Facilitatory effect of Ins(1,4,5)P₃ on store-operated Ca²⁺-permeable cation channels in rabbit portal vein myocytes

Abstract

In rabbit portal vein smooth muscle cells, store-operated Ca²⁺-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca²⁺ stores but also may be stimulated in a store-independent manner by noradrenaline acting on α-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 μm Ins(1,4,5)P₃ in the patch pipette solution increased whole-cell currents evoked by the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at −80 mV. In the cell-attached configuration the cell-permeable Ca²⁺ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 μm Ins(1,4,5)P₃ increased open channel probability (NP_o) by approximately 3-fold. Ins(1,4,5)P₃ also produced a similar increase in NP_o of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P₃ on increasing PDBu-evoked SOC activity was about 0.4 μm. The facilitatory effect of Ins(1,4,5)P₃ was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P₃ was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P₃ and Ins(1,4)P₂, a metabolite of Ins(1,4,5)P₃, but was not inhibited by the classical Ins(1,4,5)P₃ receptor antagonist heparin. Finally Ins(1,4,5)P₃ also increased NP_o of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P₃ facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.

Store-operated channels (SOCs) are Ca²⁺-permeable cation channels located in the plasma membrane which are activated in response to depletion of intracellular Ca²⁺ stores, namely the sarcoplasmic (SR) or endoplasmic reticulum. It is considered that influx of Ca²⁺ ions through SOCs affect many important cell processes such as secretion, gene regulation and apoptosis (Parekh & Penner, 1997). In smooth muscle there is considerable evidence that SOCs have an important function in producing muscle contraction and cell proliferation (see reviews by McFadzean & Gibson, 2002; Albert & Large, 2003a). In vascular smooth muscle SOCs have been recorded at the single channel level by Trepakova et al. (2001) and Albert & Large (2002a) and comparison of the biophysical properties of SOCs described in these two studies indicates that there may be more than one class of SOC in smooth muscle (Albert & Large, 2002a, 2003a).

An intriguing question concerns the activation mechanism of SOCs and several hypotheses have been put forward (see Parekh & Penner, 1997). One proposal is the conformational coupling model in which inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) receptors on the intracellular Ca²⁺ store directly couple to SOCs to initiate channel opening. With regard to this hypothesis it has been shown that Ins(1,4,5)P₃ receptors interact directly with a member of the canonical transient receptor potential proteins, TRPC3 (Kiselyov et al. 1998, 1999; Ma et al. 2000) and TRPC molecules are potential candidates for SOCs (e.g. see Clapham, 2003). A second scheme is that a diffusible factor (calcium influx factor, CIF) is released from internal Ca²⁺ stores in response to Ca²⁺ depletion to stimulate SOCs. Recently it has been suggested that CIF displaces calmodulin from Ca²⁺-independent phospholipase A₂(iPLA₂) which leads to activation of iPLA₂ and production of lysophospholipids causing SOC opening in cultured aortic myocytes (Smani et al. 2004).

In freshly dispersed rabbit portal vein smooth muscle cells we have demonstrated that SOCs have multi-modal gating mechanisms. Accordingly SOCs are activated by agents such as cyclopiazonic acid (CPA; an inhibitor of the SR Ca²⁺-ATPase) and BAPTA-AM which deplete intracellular Ca²⁺ stores. In addition SOCs were stimulated by noradrenaline acting on α-adrenoceptors and also by diacylglycerol (DAG). SOCs were activated by noradrenaline and DAG in isolated outside-out patches in which there appeared to be no functional internal Ca²⁺ stores, i.e. this is store-independent activation of SOCs (Albert & Large, 2002b). A common feature of these two gating pathways is the involvement of protein kinase C (PKC) since PKC inhibitors blocked SOC activation by CPA, BAPTA-AM and the store-independent pathway stimulated by noradrenaline. Therefore it was proposed that a PKC-mediated phosphorylation process has a central role in SOC activation in freshly dispersed rabbit portal vein myocytes (Albert & Large, 2002b). In addition we have recently demonstrated that a phosphorylation process is also involved in an inhibitory pathway in which β-adrenoceptors inhibit SOC activity via a cAMP-dependent protein kinase (Liu et al. 2005).

The present study was initiated to investigate whether Ins(1,4,5)P₃ modulates SOC activity in rabbit portal vein smooth muscle cells in order to ascertain whether there was evidence for the conformational-coupling model in this preparation. The present data show that Ins(1,4,5)P₃ does not activate SOCs but greatly facilitates channel opening produced by Ca²⁺ store depletion. In addition Ins(1,4,5)P₃ potentiates SOC activity evoked by PKC activators and a PKC catalytic subunit in isolated inside-out patches, the store-independent pathway, and does not appear to involve the classical Ins(1,4,5)P₃ receptor on the SR.

Methods

Cell isolation

New Zealand White rabbits (2–3 kg) were killed by an i.v. injection of sodium pentobarbitone (120 mg kg⁻¹) in accordance with the UK Animals (Scientific Procedures Act) 1986 and the portal vein was removed, dissected free of connective tissue and fat before being cut into strips and enzymatically dispersed as previously described (Liu et al. 2005).

Electrophysiology

Whole-cell and single cation channel currents were recorded with a HEKA EPC-8 patch clamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany) at room temperature using whole-cell recording, cell-attached and inside-out patch configurations of the patch clamp technique (Hamill et al. 1981). Patch pipettes were manufactured from borosilicate glass and were fire polished, and we used pipettes with resistances of about 6 MΩ for whole-cell and between 10 and 15 MΩ for cell-attached and inside-out patch recording when filled with patch pipette solution. To reduce ‘line’ noise the recording chamber (vol. ca 150–200 μl) was perfused using two 10 ml syringes, one filled with external solution and the other used to drain the chamber, in a ‘push and pull’ technique. The external solution could be exchanged twice within 30 s. Whole-cell currents were evoked by applying voltage ramps from −150 mV to +100 mV (0.5 V s⁻¹) every 20 s from a holding potential of 0 mV and filtered at 1 kHz (−3 db, low pass 4-pole Bessel filter, HEKA EPC-8 patch clamp amplifier) and sampled at 5 kHz (Digidata 1322A and pCLAMP 9.0 Software, Axon instruments, Inc., Union City, CA, USA). Control current–voltage relationships (I–V) were measured about 1 min after whole-cell configuration was obtained and then after approximately 5 min CPA was applied. Experiments were only continued if the control whole-cell currents were stable. CPA-evoked I–V relationships were measured at the peak of the response. When recording single channel currents the holding potential was routinely set at −80 mV and to evaluate I–V characteristics of single channel currents the membrane potential was manually changed between −140 mV and 0 mV.

Single channel currents were initially recorded onto digital audiotape (DAT) using a Bio-Logic DRA-200 digital tape-recorder (Bio-Logic Science Instruments, Claix, France) at a bandwidth of 5 kHz (−3 db, low pass 4-pole Bessel filter, HEKA EPC-8 patch clamp amplifier) and a sample rate of 48 kHz. For off-line analysis single cation channel records were filtered at 100 Hz (−3 db, low pass 8-pole Bessel filter, Frequency Devices, model LP02, Haverhill, MA, WA) and acquired using a Digidata 1322A and pCLAMP 9.0 software at a sampling rate of 1 kHz. Data were captured with a Pentium III personal computer.

Single channel current amplitudes were calculated from idealized traces of at least 10 s in duration using the 50% threshold method with events lasting for > 6.664 ms (2 × rise time for a 100 Hz (−3 db) low pass filter) being excluded from analysis (Colquhoun, 1987). Figure preparation was carried out using Origin software (version 6.0; OriginLab Corp., Northampton, MA, USA) where inward channel currents were shown as downward deflections. The number of channels in a patch was unknown and therefore open probability (NP_o) was calculated using the equation: NP_o = total open time of all channel levels in the patch/sample duration.

Solutions and drugs

The bathing solution used in whole-cell recording experiments was K⁺ free and contained (mm): NaCl (126), CaCl₂ (1.5), Hepes (10), glucose (11), 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) (0.1), niflumic acid (0.1) and nicardipine (0.005), pH adjusted to 7.2 with NaOH. The pipette solution used for whole-cell recording was also K⁺ free and contained (mm): CsCl (18), caesium aspartate (108), MgCl (1.2), Hepes (10), glucose (11), BAPTA (10), CaCl₂ (4.8) (free internal Ca²⁺ concentration approximately 100 nm as calculated using EQCAL software), Na₂ATP (1), NaGTP (0.2), pH 7.2 with Tris. In cell-attached patch experiments the membrane potential was set to approximately 0 mV by perfusing cells in a KCl external solution containing (mm): KCl (126), CaCl₂ (1.5), Hepes (10) and glucose (11), pH to 7.2 with 10 m NaOH. Nicardipine at 5 μm was also included to prevent smooth muscle cell contraction by blocking Ca²⁺ entry through voltage-dependent Ca²⁺ channels. The composition of the bathing solution used in inside-out experiments (intracellular solution) was the same as the pipette solution used for whole-cell recording except that 1 mm BAPTA and 0.48 mm CaCl₂ were included (free internal Ca²⁺ concentration approximately 100 nm). The pipette solution used for both cell-attached and inside-out recording (extracellular solution) was K⁺ free and contained (mm): NaCl (126), CaCl₂ (1.5), Hepes (10), glucose (11), TEA (10), 4-AP (5), iberiotoxin (0.0002), DIDS (0.1), niflumic acid (0.1) and nicardipine (0.005), pH adjusted to 7.2 with NaOH. Under these conditions voltage-gated Ca²⁺ currents, K⁺ currents, swell-activated Cl⁻ currents and Ca²⁺-activated conductances are abolished and non-selective cation currents can be recorded in isolation. All drugs including the PKC catalytic subunit were purchased from Sigma (UK). The PKC catalytic subunit was prepared by tryptic digestion of PKC to harvest a catalytic subunit which does not require Ca²⁺ for activation and is not specific for any PKC isoforms. The values are the mean of n cells ± s.e.m. Statistical analysis was carried out using Student's t test with the level of significance set at P < 0.05.

Results

Effect of Ins(1,4,5)P₃ on whole-cell currents evoked by cyclopiazonic acid (CPA) in rabbit portal vein myocytes

In our initial experiments we investigated whether whole-cell SOC currents evoked by CPA, which depletes internal Ca²⁺ stores by inhibiting the SR Ca²⁺-ATPase, were regulated by Ins(1,4,5)P₃.

Figure 1Aa shows in a control experiment that bath application of 20 μm CPA induced an increase in whole-cell conductance which was manifest as increases in membrane currents evoked by voltage ramps from −150 mV to +100 mV from a holding potential of 0 mV. Figure 1Ab shows the effect of 20 μm CPA on the current at −80 mV and illustrates that CPA induced a mean net increase of −33 ± 7 pA (n = 7). Figure 1Ba and b shows that inclusion of 1 μm Ins(1,4,5)P₃ in the patch pipette solution significantly increased CPA-evoked whole-cell currents (cf. Fig. 1Aa and b). Figure 1Bb illustrates that in the presence of 1 μm Ins(1,4,5)P₃ the mean peak current at −80 mV induced by CPA was −82 ± 11 pA (n = 7) which was significantly greater then in the absence of Ins(1,4,5)P₃ (P < 0.01). Figure 1C shows mean normalized current–voltage (I–V) relationships of CPA-evoked whole-cell currents recorded in the presence and absence of 1 μm Ins(1,4,5)P₃ in the patch pipette solution which had similar properties with reversal potentials (E_r) of about +25 mV.

Figure 1. Effect of Ins(1,4,5)P₃ on CPA-evoked whole-cell currents.

Aa, control cell showing that bath application of 20 μm CPA evoked an increase in whole-cell conductance. b, mean effect of 20 μm CPA on whole-cell current at −80 mV. Ba and b, effect of including 1 μm Ins(1,4,5)P₃ in the patch pipette solution on CPA-evoked whole-cell currents. In Aa and Ba the holding potential was 0 mV and whole-cell currents were evoked by voltage ramps from −150 mV to +100 mV and downward deflections represent inward current. Note the larger mean current at −80 mV prior to application of CPA in Bb compared to Ab and also that Ab has a different current scale than Bb. * denotes when whole-cell configuration was obtained. C, mean normalized current–voltage relationships of CPA-evoked whole-cell currents in the presence (n = 7) and absence (n = 7) of 1 μm Ins(1,4,5)P₃ in the patch pipette solution. Whole-cell currents were normalized to the mean amplitude of CPA-evoked whole-cell currents at −80 mV recorded in the absence of Ins(1,4,5)P₃ in the patch pipette solution.

An interesting observation was that inclusion of 1 μm Ins(1,4,5)P₃ in the patch pipette solution increased the resting whole-cell conductance prior to application of CPA (cf. Fig. 1Ab and Bb). Comparison of currents in the absence and presence of 1 μm Ins(1,4,5)P₃ in the patch pipette solution, respectively, showed that mean holding currents at −80 mV were significantly increased from −38 ± 8 pA (n = 7) to −102 ± 21 pA (n = 7, P < 0.001).

These data clearly show that Ins(1,4,5)P₃ facilitates CPA-evoked whole-cell currents in rabbit portal vein myocytes. Moreover these data indicate that Ins(1,4,5)P₃ enhances a resting holding current in portal vein which may be due to Ins(1,4,5)P₃ causing release of Ca²⁺ ions from the SR to activate SOCs or by Ins(1,4,5)P₃ enhancing a constitutively active cation current present in portal vein myocytes.

Ins(1,4,5)P₃ potentiates store-operated single channel activity evoked by depleting internal Ca²⁺ stores in rabbit portal vein myocytes

In order to verify an effect on SOCs we studied the action of Ins(1,4,5)P₃ on single channel currents which we have previously described in both cell-attached and inside-out patches (Albert & Large, 2002a; Liu et al. 2005). Therefore we investigated whether store-operated channel currents (SOCs) activated by BAPTA-AM, a cell-permeant Ca²⁺ chelating agent which passively depletes internal Ca²⁺ stores without inducing a rise in internal Ca²⁺ concentration ([Ca²⁺]_i), were also regulated by Ins(1,4,5)P₃. Figure 2 illustrates a typical experiment examining the effect of Ins(1,4,5)P₃ on SOCs in rabbit portal vein myocytes.

Figure 2. Effect of Ins(1,4,5)P₃ on store-operated channel currents (SOCs) evoked by BAPTA-AM.

A, bath application of 50 μm BAPTA-AM induced channel currents in cell-attached patch held at −80 mV. At time point denoted by * the patch was excised into an isolated inside-out patch configuration and the cell-attached bathing solution was replaced by an inside-out bathing solution (see Methods). Bath application of 1 μm Ins(1,4,5)P₃ potentiated BAPTA-AM-induced channel activity. a–c show channel currents on a faster time scale from positions on the traces shown with dotted lines. B, amplitude histograms of channel currents induced in the presence of BAPTA-AM (cell-attached, a), BAPTA-AM (inside-out, b) and BAPTA-AM in the presence of Ins(1,4,5)P₃ (inside-out, c). C, mean data of NP_o in experiments illustrated in A. n = 9, **P < 0.01.

Figure 2A shows that bath application of 50 μm BAPTA-AM induced SOC activity in a cell-attached patch at a holding potential of −80 mV. Figure 2Aa shows BAPTA-AM-evoked channel currents on a faster time scale and Fig. 2Ba illustrates that the amplitude histogram of these channel currents could be fitted with four Gaussian curves with peak values of 0, −0.22 pA, −0.42 pA and −0.61 pA representing a closed state, a unitary channel current amplitude of −0.22 pA and at least three channels in the patch. In nine cell-attached patches BAPTA-AM-evoked SOCs had a mean unitary amplitude of −0.18 ± 0.02 pA and a mean open probability (NP_o) of 0.401 ± 0.053 at −80 mV (Fig. 2C). The properties of BAPTA-AM-induced channel currents are similar to properties of SOCs previously described in rabbit portal vein myocytes (Albert & Large, 2002a; Liu et al. 2005) suggesting that they are the same channels.

Figure 2A shows that after BAPTA-AM-evoked channel activity reached a maximum and stable level the cell-attached patch was then excised (*) to form an isolated inside-out patch and the bathing solution for cell-attached patch recording was replaced with an inside-out bathing solution (see Methods). On excision from cell-attached to inside-out patch configuration BAPTA-AM-evoked channel activity remained at a similar level for at least 5 min (Fig. 2A and C) and also had a similar amplitude histogram (Fig. 2Bb) indicating that patch excision did not alter channel activity or channel properties. Subsequently bath application of 1 μm Ins(1,4,5)P₃ to the cytoplasmic surface of an inside-out patch induced marked potentiation of SOC activity by about 3-fold (Fig. 2A and C). Moreover the amplitude histogram of BAPTA-AM-evoked channel currents in the presence of Ins(1,4,5)P₃ (Fig. 2Bc) had similar peak values as those histograms in the absence of Ins(1,4,5)P₃ (Fig. 2Ba and b) suggesting that Ins(1,4,5)P₃ increased the activity of the same cation channel currents.

These data indicate that Ins(1,4,5)P₃ has a pronounced potentiating effect on SOCs evoked by depletion of internal Ca²⁺ stores in rabbit portal vein myocytes and since this effect of Ins(1,4,5)P₃ is observed in inside-out patches the binding site for Ins(1,4,5)P₃ is likely to be at, or close to, the channel protein.

Ins(1,4,5)P₃ potentiates SOC activity induced by the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu)

We have previously shown that PDBu activates SOCs through a store-independent mechanism (Albert & Large, 2002b; Liu et al. 2005) indicating that a protein phosphorylation mechanism involving PKC plays a central role in activating SOCs in rabbit portal vein myocytes. We therefore investigated whether Ins(1,4,5)P₃ also had a potentiating effect on PDBu-evoked SOCs in inside-out patches.

In initial experiments we bath applied 1 μm Ins(1,4,5)P₃ alone to inside-out patches and in seven patches Ins(1,4,5)P₃ did not evoke any channel activity suggesting that Ins(1,4,5)P₃ on its own did not activate SOCs (e.g. see Fig. 4B).

Figure 4. Effect of Ins(1,4,5)P₃ on the latency and activation rate of PDBu-evoked channel activity in inside-out patches.

A, typical activation time course of channel currents evoked by bath application of 1 μm PDBu in an inside-out patch. B, activation of PDBu-evoked channel currents after pretreatment with 1 μm Ins(1,4,5)P₃ for about 2 min. Note that in the presence of Ins(1,4,5)P₃ PDBu activated channel currents in less than 15 s compared to over 2 min in the absence of Ins(1,4,5)P₃ shown in B. C, mean data of the above experiments. n = 6–30 patches, **P < 0.01.

Figure 3A shows that bath application of 1 μm PDBu induced channel activity in inside-out patches held at −80 mV after about 2–3 min. In 30 inside-out patches PDBu-evoked channel activity had a mean NP_o value of 0.408 ± 0.089 and PDBu-evoked channel currents had a mean unitary amplitude of −0.18 ± 0.03 pA at −80 mV. These data are similar to channel currents induced by PDBu in cell-attached, outside-out and inside-out patches previously described in rabbit portal vein myocytes (Albert & Large, 2002b; Liu et al. 2005).

Figure 3. Effect of Ins(1,4,5)P₃ on PDBu-evoked channel currents in inside-out patches.

A, bath application of 1 μm Ins(1,4,5)P₃ enhanced channel activity induced by 1 μm PDBu in an inside-out patch. Channel currents are illustrated on a faster time scale from positions on the trace defined with dotted lines. B, mean pooled I–V relationship of PDBu-evoked channel currents in the presence of Ins(1,4,5)P₃ showing these channels had a unitary conductance of 2.2 pS and an extrapolated E_r of +24 mV (n = 4 for each point). C, concentration–response curve of Ins(1,4,5)P₃ concentration against PDBu-evoked channel activity in inside-out patches. Each point represents at least 6 patches.

Figure 3A shows that bath application of 1 μm Ins(1,4,5)P₃ potentiated PDBu-evoked channel activity in inside-out patches held at −80 mV. In eight inside-out patches, bath application of 1 μm Ins(1,4,5)P₃ significantly increased mean NP_o values of PDBu-induced channel activity from 0.441 ± 0.184 to 1.267 ± 0.428 (P < 0.05) representing an increase of about 3-fold. The Ins(1,4,5)P₃-induced potentiation of PDBu-evoked channel activity was reversible (Fig. 3A) and did not desensitize with channel activity in the presence of Ins(1,4,5)P₃ having a mean NP_o of 1.094 ± 0.375 (P > 0.05) 3 min after the peak response.

Figure 3B shows the mean pooled I–V relationship of PDBu-induced channel currents in the presence of 1 μm Ins(1,4,5)P₃ which had an unitary conductance of 2.2 pS and an extrapolated E_r of +24 mV, which is similar to the values for PDBu-evoked channel currents recorded in the absence of Ins(1,4,5)P₃ previously described (Albert & Large, 2002b; Liu et al. 2005) suggesting that Ins(1,4,5)P₃ enhanced the activity of the same cation channels.

Figure 3C shows a concentration–response curve of Ins(1,4,5)P₃ concentration against PDBu-induced channel activity in inside-out patches held at −80 mV and illustrates that the effective concentration of Ins(1,4,5)P₃ producing 50% of maximal potentiation of PDBu-evoked channel activity was about 0.4 μm (EC₅₀).

Ins(1,4,5)P₃ increases activation rate of PDBu-induced channel currents

The above results show that Ins(1,4,5)P₃ increases the probability of opening of SOCs evoked by store depletion and by phorbol esters and next we investigated whether the rate of activation of SOC was also modulated by Ins(1,4,5)P₃.

Figure 4 shows responses to bath application of 1 μm PDBu in inside-out patches held at −80 mV in the absence (Fig. 4A) and presence of 10 μm Ins(1,4,5)P₃ (Fig. 4B) in the bathing solution. Comparison of Fig. 4A and B shows that pretreatment with 10 μm Ins(1,4,5)P₃ reduces the onset latency (time from application of PDBu to onset of channel activity) from about 2 min to less than 15 s and reduced time to maximum activation (time from application of PDBu to peak channel activity) from about 3 min to about 30 s. Mean data for these experiments are shown in Fig. 4C.

These data show that in addition to potentiating the activity of PDBu-evoked channel currents, Ins(1,4,5)P₃ greatly increases the rate of activation of SOCs by phorbol esters.

Effect of heparin, 3-Fluoro-Ins(1,4,5)P₃ and Ins(1,4)P₂ on PDBu-evoked channel activity

The above data suggest that in portal vein myocytes Ins(1,4,5)P₃ enhances SOC activity evoked by store-dependent and -independent mechanisms and therefore our next series of experiments investigated potential mechanisms involved in these responses to Ins(1,4,5)P₃. It is well recognized that a major role of Ins(1,4,5)P₃ in vascular smooth muscle is to release Ca²⁺ from the SR by binding to heparin-sensitive binding sites on Ins(1,4,5)P₃ receptors present on the SR. Therefore we investigated the effect of heparin on Ins(1,4,5)P₃-induced potentiation of PDBu-evoked channel activity in inside-out patches.

Figure 5A and B shows that bath application of 1 mg l⁻¹ heparin had no effect on PDBu-induced channel activity and also did not affect Ins(1,4,5)P₃-induced potentiation of PDBu-evoked responses. Bath application of 1 mg ml⁻¹ heparin alone in unstimulated inside-out patches did not evoke SOC activity (n = 4). These data indicate that the action of Ins(1,4,5)P₃ on SOC activity is via a heparin-insensitive binding site that is unlikely to involve the classical Ins(1,4,5)P₃ receptor on the SR.

Figure 5. Effect of heparin on Ins(1,4,5)P₃-induced potentiation of PDBu-evoked channel activity in inside-out patches.

A and B illustrate, respectively, original trace and mean data showing that bath application of 1 mgl⁻¹ heparin had no effect on Ins(1,4,5)P₃-induced potentiation of PDBu-evoked channel activity. n = 6, *P < 0.05.

We next investigated whether Ins(1,4,5)P₃ itself or a metabolite product of Ins(1,4,5)P₃ facilitated SOC activity by studying the effect of a metabolically stable analogue of Ins(1,4,5)P₃, 3-fluoro-Ins(1,4,5)P₃, and a breakdown product of Ins(1,4,5)P₃, Ins(1,4)P₂, on PDBu-evoked channel activity in inside-out patches. Figure 6A and B shows that bath application of 10 μm 3-fluoro-Ins(1,4,5)P₃ markedly potentiated PDBu-evoked channel activity by about 3-fold. Moreover in nine inside-out patches bath application of 1 μm Ins(1,4)P₂ also significantly increased the mean peak NP_o value of PDBu-induced channel activity by about 2-fold (Fig. 6B).

Figure 6. Effect of 3-fluoro-Ins(1,4,5)P₃ and Ins(1,4)P₂, on PDBu-evoked channel activity in inside-out patches.

A shows that bath application of 1 μm 3-fluoro-Ins(1,4,5)P₃ enhanced channel activity induced by 1 μm PDBu. B, mean data showing that 3-fluoro-Ins(1,4,5)P₃ (n = 6) and 1 μm Ins(1,4)P₂ (n = 9) increased PDBu-evoked channel activity by about 3- and 2-fold, respectively (*P < 0.05).

Ins(1,4,5)P₃ potentiates channel activity evoked by a PKC catalytic subunit

Previously we have demonstrated that protein kinase C (PKC) has a central role in SOC activation in portal vein myocytes (Albert & Large, 2002b) and the next experiments investigated the effect of Ins(1,4,5)P₃ on channel activity induced by a catalytic subunit of PKC.

Figure 7A shows that bath application of 0.1 U ml⁻¹ PKC catalytic subunit evoked channel activity in an inside-out patch held at −80 mV and in six inside-out patches this activity had a mean NP_o value of 0.495 ± 0.223 and a mean activation latency of 88 ± 34 s. Figure 6Aa shows that the amplitude histogram of PKC catalytic subunit-evoked channel currents could be fitted by four Gaussian curves with peaks of 0, −0.21 pA, −0.41 pA and −0.62 pA representing a closed state, a single unitary channel current amplitude and at least three channels in the patch. In six inside-out patches PKC catalytic subunit-induced channel currents had a mean unitary amplitude of −0.19 ± 0.01 pA indicating that the PKC catalytic subunit activated the same SOCs as previously described (Albert & Large, 2002a; Liu et al. 2005).

Figure 7. Effect of Ins(1,4,5)P₃ on channel activity evoked by a PKC catalytic subunit in inside-out patches.

A, bath application of 0.1 U ml⁻¹ of a PKC catalytic subunit evoked channel activity that was increased by subsequent bath application of 1 μm Ins(1,4,5)P₃. Amplitude histograms of PKC catalytic subunit-evoked channel currents are shown in the absence (a) and presence (b) of 1 μm Ins(1,4,5)P₃. B, mean data of PKC catalytic subunit-induced channel activity in the absence and presence of Ins(1,4,5)P₃. n = 6, *p < 0.01.

Figure 7A and B illustrates that bath application of 1 μm Ins(1,4,5)P₃ significantly potentiated channel activity induced by PKC catalytic subunit by about 3-fold and the similar amplitude histogram of PKC catalytic subunit-evoked channel currents recorded in the presence of Ins(1,4,5)P₃ shown in Fig. 7Ab) indicates that Ins(1,4,5)P₃ enhanced the activity of the same channels opened by the PKC catalytic subunit.

Discussion

The present study shows that Ins(1,4,5)P₃ markedly potentiates SOC activity in freshly dispersed rabbit portal vein myocytes. The facilitatory effect of Ins(1,4,5)P₃ was manifest as an approximately 3-fold increase in the amplitude of CPA-evoked whole-cell current. Moreover Ins(1,4,5)P₃ produced a similar increase in probability of channel opening and was also manifest as a large reduction in the latency and time to maximum NP_o of activation by PDBu. In isolated inside-out patches the addition of Ins(1,4,5)P₃ on its own did not cause channel opening but required prior SOC stimulation by depletion of Ca²⁺ stores or by application of phorbol esters/PKC catalytic subunit. The effect of Ins(1,4,5)P₃ was mimicked by the stable analogue 3-fluoro-Ins(1,4,5)P₃ and the breakdown product Ins(1,4)P₂ suggesting that Ins(1,4,5)P₃ and its metabolites are capable of facilitating SOC activity. In addition the potentiating effect of Ins(1,4,5)P₃ was not mediated by the classical heparin-sensitive binding site. Therefore this represents a novel effect of Ins(1,4,5)P₃ on SOC activity which is likely to have important physiological consequences.

An interesting observation was that dialysis of the unstimulated cell with Ins(1,4,5)P₃ increased the resting membrane conductance. This may be explained by Ins(1,4,5)P₃ causing depletion of Ca²⁺ from the SR and subsequent activation of SOCs prior to addition of CPA. Alternatively this observation may indicate significant constitutive activity of either SOCs which has been reported previously (Albert & Large, 2002a) or spontaneous TRPC6-like channels (Albert & Large, 2001 and see later).

Mechanism of action of Ins(1,4,5)P₃ on SOC activity

The results suggest that the potentiating effect of Ins(1,4,5)P₃ on SOC activity may not be mediated by receptors on the SR. In most experiments the effects of Ins(1,4,5)P₃ were observed when the agent was applied to excised inside-out patches. With BAPTA-AM SOC activity was initiated in the cell-attached mode prior to detaching the membrane patch and adding Ins(1,4,5)P₃ while with PDBu and PKC catalytic subunit the inside-out patch was formed before stimulating channel activity. These results show that the activation mechanism and the facilitatory site for Ins(1,4,5)P₃ are preserved in the inside-out patch configuration. Previously we have demonstrated that CPA, which initiates SOC activity by acting on the SR, does not activate SOCs in outside-out patches in which noradrenaline does initiate channel opening (Albert & Large, 2002b). This shows that functional SR is unlikely to be present in outside-out patches which are considerably larger than inside-out patches. Also the lack of an inhibitory effect of heparin against the potentiating effect of Ins(1,4,5)P₃ argues against the involvement of the classical Ins(1,4,5)P₃ receptor in the effect described in this paper. Therefore although Ins(1,4,5)P₃ increases SOC activity the present results do not support a pivotal role for conformational coupling between the Ins(1,4,5)P₃ receptor and the channel underlying the response observed in the present work or the SOC gating mechanism in freshly dispersed rabbit portal vein myocytes.

This study also shows that a metabolite of Ins(1,4,5)P₃, Ins(1,4)P₂, also potentiated channel activity although with a lower potency than Ins(1,4,5)P₃, suggesting that the physiological metabolite of Ins(1,4,5)P₃ can also have a similar facilitatory effect on SOCs in this preparation.

The degree of potentiation by Ins(1,4,5)P₃ on SOC activity induced by PDBu and PKC catalytic subunit was quantitatively similar to that observed on BAPTA-AM-induced channel activity. Together with the observation that PKC inhibitors reduce SOC activity evoked by CPA, BAPTA-AM, PDBu and noradrenaline by a similar amount (Albert & Large, 2002b) the present results provide further support for the central role of PKC in stimulating SOC activity. Furthermore the data with a PKC catalytic subunit in inside-out patches suggest that the effects of Ins(1,4,5)P₃ occur close to, or at, the channel protein itself.

Comparison of effects of Ins(1,4,5)P₃ on SOC activity and TRPC6-like channels in rabbit portal vein myocytes

In rabbit portal vein smooth muscle cells there is a second Ca²⁺-permeable membrane conductance (I_cat) evoked by noradrenaline (Byrne & Large, 1988) and strong evidence has been provided to support the proposal that TRPC6 channel protein is an important component of noradrenaline-evoked I_cat in this preparation (Inoue et al. 2001). Previously we have shown that Ins(1,4,5)P₃ has similar effects on TRPC6-like channels in portal vein (Albert & Large, 2003b) to those described on SOCs in the present work. However it should be noted that the SOCs described in the present work are not the TRPC6-like I_cat for several reasons. First, I_cat is not activated by agents that deplete internal Ca²⁺ stores (Byrne & Large, 1988; Wang & Large, 1991). Secondly, I_cat is not activated by phorbol esters (Helliwell & Large, 1997) and finally the unitary conductance of TRPC6-like I_cat (about 23 pS, Albert & Large, 2001) is about 10-fold greater than that of SOCs described in this paper and previous work (Albert & Large, 2002a,b; Liu et al. 2005). Nevertheless the similarity between the effects of Ins(1,4,5)P₃ on SOCs and TRPC6-like channels suggest a possible molecular commonality and in regard to this point it is interesting that Ins(1,4,5)P₃ does not potentiate human or murine TRPC6 channel currents expressed in HEK293 cells (Estacion et al. 2004; Shi et al. 2004).

Physiological implications of the effect of Ins(1,4,5)P₃

In vascular smooth muscle many vasoconstrictor agents, e.g. noradrenaline, activate G-proteins with subsequent stimulation of phospholipase C to produce Ins(1,4,5)P₃ and diacylglycerol (DAG, Ohanian et al. 1998). Ins(1,4,5)P₃ releases Ca²⁺ from the SR to produce contraction and activate Ca²⁺-activated membrane conductances (Large, 2002). The subsequent activation of SOCs is brought about not only by depletion of internal Ca²⁺ stores but also by the production of DAG, which induces channel opening via PKC in a store-independent manner (Albert & Large, 2002b). The present study shows that Ins(1,4,5)P₃ also increases SOC activity and hence Ca²⁺ influx by a direct action on the channel irrespective of whether the channel is stimulated by DAG or store depletion. The equilibrium constant for this effect of Ins(1,4,5)P₃ was approximately 0.4 μm, which is within the estimated intracellular concentration of Ins(1,4,5)P₃ in stimulated (1–20 μm) and unstimulated cells (0.1–3.0 μm, Kaftan et al. 1997), and therefore this effect of Ins(1,4,5)P₃ is likely to be important physiologically.