Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030
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ABSTRACT |
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Heterologous expression of a variety of membrane proteins in Xenopus oocytes sometimes results in the appearance of a hyperpolarization-activated inward current. The nature of this current remains incompletely understood. Some investigators have suggested that this current is a Cl current, whereas others have identified it as a nonselective cation current. The purpose of this investigation was to characterize this current in more detail. The hyperpolarization-activated inward current (IIN) present in native oocytes was composed of a current carried at least partly by Ca and Mg under physiological ionic conditions plus a Ca-activated Cl current. The Ca-activated Cl current was blocked by chelation of cytosolic Ca with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. When Cl currents were blocked, the cation current could be carried by Ca, Mg, or Co, but not appreciably by Ba, Mn, or Cd. IIN was stimulated by intracellular acidification. The properties of IIN were quite different from those of the store-operated Ca current. Heterologous expression of transient receptor potential-like gene product (TRPL), one of the members of the transient receptor potential family of putative store-operated Ca channels, apparently resulted in alteration of the voltage sensitivity of the endogenous IIN.
chloride current; magnesium; divalent cation; D-myo-inositol 1,4,5-trisphosphate; calcium chelator
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INTRODUCTION |
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XENOPUS LAEVIS OOCYTES are one of the most widely used heterologous expression systems for studying the properties of cloned ion channels (3-5, 7, 16, 22, 32, 35). Injection of oocytes with cRNA into the cytoplasm or cDNA into the nucleus results in the expression of ion channels in the plasma membrane between several hours to several days later. These cloned channels can then be studied by two-microelectrode voltage clamp or patch-clamp analysis.
Experiments on cloned channels expressed in heterologous cell types can be complicated by the existence of endogenous channels. Endogenous channels may either be upregulated in response to expression of the exogenous protein or may form heteromultimers with the exogenous channel. For example, the expression of a G protein-gated inwardly rectifying K (GIRK1) current on injection of GIRK1 cRNA into oocytes depends on the presence of an endogenous inwardly rectifying K channel called XIR (10).
A number of investigators have reported that expression of exogenous ion channels or other membrane proteins in Xenopus oocytes results in the appearance of a hyperpolarization-activated current (2, 15, 34, 36). This hyperpolarization-activated current is enigmatic. Although a very similar current is present in native oocytes (27), the exact properties of this current have been reported to depend on the properties of the expressed ion channel (15). Furthermore, although most investigators conclude that this current is a Cl current (2, 15, 27, 34), Tzounopoulos et al. (36) show that in the absence of extracellular Ca, this channel appears to behave as a nonselective cation channel.
The presence of this endogenous hyperpolarization-activated current may
affect the interpretation of expression studies. For example, recently,
Schmieder et al. (33) reported that the currents induced
by expression of the Xenopus homologue of ClC-5 (xClC-5) in
Xenopus oocytes depended significantly on the vector used. If the cRNA was flanked by Xenopus -globin 5'- and
3'-untranslated regions, the anion selectivity and sensitivity to Cl
channel blockers was different than when the cRNA contained the native
xClC-5 untranslated regions. The authors suggest that the
current induced by the
-globin-flanked cRNA was genuine xClC-5
current, whereas the current induced by the native cRNA was due to
upregulation of endogenous Cl currents.
To interpret expression studies with cloned ion channels in Xenopus oocytes objectively, particularly Cl channels, it is necessary to have a more thorough understanding of the endogenous currents in the oocyte. In this paper, we show that the hyperpolarization-activated current is primarily a cation channel that is permeable to Ca ions. The entry of Ca ions through this channel secondarily activates Ca-activated Cl currents that we and others have studied in detail (9, 17, 18, 20, 21). This channel has a number of interesting features: the channel is activated by cytosolic acidification and is highly permeable to Mg ions.
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METHODS |
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Electrophysiological Methods
Xenopus oocytes were voltage clamped with two microelectrodes using an Axon Instruments GeneClamp 500. Electrodes were filled with 3 M KCl (or 4 M potassium acetate for experiments on the store-operated Ca current, ISOC) and had resistances of 0.5-2 MMicroinjection
Oocytes were sometimes injected with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), EGTA, or IP3 using a Drummond Nanoject automatic oocyte injector. The injection pipette was pulled from glass capillary tubing in a manner similar to the recording electrodes, and was then broken so that it had a beveled tip with an inside diameter of <20 µm. We usually injected 23 nl of a 50 mM solution of K4 BAPTA or 23 nl of 100 mM Na2K2EGTA in water to give a final calculated concentration in the oocyte of 1-2 mM. The injection pipettes were usually left impaled in the oocyte for the duration of the experiment. When IP3 was injected, typically, 4.6 nl of 10 mM IP3 in H2O was injected to give a calculated oocyte concentration of ~50 µM.Solutions
Normal Ringer consisted of 123 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.8 mM MgCl2, and 10 mM HEPES (pH 7.4). Ringer (90 mM Cl) consisted of 82 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM NMDG-HEPES (pH 7.4). Ringer (23 mM Cl) consisted of 15 mM NaCl, 63 mM sodium aspartate, 2 mM MgCl2, 2 mM CaCl2, and 10 mM HEPES (pH 7.4). NMDG-Ringer consisted of 90 mM NMDG-Cl, 5 mM CaCl2, and 5 mM NMDG-HEPES (pH 7.4). NMDG-aspartate Ringer consisted of 90 mM NMDG-aspartate, 5 mM Ca(OH)2, and 5 mM NMDG-HEPES (pH 7.4). Cl-free storage solution was 108 mM sodium aspartate, 1.6 mM potassium aspartate, 10 mM sodium HEPES, 2 mM Ca(OH)2, and 2 mM MgSO4 (pH7.4).Harvesting Oocytes
All procedures were performed in accordance with the Institutional Animal Care and Use Committee and NIH guidelines. Stage V-VI oocytes were harvested from adult X. laevis females (Xenopus I) as described by Dascal (4). Xenopus were anesthetized by immersion in tricaine (1.5 g/l). Ovarian follicles were removed, cut into small pieces, and digested in 0-Ca Ringer that contained 2 mg/ml collagenase type IA (Sigma) for 2 h at room temperature. The oocytes were extensively rinsed and placed in L15 medium (GIBCO) and stored at 18°C. Oocytes were usually used between 1 and 6 days after isolation.Synthesis of cRNA
Transient receptor potential-like gene product (TRPL) in PBS was linearized with Not I restriction endonuclease. Capped cRNA was synthesized in vitro using the Ambion mMessage mMachine in vitro transcription kit and T7 RNA polymerase. cRNA was analyzed by denaturing formaldehyde gel electrophoresis and found at the expected size (~3.2 kb).Display and Analysis of Data
For display of the figures, current transients during voltage steps were often blanked for 5 ms. Data points are the mean, and error bars are ± SE. Each current-voltage (I-V) and activation curve is the average of 3-6 different oocytes. For I-V relationships, the raw data were averaged. For the activation curves, the data were normalized such that the current at a particular voltage was set as 1.0. Mathematical fits were performed using an iterative Levenberg-Marquardt algorithm. Unless otherwise noted, the interval between stimuli was 10-20 s. In tail current analysis, one would like to be able to measure the current immediately after the voltage step; however, because of the capacitative transient, we were not able to measure the instantaneous current sooner than 5-8 ms after the step. ![]() |
RESULTS |
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An Endogenous Hyperpolarization-Activated Current in Xenopus Oocytes
Hyperpolarization to
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IIN Is a Mixture of a Cation Current and a Ca-Activated Cl Current
IIN is only partially blocked by BAPTA injection.
Xenopus oocytes have a very high concentration of
Ca-activated Cl channels (9). To test whether
IIN involved a Ca-activated Cl current, we
injected the oocyte with BAPTA to a final concentration of ~1 mM.
Although this concentration of BAPTA completely blocks the Ca-activated
Cl currents (9), it only partially blocked IIN, whereas IOUT was
almost completely blocked (Fig. 2,
A and B). These data led us to hypothesize that
IIN was composed of two components:
1) a Ca-permeable cation current, and 2) a
Ca-activated Cl current that was activated as a consequence of the Ca
influx through the cation conductance.
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The reversal potential of IIN suggests multiple
ionic permeabilities.
To determine the current-carrying ionic species more rigorously, we
measured the sensitivity of the reversal potential to extracellular
[Cl] ([Cl]o). The instantaneous I-V
relationship for the inward current was determined by tail current
analysis (Fig. 3A). The
membrane was hyperpolarized to 200 mV for 1 s and then
repolarized to different test potentials. The current 8 ms after
stepping to the test potential was plotted as a function of the test
potential with 90 mM [Cl]o (Fig. 3A)
or 23 mM [Cl]o (Fig. 3B). The I-V
relationships are plotted in Fig. 3C. After correcting for
liquid junction potentials (25), the reversal potential of
the current shifted 15 mV (from
28 mV with 90 mM Clo to
13 mV with 23 mM Clo), whereas the Goldman-Hodgkin-Katz equation predicted a +35-mV shift. This suggested that the current was
carried mainly by Cl but that other ions also contributed.
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Effects of divalent cations on IIN.
To determine the ionic selectivity of the BAPTA-insensitive component
of IIN to different divalent cations, the cells
were bathed in solutions that contained 90 mM NMDG-Cl, 5 mM NMDG-HEPES, and 5 mM of the Cl salt of the indicated divalent cation. The oocyte
was injected with BAPTA to block Ca-activated Cl channels ~10 min
before the experiment. Under these conditions, there was significant
hyperpolarization-activated inward current in the presence of Ca as the
only small cation (Fig. 4A).
In contrast, there was no detectable current in the presence of Ba, Cd,
or Mn. Surprisingly, there was significant current in the presence of
Co, Mg, or a mixture of Ca and Mg.
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Ca Current Through IIN Stimulates a Cl Current
Although Figs. 3 and 4 show that IIN was carried partly by divalent cations when they were the only permeant cations present, we wanted to determine whether Ca permeated the channel under physiological ionic conditions. We believe that Ca did permeate under physiological ionic conditions because when Ca was present in the extracellular solution, and the oocyte was not injected with BAPTA, a Ca-activated Cl current activated transiently on depolarization to +20 mV after the hyperpolarizing pulse. In Fig. 1, this current is labeled IOUT, and in Fig. 2, we showed that it was blocked by BAPTA injection. This demonstrated that IOUT was Ca dependent.We hypothesized that IOUT was activated by Ca
entry that occurred during IIN. If this is
correct, we would predict that the amplitude of
IOUT would be related to the amplitude of
IIN: as more Ca entered, more Cl current would
be activated. Figure 5, A and
B, shows how increasing the duration of the 200-mV pulse increases both IIN and
IOUT. As the pulse was prolonged,
IIN became larger, and the associated
IOUT also became larger. The observation that
IOUT is blocked by BAPTA and that the amplitude
of IOUT is related to the amplitude of the
preceding IIN argues that
IOUT is activated by Ca influx through
IIN.
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Figure 5, C and D, shows that
IOUT is a Cl current because the reversal
potential changes as predicted for a pure Cl current. The instantaneous
I-V relationship for IOUT was
measured by repolarizing to +30 mV for 100 ms to activate
IOUT after a 2-s-long 200-mV pulse to drive Ca
influx. After IOUT was activated, the membrane was hyperpolarized to different potentials and the instantaneous tail
current was measured (Fig. 5C). IOUT
had a linear instantaneous I-V relationship. On average, the
reversal potential changed +32 ± 2.7 mV (n = 6)
upon changing Clo from 90 to 23 mM (Fig. 5D). This shift was close to the +35-mV shift predicted by the
Goldman-Hodgkin-Katz equation. These data show that Ca entered the
oocyte during the hyperpolarizing pulse and activated a Ca-activated Cl current.
Rebound Stimulation of IIN After Washout of Certain Divalents
Although extracellular Cd, Ba, and Mn apparently did not permeate the channel or stimulate a Cl current, IIN was greatly and transiently increased above control when these ions were washed out and replaced with a permeant divalent cation (Ca, Mg, or Co). In the experiment of Fig. 6A, the oocyte was not injected with a Ca chelator. The amplitude of IIN at
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The potentiating effect of Cd appeared to require Cd entry into the
channel because the potentiating effect of Cd was not seen when the
oocyte was stepped to less negative potentials (120 mV instead of
200 mV, Fig. 6C). Furthermore, the potentiating effect was
greatly diminished if the oocyte was held at
35 mV and not
hyperpolarized while Cd was present (Fig. 6D). This
suggested that although Cd may not carry significant current, a small
amount can enter the oocyte at these negative potentials and somehow potentiate IIN when Ca is reintroduced. Similar
potentiating effects were seen with washout of Mn and Ba, although this
was not investigated in detail. Furthermore, washout of Cd stimulated
currents in the presence of not only Ca but also Co and Mg. The
mechanisms underlying this potentiation are speculative. However, the
magnitude of the effect seems too large to explain by surface charge
effects, and the observation that the effect occurs even in the
presence of EGTA, which should chelate Cd, is paradoxical.
Stimulation of IIN With Acid
Other investigators have reported that extracellular acidification produces an increase in the hyperpolarization-activated current. However, in these studies, the manner in which extracellular acidification was produced was not precisely described. When we exposed oocytes to normal Ringer solution that had been acidified by the addition of HCl, we rarely observed a significant or reproducible increase in IIN (Fig. 7A). In contrast, when we acidified the Ringer solution with a permeant, protonated acid such as acetic acid, we found that IIN was stimulated dramatically. This suggested that intracellular acidification could activate IIN. This was supported by the finding that intracellular injection of HCl also stimulated this current (Fig. 7B). Isochronal I-V curves in the presence and absence of extracellular acetic acid are shown in Fig. 7C. Acidification increased the amplitude of IIN at all potentials. We have previously described an intracellular acid-activated Ca influx pathway in Chlamydomonas, which is involved in the deflagellation response in this organism (29, 30). Some of the properties of IIN (activation by acid, block by Gd, and lack of block by Co) are very similar to this Chlamydomonas Ca entry pathway.
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IIN Is Not ISOC
Because IIN is carried at least partly by Ca ions, we wondered whether IIN could be the ISOC activated by extreme hyperpolarization. To test this question, we compared some of the properties of IIN and ISOC in the same oocytes.Measurement of ISOC is complicated by
contamination with Cl currents.
In our earlier study (9), we stimulated
ISOC by depleting intracellular Ca stores by
IP3 injection and tried to isolate ISOC from Cl currents by intracellular injection
of BAPTA to block Ca-activated Cl currents. Despite the presence of 1 to 12 mM intracellular BAPTA, we found that in some cells the current
that we recorded was contaminated with a Cl current. This result is
shown in Fig. 8, A and
B. The oocyte was bathed in a NMDG-aspartate external solution that contained 5 mM CaCl2 and was periodically
hyperpolarized to 150 mV for 500 ms from a holding potential of
35
mV. The oocyte was injected with 46 nl of a mixture of 1 mM
2-deoxy,3-fluoro inositol 1,4,5-trisphosphate (IP3F) and
250 mM BAPTA to produce calculated intraoocyte concentrations of ~50
µM IP3F and ~12 mM BAPTA. Inward current was measured
10 ms after the step to
150 mV. The IP3F injection
initially stimulated an inward current that peaked and decayed in <2
min. This initial component was the Ca-activated Cl tail current
(9) that occurred before the BAPTA block fully developed.
This initial current was followed by a slowly developing inward
current. We previously suggested that this second component was
ISOC (9). However, we found that
when we changed extracellular [Cl], the inward current amplitude changed and its reversal potential shifted. This suggested that at
least some of the inward current was carried by Cl ions.
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ISOC is highly Ca selective.
Unlike IIN, ISOC was
highly selective to Ca, as shown in Fig.
9, A and B. In this
experiment, the oocyte was initially bathed in a solution that
contained only NMDG, aspartate, HEPES, and 5 mM Ca. Changing of the
extracellular solution to one that lacked Ca but contained 5 mM Mg and
0.1 mM EGTA abolished the inward current completely (Fig.
9A). The IP3-stimulated current strongly inwardly rectified and exhibited a reversal potential near +25 mV. The
shape of this curve closely resembled the I-V relationship that has been reported for Ca release-activated Ca current in other
cell types (12) and ISOC in oocytes
(38). The inward current was strongly dependent on the
extracellular Ca concentration (Fig. 9B). The dissociation
constant for Ca was estimated to be 2.4 mM.
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Effects of divalent cations on ISOC. To investigate the effect of different cations on the store-operated Ca channel, Ca was replaced completely with different divalent cations. The replacement of Ca with Cd, Co, or Mn rapidly eliminated the current, but replacement with Ba or Sr slowly reduced the current to an intermediate level (Fig. 9C). These effects of divalent cations on ISOC are different from the effects on IIN.
IIN is augmented by expression of TRPL, a member
of a family of putative store-operated Ca channels.
Our initial interest in IIN developed when we
noticed that this current was greatly augmented by heterologous
expression of Drosophila TRPL (28). We were
interested in TRPL because it is a member of a family of channels that
has been suggested to include store-operated Ca channels (23,
40), although this remains a rather contentious issue. We found
that injection of TRPL cRNA into oocytes never increased a current
resembling ISOC, but invariably induced a large
current with characteristics very similar to IIN
(Fig. 10). For example, in the same
batch of oocytes on the same day, in oocytes injected with 23-35
ng TRPL cRNA, the tail current at +20 mV from a 160-mV pulse was
2.05 ± 0.34 µA (n = 3, Fig. 10C,
open triangles), whereas the tail current was about seven times smaller
in uninjected oocytes with a pulse of 0.3 ± 0.2 µA
(n = 4, Fig. 10C, closed squares).
The waveform of the current and the shape of the activation curve were
virtually identical for the control and TRPL oocytes, but the
activation curve was shifted dramatically to the right on the voltage
axis for the TRPL oocytes (Fig. 10C). We hypothesize that
the IIN-like current stimulated by TRPL
expression is due to upregulation of the endogenous
IIN current.
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DISCUSSION |
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This manuscript characterizes a strong
hyperpolarization-activated current that is present in Xenopus
oocytes. The current, termed IIN, is
activated only at potentials negative to 150 mV in normal oocytes,
but is apparent at more positive potentials in oocytes that express the
TRPL ion channel. This current is composed of two components: an inward
current that is carried partly by Ca and Mg ions under physiological
conditions, and a Ca-activated Cl current stimulated by Ca entering
with the inward cation current. These two components can be separated
by injection of BAPTA to block the Ca-activated Cl current. The inward
current that remains after injection of BAPTA is not a Cl current
because the current amplitude and reversal potential are independent of extracellular [Cl]. However, the observation that the conductance in
the outward direction increases with increasing internal [Cl] (Fig.
3I) suggests that the current may also have a Cl component or is Cl sensitive. The current is present in solutions that contain only Ca, Mg, and Co as permeable cations but is not present in solutions that contain Ba, Mn, or Cd. It was not possible to determine the monovalent cation permeability of the channel because removing extracellular divalent cations produced a large increase in membrane conductance due to activation of the Ca-inactivated Cl current (38).
Characteristics of the Hyperpolarization-Activated Current
The complex composition of the hyperpolarization-activated current explains why this current has generated so much confusion in the literature. An inward current activated by strong hyperpolarization was first described in normal oocytes by Parker and Miledi (27). This is probably the same current we have described here, but Parker and Miledi (27) concluded that the current was a Ca-independent Cl current because it reversed near the Cl equilibrium potential and was stimulated by removing Ca from the extracellular solution. The stimulatory effect of Ca removal (27) can be explained because they replaced Ca with Mg, which stimulates IIN (Fig. 4). Parker and Miledi (27) showed that the inward current was blocked ~80% by 10 mM Mn atKowdley et al. (15) have described a slowly developing,
noninactivating inward current, ICl(endo), which
they concluded is a voltage-dependent anion current. This current had a
mean amplitude of 1.1 µA at 150 mV. They interpret this current as a Cl current because at physiological [Cl]o, the reversal
potential was near the Cl equilibrium potential
(ECl). Furthermore, the selectivity of the channel
followed the lyotropic anion sequence (11). However, the
reversal potential changed ~35 mV/10-fold change in
[Cl]o, which was less than expected for a pure Cl
conductance, suggesting that other conductances also contributed. The
current was not affected by replacing Na with NMDG but was augmented by removing Ca. This can again be explained by the fact that their solutions contained Mg. ICl(endo) resembled a
current that is seen in oocytes injected with phospholemman cRNA
(24). The current induced by phospholemman expression
differed in some ways from the endogenous current. For example, Kowdley
et al. (15) found that the phospholemman-induced
current was sensitive to pH and DIDS, whereas the endogenous current
was not sensitive. Furthermore, the phospholemman-induced current had
different activation kinetics than the endogenous current. Because
mutations in the putative pore-forming domain of phospholemman altered
the activation kinetics of the hyperpolarization-activated current
observed when phospholemman was expressed in oocytes, it appears that
phospholemman may form a heteromer with an endogenous subunit to
enhance ICl(endo) (15).
Shimbo et al. (34) have reported that expression of a
variety of small integral membrane proteins can modify an endogenous hyperpolarization-activated inward Cl current in Xenopus
oocytes. The endogenous current is activated only at potentials
negative to 160 mV, but in oocytes expressing the minK potassium
channel, phospholemman, influenza virus NB protein, or a
synthetic ion channel protein SYN-C, a similar current was activated at
potentials negative to
100 mV. It appears that the I-V
relationship was shifted ~60 mV in the positive direction by
expression of exogenous ion channels. The augmentation of the current
was not seen with all integral membrane proteins, because the influenza
virus M2 ion channel did not stimulate this current. Furthermore, the
Cl selectivity of the channel appeared to differ depending on the type
of channel expressed in the oocyte: the reversal potential shifted 20 mV with a 10-fold change in Clo for
phospholemman-expressing oocytes and 10 mV for oocytes expressing NB
protein. Ba reduced both the control currents and currents in
expressing oocytes ~70%. All currents were increased by decreasing
extracellular pH, which agrees with our findings but disagrees with the
results of Kowdley et al. (15) that show the endogenous
current was pH insensitive. The differences between these investigators
may reflect the methods used to acidify the extracellular solution and
the ability of the extracellular pH change to ultimately change
intracellular pH.
Tzounopoulos et al. (36) also reported that expression of
certain membrane proteins induced a hyperpolarization-activated current. They reported that the minK potassium channel, a mutant Shaker channel exhibiting voltage-dependent charge movement
but no ion permeation, an amino acid transporter, and an inward
rectifier channel all induced a hyperpolarization-activated current,
whereas the dopamine D2 receptor and -galactosidase did not. This
current was identified as a Cl current because it had a reversal
potential near ECl, and the reversal potential changed 53 mV/10-fold change in Clo. The current was stimulated by
reducing extracellular Ca. In the absence of extracellular Ca,
Tzounopoulos et al. (36) concluded that the
current behaved as a nonselective cation current because the reversal
potential of the current was similar in mixtures of K, Na, and Cs. In
contrast, we find that the current is not affected by replacing Na with
NMDG. The difference between our conclusions and those of Tzounopoulos
et al. (36) can be explained because they implicitly
assumed that the Mg present in their solutions was impermeable. If Mg
is permeant and monovalent cations are not permeant, the reversal
potential will not change when monovalent cation concentration is changed.
In conclusion, we believe that of all of these data are consistent with our interpretation that IIN is an endogenous hyperpolarization-activated current that is composed of a Ca current accompanied by a Ca-activated Cl current and possibly a small inward Ca-independent Cl current.
TRPL Expression
Our study shows that expression of TRPL results in an increase in the endogenous hyperpolarization-activated current IIN. There are several possible mechanisms by which TRPL could increase IIN. The first possibility is that TRPL could encode a homomeric channel that is very similar to the endogenous IIN channel. This possibility seems unlikely because the properties of IIN differ significantly from TRPL channels expressed endogenously in Drosophila photoreceptor (31) and expressed heterologously in other systems (e.g., 8, 13, 14, 19, 26, 37). The second possibility is that TRPL forms heteromeric channels with endogenous subunits. The third possibility is that TRPL somehow indirectly regulates the function of endogenous channels. We presently cannot distinguish between these alternatives. However, the observation that the voltage dependence of IIN is the same in native oocytes and in oocytes expressing TRPL suggests that either the properties of this channel change with expression level or that TRPL protein is a part of the channel in expressing oocytes.Our conclusions contrast with those of Gillo et al. (6), who conclude that coexpression of TRP (transient receptor potential) plus TRPL results in the expression of a store-operated current that they call IdSOC. This current was activated by depletion of intracellular Ca stores when the quantity of injected cRNA was low, but was activated constitutively and independently of store depletion when large amounts of TRPL cRNA were injected. We believe that Gillo et al. (6) may have misidentified the current stimulated by TRPL plus TRP expression. The current that they recorded was measured in the presence of 10 mM Mg and 0 mM Ca, and its waveform closely resembled IIN. The current they described differs substantially from typical ISOC in selectivity and kinetics (12) and also differs significantly from TRPL channels expressed endogenously in Drosophila photoreceptor (31). For these reasons, we think it is likely that the IdSOC current described by Gillo et al. (6) is actually the endogenous IIN current.
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ACKNOWLEDGEMENTS |
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We thank Dr. Marie Phillips for the gift of the TRPL cDNA. We thank Dr. Susan Meiergerd, who participated in some of the early experiments, for helpful suggestions and discussion. We also thank Alyson Ellingson, Amber Rinderknecht, and Elizabeth Lytle for outstanding technical assistance.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-21195, HL-50474, and GM-55276.
Address for reprint requests and other correspondence: H. C. Hartzell, Dept. of Cell Biology, 1648 Pierce Dr., Rm. 311, Emory Univ. School of Medicine, Atlanta, GA 30322-3030 (E-mail: criss{at}cellbio.emory.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 November 1999; accepted in final form 5 June 2000.
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