Cloning of a Stretch-inhibitable Nonselective Cation Channel*

Makoto SuzukiDagger , Junichi Sato, Keiko Kutsuwada, Gaku Ooki, and Masashi Imai

From the Department of Pharmacology, Jichi Medical School, Minamikawachi, Tochigi 329-04, Japan

    ABSTRACT
Top
Abstract
Introduction
References

A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    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 = Sigma  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 Sigma  ntn/t, t = Sigma  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.

    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.

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.

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).

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.

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.

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.


    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.

    ACKNOWLEDGEMENTS

We thank Y. Oyama and H. Kuramochi for their excellent technical assistance and secretary work.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education and Culture of Japan, Sankyo Foundation, Takeda Foundation, and Naitoh Foundation.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) ABO15231.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Jichi Medical School, 3311-1, Yakushiji, Minamikawachi, Tochigi 329-04, Japan. Tel.: 81-285-58-7326; Fax: 81-285-44-5541; E-mail: macsuz{at}jichi.ac.jp.

    ABBREVIATIONS

The abbreviations used are: VR1, capsaicin receptor; SIC, stretch-inactivated channel; RT, reverse transcription; GFP, green fluorescence protein; CHO, Chinese hamster ovary.

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Abstract
Introduction
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