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INTRODUCTION |
The advent of patch clamp experiments has revealed the frequent
occurrence of ion channels, which were virtually not predicted by
earlier work on the macroscopic properties of membranes. Among the
various ion channels detected by patch clamp experiments, nonselective
cation channels are unique in that they are quite selective for cations
over anions but do not readily distinguish between the different
monovalent cations, especially Na+ and K+ ions
present in the physiological milieu. A number of regulatory mechanisms
have been reported for various channels. One class of nonselective
channel possesses the unique property of being activated by pressure or
stretch on the membrane and conducts Ca2+ as well as
Na+. The mechanosensitive nonselective channels have been
widely investigated and are considered to play important roles in the control of various cell functions, including cell volume regulation, smooth muscle contraction, and cardiac rhythm generation (2-6). There
are two groups of mechanosensitive nonselective channels: stretch
activated and stretch inactivated (4). Both show similar sensitivity to
a patch pipette pressure in the 10-50-mm Hg range and are inhibited by
µM concentrations of Gd3+. Even though
mechanosensitive nonselective channels pass only monovalent cations
under physiological conditions, activation of them elicits membrane
depolarization. Thus, mechanosensitive nonselective channels may
alter membrane potential or transport cations in response to cell
volume changes or stretching of the cell membrane.
Recently, the capsaicin receptor
(VR1)1 was isolated and shown
to possess the characteristics of a Ca2+-permeable
nonselective channel and sensitivity to the physical factor heat (1).
This report enticed us to clone other members of this novel group of
receptors for this channel to further investigate their properties. The
cloned channel was found to be sensitive to mechanical stimuli.
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MATERIALS AND METHODS |
Cloning of cDNA--
RNA was isolated by using the guanidine
thiocyanate method with organic extraction (Life Technologies).
mRNA was prepared by using a poly(A) column (Amersham Pharmacia
Biotech). The cDNA library was constructed using a kit (Marathon
cDNA construction, CLONTECH) with a minor
modification. To obtain homologous fragments, degenerative primer sets
were made for the amino acid alignments LFW(K)A(F)I(T)F(I) and
WKF(L)A(Q)R. Amplification conditions consisted of incubation at
94 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s for a total of 25 cycles. A fragment coding for a 108-amino acid protein, which was 93% homologous to VR1, was obtained. This was
then used as a probe in Northern blot hybridization under normal
conditions at 65 °C (7) using a ready-made membrane (Multiple Tissue
Northern blots, CLONTECH) under the protocol provided. Extensive wash was performed with 0.1 × SSC for 40 min at 42 °C.
Rapid amplification of cDNA ends was performed with an appropriate
primer set at 94 °C for 30 s and 68 °C for 4 min for a total
of 25 cycles using proof reading taq polymerase (Takara Extaq). Nested
primers were constructed for the 5'-rapid amplification of cDNA
ends protocol. A total of two fragments consisted of
stretch-inactivated channel (SIC) cDNA. The primers for the protein
coding region were then constructed and a SIC cDNA of ~2.3 kb was
recloned from the cDNA library. The cloned cDNA was ligated to
the TA cloning vector (Invitrogen), and the
Not-Sal fragment was ligated to the mammalian
expression vector pCMV-SPORT (Life Technologies). Both strands of the
SIC cDNA were sequenced by an automatic sequencer (373-S, Applied
Bio Instrument) with a Thermo Sequenase dye terminator cycle sequencing
premix. The sequence has been submitted to the GenBank (accession
number ABO15231).
Reverse Transcription (RT) and Polymerase Chain
Reaction--
Total RNA was prepared from tissues by using Trizol
(Life Technologies). RT was accomplished with recombinant avian
myelobastosis virus transcriptase (RT-AMV, Takara). Total RNA (1 µg)
was dissolved in 20 µl of buffer containing 5 mM
MgCl2, 1 mM dNTP, 2.5 µM random 9 mers, 1 unit/µl RNase inhibitor and 0.25 unit/µl recombinant avian
myelobastosis virus transcriptase. Negative controls were performed
without both RNase inhibitor and recombinant avian myelobastosis virus
transcriptase. The mixture was incubated for 30 min at 42 °C and
then was heated at 94 °C for 5 min and cooled down to 4 °C. A
part of the sample (1 µl) was added to 8 µl of buffer containing 2 mM MgCl2 and 0.25 mM dNTP.
Amplification conditions consisted of incubation at 94 °C for
30 s, 53 °C for 30 s, and 72 °C for 30 s for a
total of 25 cycles with primer sets (5'-GAAGGCCTTCCTCAGGAACA-3' and
5'-CATCAGAGACCTGTGCCGGTTT-3'). The expected amplified fragment was 420 bp.
Expression of cDNA--
A plasmid expressing green
fluorescence protein (pEGFP-N1, CLONTECH) was used
as a marker for transfection. One µg of SIC/pEGFP-N1 (2:1) was
transfected into Chinese Hamster ovary (CHO) cells grown on coverslips
using FuGENE6 (Boehringer Mannheim) that could allow cell incubation
with serum for better viability of transfected cells. For transient
transfection, CHO cells were kept in nutrient mixture Ham's F-12 (Life
Technologies) supplemented with 10% fetal bovine serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin. The day before transfection,
we seeded 1 × 105 CHO cells/35-mm culture dish on
cover glass coated by rat tail collagen. The next day, to a mixture of
2 µl of FuGENE6 and 98 µl of serum-free medium incubated at room
temperature for 5 min were then added 1 µg of plasmid SIC in
pCMV-SPORT and 500 ng of pEGFP-N1 per one transfection. Incubated at
room temperature for 15 min, lipid-DNA mixture was added to a dish
filled with 2 ml of serum-containing medium for CHO cells. Growth and
transfection were performed in 10% fetal calf serum-containing media.
The cells grown on the coverslip were used without any further
treatment for patch clamp samples.
Electrophysiology--
GFP-positive cells were visualized by
fluorescence measurement (CAM 2000 system, Jasco, Tokyo, Japan) with an
emission of 490 nm. Patch clamp recordings were carried out according
to the methods described in previous papers (8, 9). The cells were set
on a mount under a vectorial flow of solution. The solution was heated
by using DC supply (model LPD, Kikusui Electronics, Tokyo, Japan)
before the mount to regulate the temperature. Currents were recorded at
34 °C with an EPC-7 patch clamp amplifier (List Electronics). Axon
protocol version 5.5 was used to control the applied voltage and to
record the current in whole cell configuration (Clampex). The bath
solution contained (in mM) 125 NaCl, 25 NaHCO3, 5 KCl, 1.2 MgSO4, 1 Na2HPO4, 1 CaCl2, and 3 HEPES and resulted in an osmotic pressure of
310 mosm. A hypertonic solution of 350 mosm was made by adding sucrose,
and a hypotonic solution of 220 mosm was made by adding water. The
whole cell patch pipette contained a filtered solution of (in
mM) 150 K gluconate, 3 HEPES, 1 EGTA, and 20 NaCl and 100 µg/ml nystatin (pH 7.2 by NaOH).
Single channel analysis was performed. Currents were recorded with an
EPC-7 patch clamp amplifier and stored on a digital audio tape recorder
(DAT-200, Sony, Tokyo, Japan). To show the traces in long time course,
they were filtered with a low pass filter at 1 KHz to represent the
data in a pen recorder (RGJ4122, Nihon, Koden, Japan). To analyze the
probability of opening, records were sampled by software (Fetchex, Axon
version 6.0) at 10 KHz, and a decade filter was not used. The data were
transferred to another computer and analyzed by using Igor 2.01 and
Patch Analysist Pro version 1.21. The open probability was calculated
with Gaussian currents distribution analysis. Mean open probability
(NPo) was determined by 30 s recordings as:
NPo =
ntn, where N is
the number of functional channels in the patch,
Po is the single channel open probability,
n represents the state of the channel (0 = closes, 1 = one open channel, and so on), and tn is the length
of time in state n. N is the mean number of open
channels, calculated as
ntn/t,
t =
tn.
In inside-out patches, bath solution contained 3 mM HEPES
(pH 7.5) and 140 mM NaCl, KCl, or K gluconate with 140 mM NaCl in the pipette to elucidate single channel
conductance. To obtain PK/PCl/PNa,
mixtures of NaCl and KCl or K gluconate (in mM) 100:40, 70:70, and 40:100 were used for a bath solution. Selectivity as indicated by the value PCa/PNa was calculated
(10) by measurements of the reversal potential in exchanges of bath
solution from 100 to 10 mM CaCl2 through 50 mM CaCl2 with 140 mM NaCl in a
pipette. For measurements of Ca permeability for attached patches, the bath contained 140 mM NaCl, 1 mM
CaCl2, and 3 mM HEPES with 100 mM
CaCl2 in the pipette.
In cell-attached patches to investigate mechanosensitivity, a pipette
was filled with 140 mM NaCl and 3 mM HEPES. The
membrane potential of the cell was not corrected in the holding
potential. Pipette pressure was regulated by attaching an air
connection with a manometer, and the negative pressures were altered
manually. We carefully adjusted the pressure within ±2 mm Hg of that desired.
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RESULTS |
Isolation of SIC--
Fig.
1A shows a Northern blot
obtained using the VR1 homologous probe. A band with a size of ~2.3
kb was stained positive in the kidney and liver. Using the rapid
amplification of cDNA ends procedure, we successfully isolated the
homologue from the constructed kidney cDNA library (SIC). An
alignment of the SIC amino sequence is shown in Fig. 1B. SIC
starts at the 308th amino acid of VR1 and has different C-terminal and
5' untranslated regions. The SIC cDNA encodes a 563-amino acid
protein in which the six transmembrane segments and pore common to
VR1 are preserved. A polymerase chain reaction on the DNA fragment
coding for the C-terminal region by using primers
(5'-TCAGAGACCTGTGCCGGTTT-3' and 5'-TTATTTCTCCCCTGGGACCA-3') indicated
that a 4-kb intron exists between a VR1 and SIC common region and a
SIC-specific alignment (data not shown). The SIC N-terminal region
contains one ankyrin repeat domain, whereas VR1 has three. Thus, SIC is
an alternative splice variant, lacking a part of the N-terminal region
of VR1, with a different C-terminal region compared with VR1 amino acid
alignments. To elucidate further the tissue localization, RT-polymerase
chain reaction was performed using several tissues. The expected
amplification was obtained in liver and kidney.

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Fig. 1.
Tissue distribution and amino acid alignment
of SIC. A, distribution of SIC mRNA in tissues. Northern
blot analysis of the expression of SIC mRNA was performed. Two µg
of RNA were charged per lane. The migration positions of the RNA size
markers are indicated on the left. A major RNA band of
~2.3 kb corresponding to that of SIC is indicated by an
arrow. B, Amino acid alignment of SIC. The SIC
protein is identical to VR1 from the 308th methionine to the 687th
valine and contains one ankyrin repeat, six transmembrane segments, and
a region susceptible of forming a pore. Italic letters
denote SIC amino acids. The degenerative primer set used is
underlined. C, upper panel, extensive wash was
performed on the Northern blot membrane. The arrow indicates
2.3 kb. Lower panel, RT-polymerase chain reaction was
performed. RT( ), results without RT reaction using renal
RNA. The arrow indicates a predicted amplification of 420 bp.
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Expression of SIC in Whole Cellular Currents--
SIC cDNA was
ligated to an expression vector and transfected into mammalian CHO
cells. The GFP expression vector was cotransfected to identify single
cells expressing the construct. When SIC was successfully expressed, as
indicated by the green color of the cells, most of the cells appeared
round. This is frequently observed for cells expressing high Ca
permeability (1) or nonselective currents (11), indicating perhaps
damage to the cells. We usually performed electrophysiological
measurements within the 24-h period after transfection to avoid any
damage caused by overexpression. To normalize the currents, round cells
of 9-15 µm in diameter were selected as a patch clamp specimen (10).
Fig. 2 shows the representative traces of
the SIC-induced current evoked by voltage steps using tight seal whole
cell patches. Unlike the VR1-induced current, SIC did not require any
vanilloids for the expression of a large current. SIC induced
long-lasting currents in the 1-10-nA range after
100 to +80 mV
stimuli, whereas GFP alone induced a <0.2-nA current. The reversal
potential was approximately +20 mV. The current-voltage curve was
outwardly rectified, and time-dependent fluctuation was not
observed in negative voltage, although time-dependent increments during 1-s pulses were observed at more positive voltages. Alteration of cell volume in response to exposure to solutions with
varying osmolarities was confirmed by visual inspection under a
microscope, and then the currents were measured. The characteristics of
the outward rectification were preserved, but the amplitude was
apparently altered. The amplitudes of the currents were increased by
hypertonicity but were decreased by hypotonicity.

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Fig. 2.
Whole cellular currents of SIC. Upper
panel, whole cellular currents of 1-s duration with voltage
increments of 10 mV in the range of 100 to +90 mV were recorded in
steady state (left), in hypertonic solution
(center), and in hypotonic solution (right).
Recording was obtained in a series of exchanges of solution and started
~10 s after exchanging the solution with an identity of a cell volume
change. Lower panel, representative current-voltage
relations of the SIC currents in a series of experiments.
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A summary of the changes in conductance is shown in Fig.
3. Conductances were expressed by the
magnitude of the current observed between
100 and 0 mV. The
conductance was significantly increased by hypertonicity, whereas it
was significantly decreased by hypotonicity. Addition of 100 µM GdCl3 at the end of a series of
experiments led to a further drop in conductance but did not lead to
the level of conductance of the unexpressed control. The temperature of the bath solution was maintained at 34 °C. Because VR1 is sensitive to heat, the bath was heated to 47 °C. Although this induced an increment in the conductance, the cells were ruptured as a result of
this treatment. Hence, we did not evaluate whether the SIC channel is
still sensitive to heat.

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Fig. 3.
Conductance of SIC in response to cell volume
changes and GdCl3. The experiment was designed to
allow a series of exchanges of solution ranging from isohypertonic
(Iso Hyper) to isohypotonic (Iso Hypo).
GdCl3 (Gd) was added at 100 µM to
the most isohypotonic solution. The conductance (current-voltage slope
in 100 to 0 mV) of this series are plotted with the individual.
Significance: **, p < 0.001 was obtained from
comparison by exchange into an anisotonic solution (paired t
test).
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Expression of SIC in Single Channel Analysis--
Single channel
analysis was performed (12). A channel with a large unit conductance
was observed when the pipette was attached to the membrane. Conductance
was measured with isolated patches in equivalent NaCl/NaCl
(Na/gluconate) solutions (Fig. 4), and the current distribution, which was fitted well by Gaussian analysis, gave a value of a single channel current. Current-voltage relation at
~0 mV gave the conductance of 250 pS (n = 6). The
channel had complex kinetics with a mixture of rapid bursting and long
opening, which was observed in the recording with a long time scale. An exchange of the bath NaCl solution with an NaCl/KCl or KCl/K gluconate mixture revealed PNa/PK/PCl
selectivity to be 1:0.3:0.08. For example, reversal potentials of 140 mM NaCl in pipette to 140:0, 100:40, 70:70, and 40:100,
NaCl/KCl in bath solutions were 30, 17.3, 10.8, and 5.6 mV,
respectively. The results indicated permeability ratio of K over Na as
0.3. Addition of GdCl3 to a final concentration of 20 µM inhibited long opening and resulted in rapid bursting of the channel, which could be described as "flickery." After washing out of the reagents, the normal characteristics of channel opening were recovered.

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Fig. 4.
Single channel of SIC in NaCl. Traces of
the single channel of SIC in symmetrical NaCl are shown at the
left. The bar indicates each current gap with the
state of the channel (0 = close, 1 = one open, 2 = two
opens, and so on). Voltage is expressed as membrane voltage (negative
values of the holding voltage). The bottom trace was
obtained in a bath with 20 µM GdCl3 at +30
mV. The trace at 20 mV with a long time scale is shown in a
box. The relationship of single channel amplitude to
membrane voltage obtained from the mean value of four observations is
shown at the right.
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The Ca permeability of the single SIC channel was examined.
Cell-attached patches with 100 mM CaCl2 as the
pipette solution were constructed to measure the slope of the
conductance around the holding potential of 0 mV. Fig.
5 shows the traces and mean amplitudes at
various voltages. In contrast to that observed with monovalent cations,
the single channel was composed mainly of rapid bursting without any
durable opening. The single channel conductance obtained by the slope
in current-voltage relation at ~0 mV was 40 pS (n = 4). PCa/PNa was measured by exchanging the bath
solution after making an isolated membrane; reversal potentials of 140 mM NaCl in pipette to 10, 50, and 100 CaCl2 in
bath solutions were 29, 13.2, and 7 mV, respectively, giving a
permeability ratio of 0.24. This is a quite low compared with the value
of 9.6 for VR1.

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Fig. 5.
Single Ca2+ channel of SIC.
Traces of the SIC single channel were obtained in the attached cell
configuration with CaCl2 in the pipette (left
panel). This is only one representative free from contamination of
Na current. Voltage is expressed as the holding voltage. The trace at
20 mV with a long time scale is shown in a box. The
relationship of single channel amplitude to the holding voltage is
shown in the right panel.
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To investigate the relationship between tension and channel
opening, negative pressure was applied through a cell-attached pipette
filled with 150 mM NaCl. Fig.
6 shows representative traces and the
results of an analysis using four patches at the holding potential of 0 mV. The resting membrane potential was not considered. The chances of
channel opening and the number of open channels were diminished in a
dose-dependent manner by applying a series of negative
pressures and were all but abolished at pressures less than
40 mm Hg.
After normalization of the pressure, channel opening partially
recovered, although this took >1 min. These data directly suggest that
the channel belongs to a class of stretch-sensitive channels.

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Fig. 6.
Effect of stretch on the channel
opening. A continuous trace of the SIC single channel from a
cell-attached patch with graded suction was obtained. The data used for
calculation of Po are shown in the upper
panel. The settings are described under "Materials and
Methods." Pressure applied is indicated above the trace. The
lower panel shows the mean ± S.E. of the calculated
Po plotted against the pressure.
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DISCUSSION |
The present study was designed to clone a mechanosensitive
nonselective cation channel. The SIC encoding channel is cell volume sensitive and belongs to a group of stretch-inactivated nonselective channels. However, its electrophysiological parameter, conductance, does not match those of previously reported ones, which were found in
skeletal myotubes, smooth muscle, and neuronal cells (12-16). On the
other hand, in rabbit renal connecting tubules, the luminal membrane
possesses a 170-pS Na+- and Ca2+-permeable
channel (16), but it is stretch activated. A stretch-inactivated Ca2+ conducting channel has been reported in skeletal
myotubes (12). The channel is more active in myotonic model animals, in
which it causes Ca overflow leading to dystrophy (12). This channel is
blocked by Gd3+, but the 8-pS conductance of Ca is smaller
than that of SIC. Because the single channel conductance of SIC is
large, even the expression of a small number of channels per cell would
be sufficient to conduct a current.
The same characteristics have been observed during the blocking of
stretch-activated as well as stretch-inactivated nonselective channels
(3, 12). As reported earlier, stretch activated nonselective channels
are quite sensitive to Gd3+, in which concentrations of
5-10 µM are enough to block channel opening completely
(3), whereas Gd3+ at the same dosage neither blocks
completely nor reduces the current amplitude in stretch-inactivated
channels (13). Thus, stretch-inactivated channels may be generally less
sensitive to Gd3+. SIC currents were not completely
abolished in the whole cell configuration (Fig. 3). The SIC channel was
less sensitive to Gd3+, inasmuch as 20 µM
Gd3+ was not sufficient to completely block the currents,
although the flickering blockade was similar. On the other hand,
response to tension was similar to that of the stretch-activated
channel, in which SIC was influenced by
40 mm Hg. Therefore, the
present SIC is actually a member of the stretch-inactivated group of
channels that display the same sensitivities to stretch and similar
sensitivities to Gd3+ but dissimilar conductance to those
observed by previous techniques.
There may be a quantitative discrepancy between the effect of cell
volume change and of membrane stretch. Based on an assumption (17),
60% cell volume change corresponds to the membrane tension of 6 cm of
H2O, ~0.5 mm Hg. We changed the osmolarity from 310 to
220 mosm, corresponding to 30% cell volume difference. It indicated 0.25 mm Hg of suction, not reaching an inhibitory effect by the single
channel study.
A physiological role for SIC in the kidney may be considered.
Glomerular blood flow and intratubular urinary flow exhibit oscillatory
changes. Therefore, the glomerular or tubular cells are usually exposed
to mechanical stress (18). Studies with the cell volume-sensitive SIC
channel may reveal the existence of a variety of channels with similar
molecular structures that function in response to these types of
mechanical stress.
SIC is a variant of VR1 and shares the same transmembrane and pore
alignments with different electrophysiological properties. Conductance
could be different even though possessing the same transmembrane and
pore alignments. One example is the BKCa channel family. This family
has many spliced variants that display only differences in their C
tailing regions. Although the transmembrane segments and pore region
are identical, the electrophysiological characteristics of these
variants are different (19, 20). In fact, we observed longer positive
staining in skeletal muscle than in kidney and shorter staining in the
kidney and testis using Northern blotting, suggesting that still other
variants may exist with different electrophysiological characteristics.
Selectivity to Ca2+ was also different between VR1 and SIC.
SIC-expressed cells became permeable to bath or medium
Ca2+, whereas VR1 expressed cell did not. The
Ca2+ moiety around the channels before the experiments was
therefore different, which might lead to a difference of Ca/Na
permeability in these channels.