©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Monovalent Cation Channel Activated by Inositol Trisphosphate in the Plasma Membrane of Rat Megakaryocytes (*)

Baggi Somasundaram (§) , Martyn P. Mahaut-Smith (¶)

From the (1)Physiological Laboratory, Downing Street, Cambridge, CB2 3EG, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The activation of a monovalent cation current was studied in rat megakaryocytes using patch clamp techniques combined with photometric measurements of intracellular concentrations of Ca ([Ca]) and Na. ADP evoked a release of [Ca] and transiently activated a monovalent cation-selective channel, which, at negative potentials and under physiological conditions, would be expected to carry an inward Na current. The single channel conductance, estimated by noise analysis from whole cell currents at -50 to -60 mV was 9 picosiemens. Thapsigargin-induced [Ca] increases failed to stimulate the monovalent cation current, suggesting that neither [Ca] nor the depletion of internal Ca stores were activators of this conductance. However, buffering of [Ca] changes with 1,2-bis-(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid showed that both activation and inactivation of the current were accelerated by a rise in [Ca]. The monovalent cation conductance was activated by internal perfusion with inositol 1,4,5-trisphosphate, both in the presence and in the absence of a rise in [Ca]. Internal perfusion with inositol 2,4,5-trisphosphate, the poorly metabolizable isomer of inositol trisphosphate, similarly activated the monovalent cation current, whereas 1,3,4,5-tetrakisphosphate neither activated a current nor modified the ADP-induced monovalent current. Heparin, added to the pipette, blocked activation of the channel by ADP. The intracellular concentration of Na, monitored by sodium-binding benzofuran isopthalate, increased by 10-20 mM in response to ADP under pseudophysiological conditions. We conclude the existence of a novel nonselective cation channel in the plasma membrane of rat megakaryocytes, which is activated by IP and can lead to increases in cytosolic Na after stimulation by ADP.


INTRODUCTION

Megakaryocytes are large cells located in the bone marrow that are responsible for producing blood platelets, yet little is known of the cellular mechanisms underlying their function. Uneyama and co-workers (1, 2) have shown that rat megakaryocytes possess a novel purinergic receptor, which recognizes the ionized forms of ATP and ADP. Stimulation of this receptor leads to sustained oscillations of intracellular Ca concentration ([Ca])()and activation of Ca-dependent K channels. We have recently found that ATP also activates Na and Ca-permeable channels in rat megakaryocytes via two distinct classes of purinergic receptor(3) . One receptor is activated by ATP but not noticeably by ADP and causes rapid, transient opening of a nonselective cation channel. A second purinoceptor is stimulated by ATP and ADP and activates both a monovalent cation-selective channel and a channel highly selective for Ca. Stimulation via this second receptor also causes a release of Ca from intracellular stores and is most likely the same receptor as that responsible for the generation of Ca oscillations in the experiments of Uneyama et al.(1) . The Ca-selective conductance is activated by depletion of internal Ca stores via an as yet undetermined messenger and is indistinguishable from the store-regulated Ca current found in other nonexcitable cell types(4, 5, 6) . The monovalent cation-selective current is also activated at the time of internal Ca release via IP or another inositol lipid metabolite(3) . This channel is particularly interesting because, at resting membrane potentials, the current will be mostly carried by Na, and changes in [Na] have been proposed to play a role in the spreading reaction in megakaryocytes(7) . The spreading reaction may represent the physiological mechanism whereby megakaryocytes invade the bone sinusoids to reach the blood vessels and release platelets(8) . In the present study we have used a combination of patch clamp and fluorescent indicators of [Ca] and [Na] to study the monovalent cation-selective current activated by ADP and inositol phosphates in the plasma membrane of rat megakaryocytes.


MATERIALS AND METHODS

Preparation

Adult male Wistar rats weighing 200-300 g were killed by cervical dislocation. Bone marrow from the femoral and tibial bones was removed by gentle lavage using a standard external solution containing 20 mg ml apyrase and 0.1% bovine serum albumin. After filtration through a fine cotton mesh, the suspension was spun and washed twice before storage in the same standard solution. Megakaryocytes were distinguished from other bone marrow cells by their distinctive size (30-60 µm) and multilobular nucleus. Recordings were made at room temperature (20-23 °C) within 3-24 h of isolation.

Solutions

The standard external solution contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl, 2 mM CaCl, 10 mM glucose, 10 mM HEPES (pH 7.4 adjusted with Tris). K-, Na-, and Ca-free external media were obtained by replacing these ions with Cs, NMDG, and Mg, respectively. For low Cl external solutions, all Cl, except for that added with the divalent cation salts, was replaced by gluconate. In conventional whole cell patch recordings, Cs replaced K in order to block K currents and contained 140 mM cesium gluconate, 5 mM NaCl, 2 mM MgCl, 10 mM HEPES, 0.2 mM NaGTP, 0.05 mM Kfura-2, (pH 7.4 adjusted with Tris). 0.1 mM (NH)SBFI replaced the Kfura-2 in experiments where [Na] and membrane current were measured simultaneously. For internal dialysis of inositol phosphates, the pipette tip was dipped in inositol phosphate-free pipette solution and then backfilled with pipette solution containing 10 µM inositol 1,4,5-trisphosphate (1,4,5-IP), 50 µM 2,4,5-IP, or 20 µM inositol 1,3,4,5-tetrakisphosphate (IP). Highly calcium-buffered pipette solution was obtained by replacing 40 mM cesium gluconate with 10 mM CsBAPTA. In nystatin-perforated patch recordings, the pipette contained 100 mM KCl, 40 mM KSO, 1 mM MgCl, 10 mM HEPES (pH 7.4 adjusted with Tris), 150 µg ml nystatin. In these perforated patch experiments, cells were loaded with SBFI by incubation with 10 µM SBFI-acetoxymethyl ester with 0.04% pluronic acid for 60 min at room temperature. Fura-2, SBFI, CsBAPTA and pluronic F-127 were from Molecular Probes, Inc. (Eugene, OR). 1,4,5-IP, 2,4,5-IP, and IP were a gift of Dr. R. F. Irvine (Biotechnology and Biological Sciences Research Council, Babraham Institute, Cambridge, UK). All other reagents were from Sigma. A pressure injector was used to administer agonists from a patch pipette placed 150 µm from the cell. The time delay for arrival of agonists at the cell was measured and subtracted.

Electrophysiology

Patch clamp experiments were performed in conventional whole cell (9) or nystatin-perforated patch (10) configurations by means of an Axopatch 200A patch clamp amplifier (Axon Instruments, Inc., Foster City, CA). Pipettes were pulled from borosilicate glass tubing (Clark Electromedical Instruments) and had filled resistances of 2-3 megaohms. Series resistances were in the range of 7-30 megaohms, and 40-60% series resistance compensation was employed. Membrane currents during voltage ramps (0.6 mV/ms) were filtered at 2 kHz and sampled at 100 µs using Axon Instruments hardware (Digidata 1200) and software (pClamp). Where stated, voltage ramps prior to agonist application were used to subtract linear leak currents from ADP-evoked currents. A holding potential of -40 mV was used in many experiments because this was close to the membrane potential measured in perforated patch experiments under pseudophysiological conditions (see Fig. 6). A less negative potential permitted longer recordings, thus -20 mV was used in experiments where large currents were detected; however, holding potentials in the range of -70 to 40 mV were used in some experiments, depending upon the conditions, to check for measurable currents. For noise analysis, currents were filtered at 0.5 kHz and sampled at 2.5 kHz. Currents were also acquired at a rate of 60 Hz (filtered at approximately 30 Hz) by the Cairn spectrophotometer (see below) for simultaneous display alongside the fura-2 and SBFI fluoresence and signal-indicating agonist injection. Liquid junction potentials were measured by reference to a 3 M KCl agar bridge, and membrane potentials were adjusted accordingly in conventional but not nystatin whole cell recordings.


Figure 6: IP- and ADP-evoked [Na] changes. A, whole cell current (middle trace) and [Na] (bottom trace) during internal perfusion of 10 µM Ins-1,4,5-P. The top trace shows the membrane potential. [Na] is indicated by the SBFI 340/380 nm excitation ratio on a linear scale. External solution was nominally Ca-free with 3 mM 4-aminopyridine saline. B, membrane potential (lower trace) and [Na] (upper trace) during a nystatin whole cell recording under current-clamp mode. SBFI 340/380 nm excitation ratio has been calibrated for [Na] as described under ``Materials and Methods.'' Membrane potential and [Na] axes are both linear over the range shown.



Fluorescence Recordings

Fura-2 and SBFI fluoresence measurements were made by single cell photometry using a Cairn spectrophotometer system (Cairn Research Ltd., Kent, UK) coupled to a Nikon Diaphot inverted microscope. Excitation light passed through a spinning filter wheel assembly containing four 340-nm and two 380-nm bandpass excitation filters. Emitted light (400-600 nm) was selected by two dichroic filters and further filtered by a 485-nm long pass filter. The combined output from all 340 and 380 nm excitation filters provided a 340/380 nm ratio for each revolution of the filter wheel. The signal was then averaged to obtain a ratio value every 67 ms. Background and cell autofluoresence was measured in the cell-attached recording mode and subtracted to give fura-2 or SBFI fluoresence. [Ca] was calculated according to Grynkiewicz et al.(11) using a dissociation constant for fura-2 of 250 nM(12) .

Under conditions where SBFI was introduced into the cell through the recording pipette, background-corrected 340/380 ratios were used to provide an indication of [Na] changes. In experiments where SBFI was loaded from its acetoxymethyl ester, [Na] was clamped at different levels by perfusing solutions of known extracellular Na concentrations in the presence of a mixture of sodium ionophores (5 µM each of gramicidin, nigericin, and monensin; Ref. 13). The extracellular solutions were made from appropriate mixtures of high Na and K solutions. The former consisted of 110 mM sodium gluconate, 30 mM NaCl, 2 mM CaCl, 1 mM MgCl, 10 mM Na-HEPES (pH 7.4). The high K solution was identical except for substitution of all Na for K. A plot of SBFI 340/380 nm intensity ratio versus [Na] was linear in the range of 0-40 mM. The experimental 340/380 ratio values fell within this linear range, and therefore [Na]values were directly obtained from the calibration curve.


RESULTS

ADP Evokes Calcium Release and Activates a Nonselective Current

At negative potentials and under conditions that blocked K currents (see ``Materials and Methods''), 5 µM ADP activated a transient inward current and a concurrent large increase in [Ca] (Fig. 1A). The initial [Ca] increase reached a peak of 0.5-1.5 µM within 1-2 s and then returned to basal levels (approximately 50-100 nM) in the continued presence of ADP or was followed by further smaller increases in [Ca] that sometimes fused to give a plateau, as shown in the cell of Fig. 1. In the absence of external calcium, an inward current and [Ca]increase were still activated by ADP(3) . This suggests that the rise in [Ca] is at least partly due to the release of internal stores and that the current is not selective for Ca. However, variability between cells did not allow us to quantify the extent to which Ca influx contributed to the response.


Figure 1: ADP-evoked currents and [Ca] changes. A, effect of 5 µM ADP on whole cell current at -40 mV (upper trace) and [Ca] (lower trace; linear axes) in the presence of 2 mM external Ca. The bath contained 140 mM NaCl, 5 mM CsCl saline, and the pipette contained 140 mM cesium gluconate saline with 0.05 mM fura-2. B, whole cell ramp current, plotted as a function of membrane potential before the addition of ADP (a) and during the ADP-evoked inward current (b). C, ramp current after digital subtraction of background current to display the I-V relationship of the ADP-dependent current (b-a).



To further examine the conductance changes in reponse to ADP, membrane currents were recorded during 0.6 mV/ms voltage ramps within the range of potentials -100 to 90 mV. A ramp applied prior to agonist application (Fig. 1B, trace a) was used to subtract background currents from ramp currents obtained during the ADP-evoked transient (Fig. 1B, trace b). Fig. 1C shows the difference current (b-a) representing the I-V relationship for the ADP-evoked conductance. In 140 mM Na external solution and 140 mM cesium gluconate internal solution, the I-V relationship reversed at about -5 mV, was reasonably linear over the voltage range -90 to 40 mV, and, in most cells, displayed a distinct increase in slope at more positive potentials. The I-V relationship obtained by ramps at different times during the ADP-evoked current differed only in amplitude and not in reversal potential, suggesting that the response was due to activation of a single ionic conductance.

Ionic Selectivity

The ionic selectivity of the ADP-evoked conductance was investigated by substitution of internal and external ions. In Fig. 2, ADP-evoked I-V relationships are shown for each of four ionic conditions, with the membrane currents and Ca responses at a single negative holding potential in the insets. These results were obtained from the first ADP-evoked response in four different cells and were confirmed in at least five cells for each condition. Replacement of the majority of the internal and external Cl had little effect on the current and Ca response at -40 mV or on the difference I-V relationship (Fig. 2A) compared with corresponding Cl-containing salines (see for example Fig. 1C). Therefore, the ADP-evoked conductance does not appear to be significantly permeant to anions. On the other hand, replacement of all internal and external monovalent cations with the impermeant cation NMDG abolished all current at -40 mV, in the presence of an ADP-evoked Ca response (Fig. 2B, inset). No significant current developed within the voltage range -80 to 80 mV, as shown by the overlapping ramp currents before and during the Ca response in Fig. 2B, indicating that the ADP-evoked current is carried by monovalent cations. 2 mM Ca and 1 mM Mg were present in the external media throughout, which indicates little or no permeability to divalent cations at these physiological concentrations. Increasing external Ca decreases the level of ADP(1) and abolishes the ADP response(3) ; therefore we were unable to increase the external Ca concentration to test for any underlying permeability to Ca. The absence of membrane current in the experiment of Fig. 2B was not due to direct block by NMDG or the requirement of permeant ion on both sides of the membrane, because current was activated by ADP unidirectionally when either the external or internal NMDG was replaced by Cs (Fig. 2, C and D). In the presence of symmetrical 140 mM Cs, the ADP-evoked I-V relationship was similar to that observed in Na/Cs salines and reversed at about -5 mV (not shown), indicating similar Na and Cs permeabilities.


Figure 2: Ionic selectivity of the ADP-evoked current. The I-V relationships of the ADP-evoked current were obtained by subtraction of background currents as described in Fig. 1. The major external and internal ions were, respectively: A, sodium gluconate and cesium gluconate; B, NMDG chloride and NMDG chloride; C, cesium gluconate and NMDG chloride; D, NMDG chloride and cesium gluconate. The external solution also contained 2 mM CaCl, 1 mM MgCl, 10 mM HEPES, 10 mM glucose, and the internal solution contained 2 mM MgCl, 0.2 mM GTP, 0.05 mM fura-2. The insets show 10-s ADP-evoked whole cell voltage clamp currents (upper trace) and [Ca] (lower trace); the vertical bars represent 50 pA of current (upper trace) and 0-0.5 mM [Ca] (lower trace), respectively, and the horizontal bar represents 10 s (upper trace). The holding potentials were -40 mV in A, B, and C and 40 mV in D.



Single Channel Activity

Clear single channel events could not be clearly resolved during the off-phase of the ADP-evoked whole cell current. This was due to the noise generated by the high capacitance of the megakaryocyte (20-100 picofarads), although it also indicates that the ADP-evoked events are of relatively short duration(14) . We therefore turned to noise analysis to obtain an estimate of single channel conductance. When the number of channels opening is small, the variance () of the current is linearly related to the mean current with a slope equal to the single channel current(15, 16) . For this analysis we used cells that displayed a small ADP-evoked current, as shown in Fig. 3A. This reduced response was most likely the result of receptor desensitization (e.g. by ADP and ATP from damaged cells during the cell preparation) rather than a low number of total channels. As expected, the variance of the mean of the whole cell current increased in response to ADP (Fig. 3A, lower record). A plot of variance against the mean current during the ADP response could be well fitted by a linear relationship with a slope of 0.49 pA. The holding potential was -60 mV, and the reversal potential under these conditions was -5 mV, giving a single channel conductance of approximately 9 picosiemens. Within the range of holding potentials -50 to -60 mV, the average conductance was 8.6 ± 0.4 picosiemens (n = 3). This analysis assumes the existence of a uniform single channel conductance and that all channels open independently. The estimate must be considered a lower estimate for the single channel conductance, and, in practice, direct measurements of channel conductance are higher(17) .


Figure 3: Noise analysis of the ADP-evoked Na current. A, whole cell current (lines) and variance of the mean current () recorded at -60 mV in response to a brief application of 2 µM ADP. Current was low-pass filtered at 100Hz for display purposes only. B, plot of variance of the mean current during the ADP-evoked current. Variance and mean current were calculated for three 204.8-ms periods every 2 s (current low-pass filtered at 2.5 kHz and sampled at 0.5kHz). The solid line is the result of a linear regression fit and has a slope of 0.49 pA.



Mechanism of Activation

The close association of the ADP-evoked current with the rise in [Ca]suggested that this current may be modulated by Ca or a Ca-dependent process. To test this, the current was activated by ADP when internal Ca levels were strongly buffered by 10 mM BAPTA in the pipette saline. In order to eliminate the store-dependent inward current that is amplified under such conditions(3) , Ca was omitted from the external saline. Fig. 4A compares the [Ca] changes and membrane currents activated by a 30-s application of 5 µM ADP in normal (Fig. 4Ai) and enhanced (Fig. 4Aii) calcium buffering in nominally Ca-free salines. With low buffering, the current was activated 0.6 ± 0.3 s (n = 10) after ADP application, peaked within 1-3 s and inactivated to 10% of peak current after 3.5 ± 2 s (n = 10). In most cells tested the current was inactivated well before [Ca] returned to basal levels (Fig. 4Ai). In the absence of any increase in [Ca], ADP could still activate an inward current, although its kinetics were very different (Fig. 4Aii). The current was activated more slowly, reaching a peak after 5-10 s, and was inactivated more slowly (time to 10% of peak current was 18.5 ± 6 s; n = 6) compared with the currents activated in the unbuffered cells. This implies that Ca or a Ca-dependent process, although not required for activation of the ADP-evoked current, accelerates the rate of both activation and inactivation. In order to test if this current could be activated by a rise in [Ca] alone, [Ca] was continuously elevated to micromolar levels using the endoplasmic Ca-ATPase inhibitor thapsigargin (Fig. 4B). This agent results in a permanent loss of Ca from IP-sensitive stores and store-dependent (capacitative) calcium entry(18, 19) . The absence of any current in the megakaryocyte after thapsigargin treatment in Ca-free saline suggests that the ADP-evoked current cannot be triggered by an increase in [Ca] alone; neither is this current activated as a result of depletion of internal Ca stores.


Figure 4: Kinetics and Ca -dependence of the ADP-evoked current. Simultaneous recordings of membrane current (top traces) and [Ca] (lower traces) during exposure to 5 µM ADP (Ai, Aii, and C) or 1 µM thapsigargin (B). [Ca] axes are linear. Application of ADP was identical for Ai and Aii. External saline was 140 mM NaCl and was nominally Ca-free. Internal salines were 140 mM cesium gluconate saline with 0.05 mM fura-2 (Ai, B, and C) or 80 mM cesium gluconate saline with 10 mM CsBAPTA, 0.05 mM fura-2 (Aii) (see ``Materials and Methods'' for full details of saline composition). Holding potential was -40 mV in A and C and -70 mV in B.



Repeated exposures to ADP could reactivate the monovalent cation current, provided there was an interval of 1-2 min between successive applications. Under conditions of high internal Ca buffering, thus removing Ca-dependent inhibition of the current, both activation and inactivation of the current became progressively slower with repeated ADP additions, as shown in the experiment of Fig. 4C. This suggested that dialysis of the cytoplasm removes factors responsible for activation and inactivation of the current. These factors may, for example, generate and metabolize the second messenger involved in channel activation.

Inositol Phosphate-activated Currents

Release of internal Ca in nonexcitable cells appears to ubiquitously involve an increase in cytoplasmic IP levels and IP-dependent Ca stores(20) . IP is therefore a candidate for the second messenger involved in the activation of the monovalent cation channel in the rat megakaryocyte. A previous study provided preliminary evidence for a role for IP, because dialysis with 1,4,5-IP activated a monovalent cation current with similar characteristics to that activated by ADP(3) . However, it was not shown whether IP was acting alone or in synergism with other second messengers. Fig. 5A compares the currents activated by dialysis of 1,4,5-IP with (5Ai) and without (5Aii) an increase in intracellular Ca levels. For these dialysis experiments, 3 mM 4-aminopyridine was added to the external medium to accelerate the blockade of K currents. As shown by the current-voltage relationships acquired at three timepoints during each experiment, a current similar to that observed with ADP was activated by 1,4,5-IP and did not require an increase in [Ca].


Figure 5: Effect of inositol phosphates and heparin on IP and ADP-evoked currents. A continuous recording of membrane current (upper traces) and [Ca] (lower traces) is shown during internal perfusion of 10 µM Ins-1,4,5-IP (A), 50 µM Ins-2,4,5-IP (B), 20 µM IP (C), and 10 mg/ml heparin (D). 5 µM ADP was applied externally in B, C, and D at the times indicated. In A, Bi, C, and D, I-V relationships obtained by 0.6-mV/ms voltage ramps are displayed at up to three time points (a, b, and c) indicated on the continuous current recording. In A and B, the continuous records of membrane current and [Ca] start immediately after the block of voltage-gated K currents, whereas in C and D, the first 90 s and 5 min of the whole cell recording, respectively, are not shown. Holding potential was -20 mV in A and Bi and -40 mV in Bii, C, and D; in Bi, the cell was depolarized to 0 mV for approximately 17 s between ramps at B and C. Current scale bars are 100 pA in A, B, and C and 50 pA in D. [Ca] axes are linear. The pipette saline in Aii and Bii contained 10 mM CsBAPTA to increase the cytosolic Ca buffering power. The external saline was 140 mM NaCl, 5 mM CsCl saline throughout which was nominally Ca-free in A and B but contained 2 mM CaCl in C and D. 3 mM external 4-aminopyridine was also present in A and B.



1,4,5-IP is rapidly metabolized to other inositol lipid products, including IP(20) . Its isomer, 2,4,5-IP is also active at IP receptors on the Ca stores, although at higher concentrations(21) , and is experimentally useful because it is a poor substrate for the 1,4,5-IP-kinase. Therefore 2,4,5-IP can be used to stimulate IP-dependent processes, whereas cytoplasmic levels of IP remain low. As shown in Fig. 5B (i, left panel), dialysis with 2,4,5-IP released internal Ca and activated the monovalent cation current in a manner indistinguishable from that seen with 1,4,5-IP. In the presence of high Ca buffering power, 2,4,5-IP evoked the inward current as expected and application of ADP failed to activate further current (Fig. 5Bii). These results suggest that IP on its own is sufficient to activate the monovalent cation current without a need for other inositol lipid products such as IP. Furthermore, following internal dialysis with 20 µM IP, no inward current was observed and subsequent exposure to ADP produced a normal response (Fig. 5C).

Block of the ADP and IP-activated Channel

1,4,5-IP-activated channels located on the membrane of internal Ca stores and those on the plasma membrane of olfactory receptor neurons are both blocked by heparin(22, 23) . Fig. 5D shows that internal perfusion of megakaryocytes with 10 mg ml heparin for 5 min virtually abolished both the [Ca] and membrane current response to ADP. Cd has also been shown to block the olfactory neuron IP-dependent plasma membrane channel(24) ; however, neither Cd nor Zn, added to the bath at 1 mM, affected the monovalent cation currents activated by ADP, 1,4,5-IP, or 2,4,5-IP (not shown). Tetrodotoxin, a blocker of voltage-dependent Na channels, was also ineffective at concentrations up to 5 µM added to the bath saline (data not shown).

ADP- and 1,4,5-IP-activated NaEntry

In physiological salines, given a normal negative resting potential, the IP and ADP-evoked current would be inward and carried mainly by Na. To detect whether this conductance can result in significant changes in [Na], the Na-sensitive indicator, SBFI(25) , was added to the pipette saline in place of fura-2 and 1,4,5-IP dialyzed from the pipette (Fig. 6A). The inward current that developed at the holding potential of -40 mV, which we have shown above to be induced by 1,4,5-IP, was associated with a gradual increase in [Na]. Depolarization to 0 or 20 mV prevented the [Na] increase, an effect that was fully reversible, although more negative potentials were required to produce similar rates of Na increase once a substantial increase in the 340/380 ratio signal had occurred.

The above whole cell patch clamp experiments represent conditions that are far from physiological and dialyze important cytoplasmic factors. We therefore turned to the nystatin-perforated patch technique to further assess the magnitude of the ADP-evoked [Na] increase. In these experiments, represented by that in Fig. 6B, a K-based pipette saline and current-clamp conditions were also used in an attempt to further mimic physiological conditions. SBFI was loaded prior to patch clamp by incubation with the acetoxymethyl ester (see ``Materials and Methods''). Application of 5 µM ADP produced a regular oscillation in membrane potential from the resting level of -40 mV to -75 mV (approximately 6 times/min), known to result from oscillations of [Ca] and activation of Ca-dependent K channels(1, 2) . In this cell, which is typical of 5 other experiments, [Na] increased gradually by 3-4 mM during the 3 min ADP application and then continued to increase after agonist removal. Further [Na] increases were observed in response to a second exposure to ADP. In 5 cells, after a 3-min application of 5 µM ADP, [Na] increased from a resting level of 15 ± 6 mM to 28 ± 13 mM, measured 2 min after removal of the agonist.


DISCUSSION

The present study demonstrates that both extracellularly applied ADP and internally perfused 1,4,5-IP evoke a monovalent cation-selective current in rat megakaryocytes. The similarity between the I-V relationships, reversal potential, stimulation of Na influx, and failure of ADP to evoke a current on top of the IP-induced response, suggests strongly that these two agents activate the same channel. Although there is no direct evidence for ADP-induced 1,4,5-IP production in megakaryocytes, ADP is known to stimulate 1,4,5-IP production in platelets (26), and both ADP and IP induce a similar [Ca] oscillation in the rat megakaryocyte (27). In addition, the lack of effect of ADP on [Ca] after internal perfusion of heparin, a known blocker of IP receptors, suggests that this agonist acts via phospholipase C to increase intracellular IP and internal Ca levels, as in many other nonexcitable cells (20, 28). The activation of the monovalent cation current by 2,4,5-IP, a poor substrate for 1,4,5-IP-kinase(21) , and not by IP is strong evidence for direct stimulation by 1,4,5-IP rather than by any of its metabolites. The rate of inactivation of the current was reduced in the absence of a [Ca] increase, which may be explained in part by slower hydrolysis of 1,4,5-IP because 1,4,5-IP-kinase is calcium-dependent(29) . Direct inactivation of the current by calcium cannot be not ruled out, although this is unlikely because, in experiments where IP was introduced directly into the cells, fluctuations in [Ca] had little or no effect on the IP-dependent current(3) . The reduced activation rate of the ADP-evoked current in the presence of BAPTA can be explained by the calcium dependence of phospholipase C activity because IP production is accelerated by an increase in [Ca](28, 30) . The lack of effect of thapsigargin-induced rise in [Ca], which does not involve an increase in IP(18, 19) , suggests that the monovalent cation current cannot be activated by [Ca] alone. The observation that ADP-evoked currents inactivated more slowly and incompletely the longer a whole cell recording was made suggests loss by dialysis of a factor responsible for current inactivation. One likely candidate for this labile factor is 1,4,5-IP kinase.

In many nonexcitable cells, including the megakaryocyte, IP stimulates a plasma membrane current indirectly by releasing Ca from internal stores(3, 4) . This pathway cannot account for the whole cell currents gated by internal perfusion of IP in this study, with low internal Ca buffering, because thapsigargin, which releases internal Ca without generation of IP(18) , failed to elicit a significant current. Furthermore, the store-regulated Ca currrent is highly selective for divalent cations, is blocked by Zn and Cd, and is amplified by buffering of internal Ca with BAPTA or EGTA(4, 31) , none of which applied to the IP-dependent response under our experimental conditions. 1,4,5-IP receptors have been found in the plasma membrane of T lymphocytes(32) , platelets (33), and olfactory cilia(34) , and channels activated by 1,4,5-IP have been identified in patch clamp recordings from Jurkat T cells(35) , A431 cells(36) , and olfactory neurons(23, 24, 37, 38) . In the T cell, the A431 cell, and insect or channel catfish olfactory neurons, only divalent cation currents were reported, implying a different selectivity from the IP-gated channel in the megakaryocyte, which conducts little, if any Ca. Nonselective cation currents were activated by IP in rat olfactory neurons, although, unlike in the megakaryocyte, these were Cd-sensitive(24) . IP also gates ion channels in the membranes of internal organelles, including the sarcoplasmic reticulum (39) and nucleus(40) , which possess a higher permeability to calcium than monovalent cations. Thus, the IP-dependent channel in the megakaryocyte may represent a new class of ion channel gated by this second messenger.

The physiological function of the ADP-evoked monovalent cation channels in the rat megakaryocyte remains speculative. We could not detect any significant Ca permeability, thus the conductance is unlikely to play a role in agonist-evoked Ca signaling. Furthermore, the rat megakaryocyte has a store-dependent influx pathway that is highly selective for Ca(3, 4) , and this is likely to account for most if not all of the Ca influx that occurs during IP-dependent Ca release(3) . Application of ADP caused a 10-20 mM increase in [Na], which, considering the large volume of the megakaryocyte, amounts to a considerable Na influx. The continued, slow increase in [Na], after removal of ADP, may be explained if IP levels remain elevated for some time. This certainly does appear to be the case because repetitive hyperpolarizations in the membrane potential, known to arise from IP-induced Ca release and activation of Ca-dependent K channels(27) , continued for several minutes after ADP application. Agonist-evoked Na influx may outlast the Ca responses if IP levels are higher at the plasma membrane, where this messenger is produced, than deeper in the cytoplasm near the Ca stores or if the threshold for activation of the plasma membrane channel by IP is lower than for that of store Ca channel. An alternative explanation is that the gradual increase in [Na] is due to slow equilibration of Na within the cytoplasm following IP-evoked Na entry. This would imply a much greater increase in [Na] next to the plasma membrane and may be detectable by confocal ratiometric measurements of SBFI fluorescence. The monovalent cationic conductance that we report here is of particular interest since a previous study by Leven et al.(7) concluded that the ADP and thrombin-evoked spreading reaction in the rat megakaryocyte, which may be a functional response leading to platelet formation, depended upon an increased Na conductance. Further work, however, is needed to assess whether the IP-activated Na influx is indeed involved in the cell spreading reaction because the present study did not assess the possible contribution of ADP-dependent stimulation of Na/Ca exchange or inhibition of Na/K exchange to the observed [Na] increase.

Platelets have little or no capacity to manufacture proteins, thus its progenitor, the megakaryocyte, must eventually express most, if not all, platelet ion channels. Therefore, the IP-dependent cation current, in addition to playing a role in the megakaryocyte, may be important for platelet signaling. With a much larger surface area to volume ratio in the platelet, this current may result in large alterations of [Na]. In fact, in human platelets, thrombin induces a relatively greater production of IP than ADP (26) and a greater increase in [Na](41, 42) . The ADP-induced rise in [Na] has been shown to be mostly via ADP-activated receptor-operated channels(421) ; however, the mechanism of the thrombin-induced Na influx is not known and may involve the channel we report here in the rat megakaryocyte if this is also expressed in human megakaryocytes.

In conclusion, we have demonstrated the existence of a plasma membrane conductance activated by 1,4,5-IP that carries Na into the cell at resting membrane potentials and may have a functional role in megakaryocyte signaling or be expressed for later use in platelet responses.


FOOTNOTES

*
This work was supported by the Biotechnology and Biological Sciences Research Council and the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

Holds a British Heart Foundation Science Lectureship.

The abbreviations used are: [Ca], cytosolic Ca concentration; SBFI, sodium-binding benzofuran isopthalate; IP, myo-inositol trisphosphate (with positional determinants of the phosphate groups as specified); IP, myo-inositol 1,3,4,5-tetrakisphosphate; BAPTA, 1,2-bis-(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; NMDG, n-methyl-D-glucamine; [Na], cytosolic Na concentration; I-V, current-voltage.


ACKNOWLEDGEMENTS

We thank Andres Floto for helpful discussion, in particular on noise analysis methods, and thank Dr. Stewart Sage for comments on the manuscript.


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