(Received for publication, November 7, 1995)
From the
Previous studies have shown that the Drosophila cation
channels designated Trp and Trpl can be functionally expressed in Sf9
insect cells using baculovirus expression vectors. The trp gene encodes a Ca-permeable channel that is
activated by thapsigargin, blocked by low micromolar
Gd
, and is relatively selective for Ca
versus Na
and Ba
. In
contrast, trpl encodes a Ca
-permeable cation
channel that is constitutively active, not affected by thapsigargin,
blocked by high micromolar Gd
, and non-selective with
respect to Ca
, Na
, and
Ba
. The region of lowest sequence identity between
Trp and Trpl occurs in the COOH-terminal domain. To test the hypothesis
that this region is responsible for the differential sensitivity of
these channels to thapsigargin, chimeric constructs of Trp and Trpl
were created in which the COOH-terminal tail region of each protein was
exchanged. The Trp construct with the Trpl COOH-tail was constitutively
active, insensitive to thapsigargin, but retained selectivity for
Ca
over Na
and Ba
.
In contrast, the Trpl construct with the Trp COOH-tail was not
constitutively active, could be activated by thapsigargin, but remained
non-selective with respect to Ca
,
Ba
, and Na
. These results suggest
that the COOH-terminal domain of Trpl plays an important role in
determining constitutive activity, whereas the COOH-terminal region of
Trp contains the structural features necessary for activation by
thapsigargin.
The Drosophila proteins encoded by the trp and trpl genes (Trp and Trpl) are thought to form
Ca-permeable cation channels responsible for
depolarization of photoreceptor cells following stimulation by light (1, 2) . Since phototransduction in Drosophila requires phospholipase C activity (for review, see (2, 3, 4) ), Trp and Trpl may be insect
homologues of the channels responsible for I
, the
elusive store-operated, capacitative Ca
entry
channels that are activated by receptor-mediated phosphoinositide
hydrolysis in mammalian non-excitable cells. The first studies
demonstrating that trp and trpl encode ion channels
were performed in a heterologous expression system using baculovirus
expression vectors(5, 6, 7, 8) .
Evaluation of whole cell membrane currents in Sf9 insect cells
following infection with recombinant baculovirus containing the cDNA
for trp and trpl under control of the polyhedrin
promoter demonstrated that trp encodes a cation channel that
is selective for Ca
over Na
, can be
activated by depletion of the internal Ca
store by
thapsigargin, and is a poor conductor of
Ba
(8) . In contrast, trpl encodes a
cation channel that is constitutively active, is relatively
non-selective with respect to Ca
,
Ba
, and Na
, is unaffected by
thapsigargin(7, 8) , but can be activated by a
receptor-mediated increase in
inositol-1,4,5-trisphosphate(6, 9) .
The functional
studies on Trp and Trpl allow for specific predictions concerning the
structural features of Trp that may be responsible for the differential
sensitivity of these two channel proteins to thapsigargin. Trp and Trpl
exhibit substantial amino acid identity in their
NH-terminal regions and in their proposed membrane spanning
segments but differ in their COOH-terminal
domains(1, 10, 11) . We hypothesized that the
COOH-terminal domain of Trp is necessary for thapsigargin sensitivity.
To test this hypothesis, chimeric proteins were created in which the
COOH-terminal domains of Trp and Trpl were exchanged, and the resulting
channels were functionally expressed using the baculovirus-Sf9 insect
cell expression system. The results demonstrate that the COOH domain of
Trp confers thapsigargin sensitivity to Trpl and that the COOH domain
of Trpl confers constitutive activity to Trp. Although the relative
selectivity of the chimeric channels for Ca
,
Na
, and Ba
appears to be unchanged
from the native channels, the sensitivity of the chimeric channels to
Gd
blockade is intermediate between that seen for Trp
and Trpl. Thus, the COOH-terminal domain may influence pore
characteristics. A preliminary report of this work has appeared in
abstract form (12) .
Figure 1:
Panel A,
schematic comparison of the Drosophila proteins Trp and Trpl.
The trp gene encodes a protein of 1275 amino acids and is
represented as three major domains: the NH-terminal region,
a central hydrophobic region that includes the putative
membrane-spanning segments S1-S6, and COOH-terminal domain. The
degree of identity between the primary amino acid sequences of the
different domains of Trp and Trpl were calculated using the GCG
analysis package and are indicated as percentages. The approximate
position in both trp and trpl of the recognition
sequence for endonuclease DraIII is indicated by the arrow. Panel B, protein expression.
SDS-polyacrylamide gel electrophoresis and immunoblotting were
performed as described under ``Experimental Procedures'' on
membrane preparations isolated from non-infected Sf9 insect cells (lane b, 10 µg of protein) or from cells infected with
recombinant baculovirus containing the cDNA for the B
bradykinin receptor (lane c, 10 µg), FLAG-Trp (lane d, 1 µg), FLAG-Trpl (lane e, 20 µg),
FLAG-Trpl/Trp (lane f, 15 µg), or FLAG-Trp/Trpl (lane
g, 2 µg). Molecular weight standards are also shown (lanes
a and h). With the exception of Trpl, which was isolated
at 30 h, all membrane preparations were isolated from infected cells at
48 h postinfection. The protein bands seen in lanes b and c arise from nonspecific binding of the avidin-conjugated
horseradish peroxidase used to visualize the biotinylated molecular
weight standards. Panel C, effect of protein expression on
basal, non-stimulated
[Ca
]
. Basal
[Ca
]
in Sf9 cells
expressing the indicated protein was estimated using fura-2
fluorescence as described under ``Experimental Procedures.''
All cells were examined at 48 h postinfection, and the values represent
the mean ± S.E. (n = 6-10). Note that the
FLAG-Trpl/Trp construct was used for these experiments; all others were
native non-FLAGed proteins.
To monitor expression at the protein
level, the nucleotide sequence encoding the FLAG epitope (DYKDDDDK) was
attached to the NH terminus of each construct
(pVL-trp, pVL-trpl, pVL-trp/trpl,
and pVL-trpl/trp) using the following general procedure.
Oligonucleotides (Ransom Hill Bioscience, Ramona, CA) were synthesized
consisting of a methionine start codon, the FLAG sequence, and several
base pairs encoding the NH
terminus of either trp or trpl. A second set of oligonucleotides was synthesized
consisting of sequence within the coding region of trp and trpl that included a unique restriction site. These
oligonucleotides were used as primer sets for amplification by
polymerase chain reaction of the FLAG-attached NH
terminus
of trp and trpl. The polymerase chain reaction
products were subcloned into plasmid pCRII (Invitrogen) and
subsequently transferred to the pVL-trp and -trpl constructs using convenient restriction sites. All constructs were
sequenced to confirm that the nucleotides encoding the FLAG epitope
were attached and that the trp and trpl coding
sequence remained in frame with the new start codon.
Previous studies have suggested that Trp and Trpl differ in
their sensitivity to thapsigargin(8) . Comparison of the
primary structure of the two proteins demonstrates that the region of
greatest difference between Trp and Trpl occurs in the COOH-terminal
domain (Fig. 1A). To test the hypothesis that this
region is responsible for the differential sensitivity of these
channels to thapsigargin, chimeric constructs were created in which the
COOH-terminal domains of the two proteins were exchanged, and the
resulting proteins were expressed using the baculovirus expression
system. The predicted molecular mass for Trp and Trpl is 142.1 and
127.6 kDa, respectively(1, 10, 11) .
Recombinant Trp and Trpl expressed in Sf9 insect cells migrate on
SDS-polyacrylamide gel electrophoresis as 152- and 127-kDa proteins,
respectively (Fig. 1B, lanes d and e). Since a major difference between Trp and Trpl is the
length of the COOH-terminal domain (Fig. 1A), the
chimeras should have a molecular weight determined by the COOH-domain
present in each construct. As seen in Fig. 1B, the
Trpl/Trp and the Trp/Trpl chimeras have apparent molecular masses of
150 and 134 kDa, respectively (lanes f and g). The
predicted molecular masses based on the cDNAs are 142.3 and 128.0 kDa
for the Trpl/Trp and Trp/Trpl chimeras, respectively. Thus, all
constructs that contain Trp sequences migrate with an apparent
molecular weight that is greater than that predicted by the DNA
sequence. The reason for this discrepancy is unknown. It seems unlikely
to be related to expression in Sf9 cells since Trp expressed in Xenopus oocytes also migrates as a 150-kDa
protein(22) . Specific protein bands were not observed in
either non-infected Sf9 cells or in cells infected with recombinant
baculovirus containing the cDNA for the human bradykinin receptor (Fig. 1B, lanes b and c). Likewise,
no specific bands were observed on Western blots of membrane proteins
isolated from Sf9 cells infected with baculovirus containing cDNA for
native (non-FLAGed) proteins (data not shown). Estimates of relative
protein expression level determined from band intensities on a number
of gels were Trpl > Trp Trp/Trpl > Trpl/Trp.
Figure 2:
Effect of thapsigargin on whole cell
membrane currents in cells expressing Trp/Trpl and Trpl/Trp chimeric
proteins. Whole cell membrane currents were recorded in Sf9 cells
infected with recombinant baculovirus containing the cDNA for Trp/Trpl (left) or Trpl/Trp (right) chimeric proteins. Current
traces shown were recorded before (control) or after application of 200
nM thapsigargin. Voltage steps (400 ms) were applied at 2-s
intervals from a holding potential of 0 mV to potentials ranging from
-100 to +60 mV. Average current-voltage relationship in the
absence () or presence (
) of thapsigargin is shown below the
respective current traces. All values represent the mean ± S.E.
current amplitudes obtained at time 200 ms during each voltage pulse (n = 16 and 17 for Trp/Trpl and Trpl/Trp,
respectively). Dotted line in the current records indicates
the zero current level.
Figure 3:
Ionic selectivity of Trp/Trpl and Trpl/Trp
chimeric channels. Whole -cell membrane currents were recorded in
baculovirus-infected Sf9 cells as described in the legend to Fig. 2. Currents were recorded under basal non-stimulated
conditions for Trp/Trpl, whereas currents were recorded in the presence
of 200 nM thapsigargin for Trpl/Trp. The average
current-voltage relationship was first determined in each cell with 100
mM sodium gluconate in both the bath and pipette solution
(). The bath solution was then changed for one containing 50
mM calcium gluconate or barium gluconate (
) as indicated
in each panel. All values represent the mean ± S.E. current
amplitudes obtained at time 200 ms during each voltage pulse (n = 4).
Figure 4:
Blockade of inward current by
Gd. Whole cell membrane currents were recorded in
baculovirus-infected Sf9 cells as described in the legend to Fig. 2. Currents were first determined in each cell with 100
mM sodium gluconate in both the bath and pipette solution.
EGTA was omitted from the bath solution for this set of experiments.
Currents were recorded under basal, non-stimulated conditions for Trpl
(
) and Trp/Trpl (
), and in the presence of 200 nM thapsigargin for Trp (
) and Trpl/Trp (
) expressing
cells. The bath solution was then changed for one containing 100 mM sodium gluconate plus the indicated concentration of
Gd
. All values represent the mean ± S.E. (n = 4-5) current amplitudes taken at 200 ms
after a step change in membrane potential from 0 to -100 mV.
Currents in the presence of Gd
were normalized to the
control current amplitudes obtained in each cell before application of
Gd
. In all cells reported, currents returned to
control levels following washout of
Gd
.
Previous studies on the functional expression of Trp in Sf9
insect cells suggest that although Trp appears to be a Ca store-operated channel that can be activated by thapsigargin, the
ionic selectivity is different from both the endogenous I
recorded in the Sf9 insect cells (8) and I
observed in several mammalian cell
types(23, 24, 25, 26) ; I
appears to be highly selective for Ca
over
Na
and to be inwardly rectifying even in the presence
of symmetrical Na
solutions(25) . Thus, Trp
may not be the insect homologue of the I
channel. Trp
does, however, appear to be a member of a large protein family found in Drosophila, Calliphora, Xenopus,
mouse(22) , and human(27, 28) , although there
is no functional information on the Trp homologues from sources other
than insect. Petersen et al.(22) recently reported
that expression of Drosophila Trp in Xenopus oocytes
following injection of cRNA gives rise to an enhanced
thapsigargin-induced increase in Ca
influx estimated
by the magnitude of the Ca
-activated Cl
current, consistent with the activation of Trp by depletion of
the internal Ca
store. Thus, it seems likely that
although Trp may not be identical to I
channels, it may
be regulated in a fashion similar to I
. Understanding
the mechanism by which Trp is regulated by thapsigargin and
identification of the the structural domain of Trp necessary for this
regulation could provide important clues to structure and function of
mammalian I
channels. Toward this end, the purpose of
the present study was to determine the general region of Trp that is
necessary or sufficient for regulation of channel activity by
thapsigargin. To accomplish this goal, we exploited the differences in
sensitivity of Trp and Trpl to thapsigargin. With exception of amino
acid residues 330-500, Trp and Trpl are very similar over the
first two-thirds of the predicted amino acid sequence. We therefore
focused our attention on the COOH-terminal domain as the region that
gives rise to the differential sensitivity of these two channel
proteins to thapsigargin.
Exchanging the COOH-terminal domains of
Trp and Trpl produced several important functional changes. First, and
most importantly, thapsigargin sensitivity was conveyed to Trpl by the
COOH domain of Trp, and the presence of the Trpl COOH-terminal domain
on Trp eliminated thapsigargin sensitivity and increased constitutive
activity. These results provide strong evidence that the region of Trp
necessary for thapsigargin sensitivity resides in the COOH-terminal
domain. This altered sensitivity of the chimeras to thapsigargin
occurred without a change in the relative permeability of the channels
for Ca, Na
, or Ba
,
suggesting that pore characteristics are predominately determined by
the first two-thirds of the protein structure and probably by the
central hydrophobic core, which contains the proposed transmembrane
segments S1-S6. However, the Trp/Trpl channel had a lower
sensitivity to Gd
compared to native Trp channels,
and the Trpl/Trp chimera had a higher sensitivity to Gd
compared to native Trpl channels. Thus, it appears that the
COOH-terminal domains of Trp and Trpl may have subtle influences on
pore characteristics. One possibility is that the COOH-terminal domain
of Trp forms an extension of the pore structure into the cytoplasm
beyond the inner leaflet of the phospholipid membrane in a fashion
analogous to the nicotinic acetylcholine receptor, where a large
extracellular domain forms the vestibule of the channel, which extends
out from the membrane structure. Presumably this region would contain
the sequence responsible for binding Gd
.
Alternatively, the COOH-terminal domain may influence the conformation
of the pore region producing subtle alterations in the Gd
binding site. A similar phenomenon has been observed in the
inwardly rectifying K
channel, where the COOH-terminal
domain appears to have a major role in specifying pore
properties(29) .
The mechanism by which the COOH-terminal
domain affects constitutive activity is unknown. This does not reflect
variation in protein expression, since a difference in basal
[Ca]
is seen between Trp and
the Trp/Trpl chimera, yet both are expressed to approximately the same
levels in the Sf9 cell. Furthermore, the constitutive activity (or lack
thereof) of the chimeras is clear from the whole cell current
recordings; the COOH-terminal domain of Trp maintains the channel in a
non-conducting state. In a fashion analogous to other channel types,
part of COOH-terminal domain of Trp may act as the ``gate,''
which is closed in the absence of thapsigargin but opens in response to
depletion of the internal Ca
store.
The present
structure-function study may also provide insight into the mechanism by
which store depletion activates surface membrane channels like
I. In this regard, there are basically two hypotheses.
The conformational coupling hypothesis suggests that close association
between the endoplasmic reticulum and the plasmalemma allows for direct
physical coupling between the inositol 1,4,5-trisphosphate receptor and
Ca
entry channels and that information concerning the
repletion status of the internal store is related to surface membrane
channels via a conformational change (30) . Alternatively, a
soluble, diffusible messenger may be generated and/or released upon
depletion of the Ca
store, which then either directly
or indirectly activates the surface membrane
channels(31, 32) . The COOH-terminal domain of Trp
contains a unique proline-rich region in which the dipeptide KP is
repeated 27 times at relatively even intervals and includes a highly
charged segment where the sequence DKDKKP(G/A)D is repeated 9 times.
Interestingly, the bacterial protein TonB also has a proline-rich
region in which a string of EP and KP repeats is thought to form a
mechanical linkage between the inner and outer bacterial
membranes(33, 34) . By analogy, the highly charged
proline-rich segment of Trp may perform the same function linking
proteins of the endoplasmic reticulum to the Trp channel.
Alternatively, another region of the COOH-terminal domain of Trp may be
responsive to depletion of the Ca
store. In this
regard, the region may contain the binding site for CIF, the putative
Ca
influx factor(31) . Another possibility
derives from studies suggesting the involvement of tyrosine kinase
activity in Ca
entry activated by depletion of the
stores (35, 36, 37, 38) . In this
regard, Trp has 5 tyrosine residues in the COOH-terminal domain at
positions 665, 687, 745, 756, and 922. The first three tyrosine
residues are conserved in Trpl. It is possible that phosphorylation of
Tyr-756 or Tyr-922 may play a role in activation or regulation of Trp
by thapsigargin. Additional chimeric constructs in which different
regions of the COOH-terminal domains of Trp and Trpl are exchanged may
help determine the specific regions necessary for thapsigargin
sensitivity, for constitutive activity, and for determination of pore
characteristics.