bath = 0 mM), the mean IP3R channel current at 20 mV fell between those observed in symmetrical 0 and 2.5 mM Mg2+ solutions (Fig. 4B) and was similar to that observed in the reversed [Mg2+] conditions, i.e., 0 mM [Mg2+]pip /2.5 mM [Mg2+]bath.

Whereas symmetrical 2.5 mM Mg2+ solution was sufficient to stabilize the channel conductance to a single-peak, Gaussian-like amplitude distribution (Fig. 2D), with 2.5 mM Mg2+ solution on only one side of the channel and 0 mM Mg2+ solution on the other, the channel conductance was still observed to vary from patch to patch with a broad, non-Gaussian amplitude distribution (SD = 0.6 pA, Fig. 2E), although the range of conductance variation was significantly reduced compared with that in symmetrical 0 mM Mg2+ solution. Changes of conductance over the course of one recording and conductance fluctuations during single-channel openings were also observed. However, 9.5 mM Mg2+ solution on just one side of the channel, in the absence of free Mg2+ on the other side, was sufficient to stabilize the IP3R channel conductance to a single-peak, Gaussian-like amplitude distribution with a standard deviation similar to that observed in symmetrical 2.5 mM Mg2+ solution (Figs. 2F and 4B).

I-V curves for the IP3R in asymmetrical [Mg2+] (Fig. 5) gave a reversal potential (Vrev) of -6.2 mV for 9.5 mM [Mg2+]pip /0 mM [Mg2+]bath and 2.0 mV for 0 mM [Mg2+]pip /2.5 mM [Mg2+]bath. By use of PK/PCl of 1:0.05 derived previously (22), PMg/PK/PCl of 2.6:1:0.05 was evaluated by applying the Goldman-Hodgkin-Katz voltage equation (16) to the Vrev values. It can be seen from the I-V curves that rectification of channel current at high Vapp occurred with Mg2+ on either side of the channel. I-V curves with similar rectification were observed in 2.5 mM [Mg2+]pip/0 mM [Mg2+]bath (data not shown) and in 0 mM [Mg2+]pip/2.5 mM [Mg2+]bath. The rectification was symmetrical with respect to Vrev (Fig. 5), regardless of the side of the channel on which Mg2+ was present.


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Fig. 5.   I-V curves for IP3R channel in solutions with asymmetrical Mg2+ concentrations. Each data set was obtained from a recording during which no change was observed in channel conductance under constant Vapp. Both I-V curves were symmetrical with respect to reversal potential (Vrev), as shown by fit of data with cubic polynomials: I = a1(Vapp - Vrev) + a3(Vapp - Vrev)3. square , 9.5 mM [Mg2+]pip/0 mM [Mg2+]bath, with Vrev = -6.2 mV from fitted curve (dashed line); open circle , 0 mM [Mg2+]pip/2.5 mM [Mg2+]bath with Vrev = 2.0 mV from fitted curve (solid line).

Behavior of the IP3R in Ba2+-containing solutions. To determine the divalent specificity of the observed effects of Mg2+, we performed similar experiments using Ba2+ instead of Mg2+. In symmetrical 2.5 mM Ba2+ solutions, IP3R channels were observed regularly with characteristics very similar to those observed in symmetrical 2.5 mM Mg2+ solutions. Of 42 patches obtained, 21 showed IP3R channel activity: 9 were single-channel patches and 7 were from one active region (23). The channels exhibited a nonlinear I-V curve very similar to that in 2.5 mM Mg2+ solution (Fig. 6A). The channel kinetics were similar to those observed in 2.5 mM Mg2+ solution, including long quiescent periods at higher Vapp (Fig. 6B). Inactivation of the channel was observed consistently, with all observed channels disappearing within 2 min of seal formation.


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Fig. 6.   I-V curve (A) and typical current traces (B) of IP3R in symmetrical solutions with 2.5 mM Ba2+. Solid line in A, 5th-order odd polynomial [I = a1Vapp + a3(Vapp)3 + a5(Vapp)5] fitted to data showing that I-V curve is symmetrical with respect to Vrev (0 mV); dotted line, 5th-order odd polynomial I-V curve of IP3R in symmetrical 2.5 mM Mg2+ solution; dashed line, linear I-V curve of IP3R in symmetrical 0 mM Mg2+ solution (same as curves in Fig. 2), shown for comparison. M, normal state; C, closed state.

In asymmetrical solutions with 2.5 mM Ba2+ solution on one side and 0 mM Ba2+ solution on the other, the nonlinear I-V curve of the channel showed a Vrev of 2.65 mV (Fig. 7, A and B), consistent with PBa/PK/PCl of 3.5:1:0.05 according to the Goldman-Hodgkin-Katz voltage equation. Changes of conductance of the same channel in one recording and continuous fluctuations of channel conductance during one channel opening (Fig. 7C) were observed with 2.5 mM Ba2+ solution on just one side of the channel, whereas 2.5 mM Ba2+ solution on both sides stabilized the channel conductance and eliminated such fluctuations.


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Fig. 7.   A: nonlinear I-V curve of IP3R in 0 mM [Ba2+]pip/2.5 mM [Ba2+]bath obtained from 1 recording during which channel conductance was stable under constant Vapp. B: I-V curve of channel near its Vrev (boxed region in A). Vrev is 2.65 mV from 5th-order polynomial fit to data (solid line). C: current trace of IP3R in 2.5 mM [Ba2+]pip/0 mM [Ba2+]bath showing spontaneous fluctuations of channel conductance under constant Vapp (-60 mV).

Effects of divalent cations on the flicker kinetic mode. Besides the normal kinetic mode of the IP3R channel, a novel flicker kinetic mode was observed previously in which the channel rapidly gates between conductance levels 27 and 78% of the main level (23). This kinetic mode was also observed in all symmetrical and asymmetrical [Mg2+] conditions represented in Fig. 4 (Fig. 8A), as well as in the presence of Ba2+ (Fig. 8B). Despite the wide range of channel conductances observed at the various Vapp and divalent cation concentrations used in these experiments, the ratios of channel currents for the two flicker states (F1 and F2) and the normal state (M) remained constant: IF2/IM = 0.78 ± 0.03 and IF1/IM = 0.27 ± 0.03, which are identical to those reported previously (23). Furthermore, the voltage dependencies of the flicker mode under all conditions were qualitatively similar to those reported previously (23), with a high positive Vapp favoring the F2 flicker state and a negative Vapp closing the channel in flicker mode (Fig. 8C).


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Fig. 8.   A: current traces of IP3R channel in 2.5 mM [Mg2+]pip/0 mM [Mg2+]bath with normal and flicker kinetic modes. Vapp was 60 mV. Data were digitized at 12.5 kHz and filtered at 5 kHz. Dashed lines, current levels for closed state (C), flicker states (F1 and F2), and normal state (M). B: current trace of IP3R in 2.5 mM [Ba2+]pip/0 mM [Ba2+]bath showing flicker and normal kinetic modes. Vapp was 20 mV. Data were digitized at 5 kHz and filtered at 1 kHz. C: inactivation of IP3R channel in flicker mode by a change in polarity of Vapp. Channel was exposed to symmetrical 0 mM Mg2+ solution. Data were digitized at 2 kHz and filtered at 1 kHz. Shift in closed-channel current during jump in Vapp was caused by background leak current (seal resistance = 6.5 GOmega ).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Permeation block of the IP3R by divalent cations. Our investigations of the single-channel properties of the Xenopus oocyte IP3R under various concentrations of free Mg2+ and Ba2+ have revealed complex interactions between the channel and divalent cations in the solutions on both sides of the channel.

First, Mg2+ and Ba2+ permeate the channel with a high permeability: PMg/PK of 2.6 and PBa/PK of 3.5, which we derived from the Vrev values under asymmetrical ionic conditions. Because PCa/PK is 8 (22), the IP3R has a permeability sequence of PCa > PBa PMg > PK in the presence of the monovalent cation K+ as the dominant charge carrier through the channel. This sequence agrees with that determined for the mammalian cerebellar IP3R reconstituted into planar lipid bilayers (3), although a somewhat different PBa/PK of 6.3 was derived on the basis of extrapolated Vrev values with 110 mM K+ and 0 mM Ba2+ on the cytoplasmic side of the channel and 55 mM Ba2+ and 0 mM K+ on the other side. This discrepancy may be caused by the very dissimilar ionic conditions used in the experiments, by the distinct membrane environments around the channels, or by intrinsic differences between mammalian and Xenopus IP3R channels. This sequence is similar to that of the ryanodine receptor channel PCa > PBa approx PMg > PK (48) under ionic conditions comparable to ours. The similar permeability sequences of the two Ca2+-release channels, particularly in light of their sequence homology in some of the putative channel-forming helices (26), suggest that the molecular characteristics of the channel pores, which are undefined, may be similar between the two channels.

Second, an increase in [Mg2+] decreases the monovalent cation conductance of the IP3R. Simultaneously, the I-V relation of the IP3R, which is linear in the absence of Mg2+, becomes rectified in the presence of free Mg2+ on either side of the channel, with dI/dV increasing with |Vapp|. These effects of Mg2+ on the K+ current through IP3R are distinct from the reduction of K+ current by Mg2+ in some large-conductance, Ca2+-activated K+ channels, where it is due to electrostatic screening by Mg2+ of surface charges around the channel pore. Such current reductions by electrostatic screening do not generate rectification in the I-V relation of the channels (21, 51). The rectified I-V relation of the IP3R in the presence of free Mg2+ is symmetrical with respect to Vrev and, therefore, cannot be fitted by a Woodhull model (53), which describes an I-V relation that is asymmetrical with respect to Vrev because of competitive block of the channel pore by impermeant ions present on one side of the channel. Thus the observed effects of Mg2+ on the monovalent cation current of the IP3R are different from the Mg2+-induced rectification of the I-V curves of ATP-sensitive K+ channels (13, 18), ACh-regulated K+ channels (17), inward rectifier K+ channels (20, 39), and some large-conductance, Ca2+-activated K+ channels (12, 55).

The effects of Mg2+ on the I-V relation of the IP3R, while being very different from those observed in various K+ channels that are impermeable to Mg2+, are reminiscent of the effects of divalent cations on the monovalent cation conductance of the ryanodine receptor (46, 47). The ryanodine receptor has a high Mg2+ permeability (48), similar to the IP3R. It has been proposed that divalent cations experience low energy barriers to entry into the pore of the ryanodine receptor, which enables them to move into the conduction pathway with relatively high permeabilities compared with monovalent cations (46, 47). However, because the divalent ions bind tightly in a potential well inside the channel pore, they permeate through the channel pore more slowly than monovalent ions. Because the ryanodine receptor is a single-ion occupancy channel (48), it is effectively nonconducting when occupied by a divalent cation (46). Thus the slow passage of divalent cations through the channel reduces the monovalent cation conductance as well. High |Vapp| alleviates the block by increasing the rate at which divalent ions move through the channel, which generates nonlinearity in the I-V relation. The IP3R is also a single-ion occupancy channel (3). The conversion of the linear monovalent cation I-V relation in the absence of divalent cations to the rectified I-V relations in their presence (Mg2+ and Ba2+) observed in the IP3R (present data) and the ryanodine receptor (46, 47) strongly suggests that this model for ionic conduction is applicable to the IP3R. These results indicate that the IP3R has a pore that possesses lower energy barriers for divalent (Ca2+ and Mg2+) than for monovalent (K+) cation entry and, therefore, higher divalent cation permeabilities and relatively stronger divalent cation binding sites, which cause divalent ion blocking of the channel.

The symmetrical nonlinear I-V relation of the IP3R in the presence of symmetrical divalent ions suggests that the energy profile experienced by divalent ions in the channel pore is symmetrical about a central axis (46). Rectification of the I-V relation occurs at positive and negative Vapp with Mg2+ present on only one side of the channel. Thus the polarity of Vapp does not affect significantly the movement of Mg2+ from either side of the channel into those binding sites that cause channel block and consequent rectification. This implies that a divalent ion binding site is located a short electrical distance from the mouth of the pore on each side of the channel. This feature is again reminiscent of the ryanodine receptor conduction pathway, which has been modeled with divalent ion binding sites 10 and 90% of the way across the potential drop through the channel besides a central binding site (46).

Because of the high concentrations of K+ and free Mg2+ in the cytoplasm and the relatively high permeability of the IP3R to Mg2+ and K+ [PCa/PMg approx  3, PCa/PK approx  8 (22)], an IP3R channel in situ must be blocked to Ca2+ flow for a significant portion of its open time because of the occupation of the channel by Mg2+ and K+ that bind in the permeation pathway. This suggests that the magnitude of the Ca2+ current passing through single open IP3R channels under physiological ionic conditions will be substantially lower than that measured in the absence of Mg2+ and K+.

Variability of IP3R conductance in the absence of divalent ions. The conductance of the IP3R exhibited considerable variability in the absence of Mg2+. The stable IP3R conductance varied from patch to patch over a wide range. It also changed over the course of a recording and sometimes fluctuated during single-channel openings. It is unlikely that such conductance variability was caused by variations in experimental conditions, since the same experimental solutions and, in many cases, the same nuclei were used in those experiments that recorded different IP3R channel conductances. In most of the recordings during which the channel conductance changed or fluctuated, the baseline closed-channel current level remained constant, indicating that seal instability was not the cause of the observed conductance modulations. Furthermore, such conductance modulations were not observed in similar experimental conditions with use of symmetrical 2.5 mM Mg2+ solution, even in current records during which the gigaohm seals were not fully stable and the baseline current varied considerably (e.g., Fig. 4A in Ref. 23). The slow kinetics of the observed conductance changes and fluctuations, therefore, suggest that in the absence of Mg2+ the IP3R can adopt a large number of kinetically stable configurations with slight differences in channel structure and conductance.

In individual patches containing multiple channels in 0 mM Mg2+ solutions, the conductances of the channels tended to be quite similar to one another. Even in those recordings in which the IP3R channel conductance changed, most of the channels changed their conductances in concert. In those recordings in which the individual channels started with dissimilar conductances, changes in channel conductances tended to make them similar by the end of the recording. In a majority of recordings that exhibited changes in conductances, the conductances increased during the recordings, until all were within the most frequently observed range of stable conductances (280-380 pS). It is possible that channel configurations corresponding to conductances in that range are energetically more favorable than others, so that the IP3R tended to adopt those configurations. Channels with conductances that remained outside the favorable range throughout a recording might have also achieved such configurations if the kinetics of this process had not been terminated by channel inactivation or seal rupture. However, this cannot account for the observation that there was a strong tendency for channels in the same membrane patch to adopt similar conductance values (±20 pS, much smaller than the range of stable conductances), especially in recordings in which the conductance was outside the stable range or did not fluctuate during the recordings.

One possible explanation is that in the absence of divalent cations to stabilize their configurations the IP3R channels observed in multichannel patches adopted configurations with similar conductances in response to similar local physicochemical variables. However, IP3R channel conductance is not sensitive to membrane stretch (unpublished observations). It is also unlikely that variable amounts of divalent cation contamination among patches can account for the variability, since the amount of divalent ions required to account for the level of variability observed would have to be so considerable (hundreds of micromoles) that it is difficult to conceive of a mechanism to generate such variable contamination. Nevertheless, the channels might be sensitive to local microscopic environmental variables, e.g., lipid composition in the local membrane patches containing the channels, despite the stable global experimental conditions maintained.

Another possibility is that IP3R channels interact in such a manner as to enable channels within a membrane patch to achieve similar conductances. Two previous observations suggest that such interactions could possibly occur. First, we previously demonstrated that the Xenopus IP3R localizes within clusters of functional channels (23), suggesting that mechanisms exist to couple channels to discrete locations and, therefore, possibly to each other. These mechanisms could involve direct interactions similar to those that organize monomers into tetrameric units (35) or indirect interactions mediated by other molecules, e.g., ankyrin (6) or actin (14), with which the IP3R is believed to associate. Second, we previously determined that IP3R channel clustering affected the kinetics of individual channel inactivation (23). Channel inactivation for channels in multichannel patches was considerably slowed compared with inactivation of channels in single-channel patches. Channel interactions may, besides altering their gating properties, enable them to coordinate their conductance levels.

Stabilization of the single-channel conductance of the IP3R by divalent cations. In addition to channel block by divalent cation permeation, interactions between Mg2+ and the IP3R are also involved in stabilizing the IP3R conductance. As [Mg2+] was increased, one configuration of the channel was progressively stabilized relative to the others, thus reducing the range of channel conductances observed. Stabilization of the channel conductance was achieved by Mg2+ from either side of the channel, indicating that 1) Mg2+-sensitive sites responsible for channel stabilization are present on both sides of the channel or 2) the Mg2+-sensitive site(s) for channel stabilization is located in or very close to the ion conduction pathway of the channel so that Mg2+ permeating through the channel from either side can access and bind to it. Modulation of the IP3R conductance has not been previously observed. However, millimolar [Mg2+] was present in previous patch-clamp studies of type 1 IP3R (22, 23, 40), and lipid bilayer reconstitution studies were performed with tens of millimoles of divalent cations on one side of the channel (3, 4). Under these experimental conditions a stable single-channel conductance, as observed, would be expected. The data from the reconstituted IP3R also suggest that other divalent cations in addition to Mg2+ must also stabilize the channel configuration. This was confirmed in the present study with Ba2+ as a substitute for Mg2+. Thus the divalent ion binding site(s) responsible for channel conductance stabilization has low specificity and low affinity (dissociation constant in millimolar range). The type 1 IP3R contains several distinct Ca2+-binding domains (36). Whereas some of these domains are likely responsible for Ca2+-dependent gating of the IP3R, others may play a structural role related to stabilization of channel conformations that contribute to conductance properties, and they may be responsible for mediating the effects of divalent cations observed in the present studies. No modulation of channel conductance was reported in a recent study of IP3R reconstituted into lipid bilayers with 200 nM free Ca2+ on the cytoplasmic side as the only divalent ions present (33). This might be due to the different isoform of IP3R or different membrane environment (bilayer vs. nuclear envelope) used in these studies.

All the effects of Mg2+ on the IP3R channel observed in our experiments were concentration dependent. It is noteworthy that they mostly occurred between 0 and 2.5 mM (blocking constant by Mg2+ ~0.6 mM), because this largely coincides with the physiological range of intracellular [Mg2+] in cells (~ 0.3-1.2 mM) (15, 28, 29, 37, 38). Importantly, acute as well as long-term changes of intracellular [Mg2+] have been observed in response to various stimuli and physiological conditions (37, 38). Thus, besides affecting the binding of IP3 to IP3R (49, 50), the effects of Mg2+ on the single-channel properties of the IP3R may therefore contribute to the control of temporal and spatial patterns of Ca2+ release from intracellular stores.

    ACKNOWLEDGEMENTS

We thank Sean McBride for experimental assistance and Susanne Pedersen for comments on the manuscript.

    FOOTNOTES

This study was supported by grants from the Medical Research Council of Canada, the National Institutes of Health, and the Cystic Fibrosis Foundation.

Address for reprint requests: J. K. Foskett, Dept. of Physiology, University of Pennsylvania, Stellar-Chance Laboratories, Rm. 313B, 422 Curie Bl., Philadelphia, PA 19104.

Received 8 December 1997; accepted in final form 6 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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