Shinshu Med J, 62⑴ :33〜44, 2014

TRPM4 Channels Mediate Hypertonicity‑induced, Ca ‑impermeable,
Non‑selective Cation Currents in a Cervical Cancer Cell Line, HeLa Cells
Simmon GOMI , Tsutomu NAKADA , Toshihide KASHIHARA
Uichi IKEDA and Mitsuhiko YAMADA ＊1) Department of Molecular Pharmacology, Shinshu University School of Medicine
2) Department of Cardiovascular Medicine, Shinshu University School of Medicine

When extracellular osmolarity exceeds intracellular osmolarity, cells initially shrink but then approach
the original cell volume by so‑called regulatory cell volume increase (RVI).RVI operates under physiologicalconditions so that impairment of RVI leads to immediate cell cycle arrest and apoptosis.In a cervical cancer
cell line, HeLa cells, extracellular hypertonicity induced non‑selective cation currents (Icurrents) whichtransported mainly Na into cells to induce RVI as assessed with the patch‑clamp method. Ion channels
mediating these currents were Ca ‑impermeable and sensitive to ﬂufenamic acid (FFA)and econazole but not
to amiloride or ruthenium red.RT‑PCR indicated that HeLa cells express transient receptor potential (TRP)
C1, C6, M 3, M4, M7, M 8, V1 and V2 subunits which form non‑selective cation channels. From these results,
we speculated that TRPM4 may mediate I
subunit signiﬁcantly inhibited I

currents. Indeed, transduction of a dominant‑negative TRPM4

currents. TRPM4 channels became insensitive to hypertonic stimulus when

intracellular Ca was strongly buﬀered.Thus,extracellular hypertonicity activates TRPM4 channels throughintracellular Ca

to induce RVI in HeLa cells.These results indicate that intra‑uterus or ‑vaginal applicationof drugs blocking TRPM 4 channels may cause antiproliferative/proapoptotic eﬀects on cervical cancer.
Shinshu Med J 62 : 33 ―44, 2014
(Received for publication August 7, 2013;accepted in revised form September 10, 2013)
Key words:transient receptor potential M4, regulatory cell volume increase, HeLa cells

Introduction

Because cell membranes are usually highly permeable to water due to the abundance of water

Cells have to avoid excessive alteration of cellchannels, aquaporins , water movement acrossvolume, which jeopardizes structural integrity andthese membranes is in large part dictated by os-

the constancy of the intracellular milieu. For

motic pressure gradients. Thus, in RVI, volume

instance, acute elevation of NaCl or urea produces

regulatory ion transporters accumulate ions in cells.

rapid arrest in all phases of the cell cycle and

The major ion transport systems involved in RVI

apoptosis .Therefore,cells continuouslyexposed to

are the Na , K , and 2Cl co‑transporters and thehypertonic extracellular ﬂuid initially shrink but

Na /H exchangers.In some cells,electrolyte accu-

then approach the original cell volume by so‑calledmulation during RVI is accomplished by activation

regulatory cell volume increase (RVI) .

of Na channels and/or non‑selective cation channels, which are much more eﬃcient than ion tran-

Corresponding author:Mitsuhiko Yamada
Department of Molecular Pharmacology,Shinshu University School of M edicine 3‑1‑1 Asahi, M atsumoto,sporters in mediating RVI and cause depolarizing
inward currents at usual cellular resting membrane

Nagano 390‑8621 Japanpotentials.The depolarization induced byNa entry

E‑mail:myamada＠shinshu‑u.ac.jp

favors Cl entry into the cell;as a result, intracel-

No. 1, 201433

Gomi.Nakada.Kashihara et al.

lular NaCl concentration and, thus, osmolarity areotherwise indicated. Modiﬁed Tyrode solution

increased.

contained (in mM):NaCl,136.5;KCl,5.4;CaCl ,1.

In HeLa cells, a cervical cancer cell line, hyper-

8;MgCl , 0.53;HEPES, 10 (Dojindo Laboratories,

tonic extracellular solution induces ﬂufenamic acid

Kumamoto,Japan);and glucose,10 (pH adjusted to(FFA) ‑sensitive, Ca ‑impermeable, non‑selective

7.4 with NaOH). Na‑bathing solution contained (incation currents (Icurrents). The major class ofmM): NaCl, 94; Na‑gluconate, 6 (Sigma‑Aldrichmolecules known to form non‑selective cation chan-Inc., St. Louis, MO, USA); MgCl , 1; CaCl , 1;

nels is “transient receptor potential”(TRP) chan-tetraethyl ammonium (TEA)‑Cl,2;and HEPES,10

nels . They are either homo‑ or hetero‑tetramers(pH adjusted to 7.5 with NaOH). NMDG‑bathingof putative six ‑transmembrane polypeptide

solution contained (in mM):NM DG,103;gluconate,

subunits. The TRP superfamily comprises six

6;TEA‑Cl, 2;and HEPES, 10 (pH adjusted to 7.5

related proteins: TRPC (canonical), TRPV (val-with HCl). Ca‑bathing solution contained (in mM):linoid), TRPM (melastatin), TRPP (polycystin),

CaCl ,69 ;gluconate,6;TEA‑Cl,2;and HEPES,10

TRPM L (mucolipin)and TRPA (ankyrin).

(pH adjusted to 7.5 with Ca(OH) ). Mannitol was

In this study,we sought to identify the molecules

added to the bathing solutions to make their os-

channels in HeLa cells. Quite recently,

molality 340 (isotonic solution)or 450 mOsmol/kg‑

Numata et al. reported that a splicing variant of

H O (hypertonic solution).Pipette solution contained

TRPM 2 that lacks part of an NUDT9 ‑H domain in(in mM): NaCl, 26; Na‑gluconate, 69 ; MgCl , 1;

the cytosolic C‑terminus forms I

channels in

TEA‑Cl,2;EGTA,1;HEPES,10;and mannitol,90

HeLa cells. Unexpectedly, however, we could not(pH adjusted to 7.3 with NaOH ;300 mOsmol/kg‑

detect the expression of TRPM 2 transcripts includ-

H O).When indicated,EGTA in the pipette solution

ing the splice variant in HeLa cells. Instead, we

was replaced with 10 mM BAPTA (Sigma‑Aldrichfound that HeLa cells abundantly expressed

Inc., MO, USA). Flufenamic acid (FFA) (Sigma‑

TRPM 4, which is well known to form FFA‑sensi-

Aldrich Inc., M O, USA), ruthenium red (RR), andtive, Ca ‑impermeable, non‑selective cation chan-econazole (ECO) were dissolved in 100% DMSO

nels ,and that transduction of the dominant nega-

at 100 mM. U73122 and U73343 were dissolved intive form of TRPM4 subunits almost completely

100% DMSO at 2 mM.These stock solutions were

suppressed I

diluted on use to the indicated concentration withforming I

currents.

Inhibition of I

currents and RVI with FFA

bathing solution. The ﬁnal

0.1% DMSO did notsensitizes HeLa cells to shrinkage ‑induced

signiﬁcantly aﬀect membrane currents of HeLa

apoptosis . FFA is a clinically used non‑steroidal

cells. Amiloride (AM L) (Sigma‑Aldrich Inc., M O,

anti‑inﬂammatory drug with an established safety

USA) was dissolved in distilled water at 10 mM

record.Therefore,a clinically important possibility

and diluted to the indicated concentration withis that intra‑uterus or‑vaginal application of FFA

bathing solution on use.

may cause antiproliferative/proapoptotic eﬀects on
cervical cancer. Identiﬁcation of the molecules forming I

channels in this study may lead to develop-

Cell culture
HeLa cells were purchased from ATCC (Manassas,VA,USA)and cultured according to the manu-

ment of more selective and potent inhibitors of I

facturerʼs instructions as previously described .

channels in cervical cancer than FFA.

Brieﬂy,theywere grown as monolayers in minimum

M aterials and M ethods
Solutions and chemicals

essential medium (MEM) supplemented with 10%
fetal bovine serum, 40 IU/ml penicillin, and 100 g/
ml streptomycin at 37 ℃ in a 95% air‑5% CO

Chemicals were purchased from Wako Pureatmosphere. For sub‑culturing, phosphate buﬀered

Chemical Industries, Ltd. (Osaka, Japan) unlesssaline containing 0.25% trypsin and 0.02% EDTA

34

Shinshu Med J Vol.62

TRPM4 channels in HeLa cells

was used to detach cells from the plastic substratum

capacitance,which represents the cell surface area.

of the culture ﬂasks.For the experiments,cells were

When indicated, rectangular voltage pulses for 500

resuspended by mechanical detachment and thenmsec to potentials between ‑100 and ＋60 mV in a 10

stored in gently stirred M EM for 15‑300 min at

mV increment were applied from ‑60 mV every 3

37 ℃ in an incubator before use.

sec.

Electrophysiology

Molecular biology

Aliquots of the suspended HeLa cells were trans-

Total RNA of HeLa cells was isolated withferred to the organ chamber on an inverted micro-

Isogen (Nippon Gene, Tokyo, Japan) according toscope (Axiovert 135 TV ; Carl Zeiss, Oberkochen,

the manufacturerʼ
s instructions.The obtained RNA

Germany)ﬁlled with modiﬁed Tyrode solution.The

was dissolved in diethyl pyrocarbonate‑treatedcurrent of isolated HeLa Cells was measured in thewater, and its concentration was measured spectro

whole‑cell conﬁguration of the patch‑clamp tech-photometricallyat 260 nm.For cDNA synthesis,5μg

nique at 35‑37 ℃ with a patch‑clamp ampliﬁer

total RNA was reverse‑transcribed with oligo(dT)(Axopatch 200B ; Molecular Devices Corp., Sun-

20 primer and SuperScript III First‑Strand Synthe-

nyvale, CA, USA) as previously described . Patchsis System for RT‑PCR (Invitrogen, Carlsbad, CA,pipettes were fabricated from borosilicate glassUSA)according to the manufacturerʼ
s instructions.

capillaries (Kimax‑51;Kimble Glass Inc.,Vineland,

To check the expression of TRP channels in HeLa

NJ, USA). Series resistance was always kept ＜7cells, PCR was performed with the above cDNA,

MΩand was routinelycompensated by〜75% using

GoTaq M aster M ix (Promega, Madison, WI, USA)an ampliﬁer.

and the previously described oligonucleotide

To measure membrane currents, the whole‑cellprimers . Among them, a pair of primers for

conﬁguration was formed in the modiﬁed Tyrode

TRPM2 was designed to detect the part of TRPM2

solution with the indicated pipette solution. Bytranscripts which was common in the wild‑typecontinuously applying triple pulses to ‑100, ‑40

TRPM2 and the splice variant of TRPM2 recently

and ＋10 mV (300 msec duration for each pulse)

identiﬁed by Numata et al. . The PCR condition

from the holding potential of ‑60 mV every 3 sec,

was as follows:30 cycles of denaturation (94 ℃,15

the external solution was switched to isotonic Na‑sec),annealing (55 ℃,30 sec)and extension (72 ℃,1bathing solution. Several minutes after the ampli-

min).

tude of membrane currents had settled at a stable

Dominant negative TRPM4 construct (deletion ofvalue, the membrane potential was clamped at ‑60

the ﬁrst 177 amino acids in the N terminus) was

mV,and a voltage ramp pulse from ‑100 to ＋60 mV

generated by PCR as previously described . The

for 1 sec was applied. Then, the external solution

cDNA coding for human TRPM 4b (DDBJ/Gen-

was switched to hypertonic Na‑bathing solution.

bank/EMBL accession number: AF497623) was

After the inward membrane currents reached thekindlyprovided byProf.Veit Flockerzi(Universitat

steady‑state value, the ramp voltage pulse wasdes Saarlandes, Homburg, Germany).

applied. When indicated, Na‑bathing solution was

Statistics

changed to NMDG‑ or Ca‑bathing solution. At theData are shown as the means ± S.E.M .Statisticalend of experiments, the external solution was swit-

signiﬁcance was evaluated with Studentʼ
s paired orched to hypertonic Na‑bathing solution containing

unpaired t‑test. For multiple comparison of data,

100 mM FFA, followed by the ramp pulse. To

analysis of variance with Bonferroniʼs test was

obtain the current‑voltage relationship,the currents

used. p＜0.05 was considered signiﬁcant.

during the ramp pulse were plotted against themembrane potential. The amplitudes of membranecurrents were normalized to cell membrane
No. 1, 201435

Gomi.Nakada.Kashihara et al.

inset). As shown in the left‑hand panel, there were

Results

small, time‑independent, and slightly outwardly

Basic features of membrane currents induced

rectiﬁed currents under isotonic conditions. The

byhypertonic Na‑bathing solution in HeLa cells

middle panel shows that hypertonic Na‑bathing

Fig. 1Aa shows membrane currents induced bysolution induced large,time‑dependent,and slightly

hypertonic Na‑bathing solution in HeLa cells (I ).

outwardly rectiﬁed currents. Diﬀerence currents

In the conventional whole‑cell conﬁguration of the

between the two conditions (i.e. I

patch‑clamp method, an isolated HeLa cell wastime‑dependent activation especially at depolarized

voltage clamped at ‑60 mV in isotonic Na‑bathing

potentials and slight inactivation and large channel

solution. Under this condition, 〜‑50 pA of steadynoises at hyperpolarized potentials. Upon repolar-

inward current was observed. After the external

ization to ‑60 mV from depolarized potentials,

solution was switched to hypertonic Na‑bathingsmall inward tail currents were induced, consistentsolution, inward currents gradually increased with

with the voltage‑dependent kinetics of I

increasing channel noise and reached a steady valueof 〜‑750 pA. I

currents) showchannels.

Ion permeability of ion channels activated bycurrents were almost fully inhib-

hypertonic solution in HeLa cells

ited by FFA, an inhibitor of non‑selective cation

Fig. 2A shows the results of experiments similar

channels. Fig. 1Ab shows the mean and SE of

to those shown in Fig. 1;however, Na in bathing

inward current density under isotonic and hyper-

solutions was replaced with NMDG, a large

tonic conditions,indicating that the hypertonic Na‑

monovalent cation impermeable through channels.

bathing solution signiﬁcantly induced large I

cur-As shown in Fig. 2Aa, substitution of Na withrents in HeLa cells.At the timings indicated by the

NMDG strongly inhibited inward currents under

numbers in parentheses in Fig. 1Aa,a voltage ramp

both isotonic and hypertonic conditions. Figs. 2Ab

pulse from ‑100 to ＋60 mV was applied. Fig. 1Ac

and Ac show the I‑V relationships obtained at theshows the relationship between membrane current

timings indicated by numbers in parentheses in Fig.

density and membrane voltage (IV relationship).

2Aa.Fig.2Ab shows that the two curves once again

Under isotonic conditions, small and slightly out-

crossed at 〜0 mV in the presence of external Na ,wardly rectiﬁed currents were observed (1)whereas

whereas Fig. 2Ac shows that the two curves did notlarge quasi‑linear currents were seen under hyper-

cross at any membrane potentials in the presence of

tonic conditions (2).These two curves crossed at 〜0

external NMDG. The diﬀerent currents shown in

mV. Under the present condition, the equilibration

Fig. 2Ad indicate that replacement of Na withpotential of Na and Cl‑ was ＋1.4 and ‑32.7 mV,

NMDG completely abolished inward but not out-

indicating that the membrane currents of HeLa

ward currents, suggesting that I

cells induced by hypertonic Na‑bathing solution

mediated by Na .

were mainly carried by Na , not Cl‑ ions. In thecurrents were

In Fig. 2B,external Na was replaced with Ca .

presence of FFA,the hypertonicity‑induced current

External Ca

was completely inhibited. Consistent with this,rents,indicating that I

diﬀerence currents between (1) and (2) or between

impermeable.Taken together,these results indicate(2) and (3) were superimposable and show that I

that I channels areCa ‑impermeable,non‑selective

currents have quasi‑linear conductance (Fig. 1Ad).

monovalent cation channels.

To analyze the voltage‑dependent kinetics of I

completely suppressed inward curchannels are virtuallyCa ‑Pharmacological proﬁle of ion channels

currents,rectangular voltage pulses for 500 msec toactivated by hypertonic solution in HeLa cells

potentials between ‑100 and ＋60 mV in a 10 mV

In some cells, AM L‑sensitive Na channels are

increment were applied from ‑60 mV every 3sec

activated in RVI . However, AML did not inhibit

under isotonic and hypertonic conditions (Fig. 1B,I

36channels in HeLa cells (Fig. 3A, D).It was also
Shinshu Med J Vol.62

TRPM4 channels in HeLa cells

Fig. 1 M embrane currents induced by hypertonic Na‑bathing solution in HeLa cells.
Ａ. a. In the conventional whole‑cell conﬁguration of the patch‑clamp method, membrane currents were
continuously measured at ‑60 mV. As indicated by the bars over the current trace, a HeLa cell was
perfused with isotonic Na‑bathing solution,hypertonic Na‑bathing solution and hypertonic Na‑bathing
solution containing ﬂufenamic acid (FFA).Three vertical spikes in the current trace((1)‑(3))are current
responses to ramp voltage pulses from ‑100 to ＋60 mV.Broken line indicates the zero current level.b.
Inward current density at ‑60 mV under the isotonic and hypertonic conditions. The graph shows themean and S.E. p＜0.05 vs. isotonic condition. c. Current‑voltage relationships measured at (1)‑(3)indicated in a. d. Diﬀerence currents between (1)and (2)or between (2)and (3).
Ｂ. Responses of membrane currents to rectangular voltage steps for 500 ms between ‑100 and ＋60 mV in
a 10 mV increment from the holding potential of ‑60 mV under the isotonic and hypertonic conditions.
Diﬀerence currents between the two conditions are also shown. Inset :a voltage pulse protocol.

No. 1, 201437

Gomi.Nakada.Kashihara et al.

Fig. 2 Ion permeability of the HeLa cell membrane under hypertonic conditions.
A and B. a. Membrane currents observed at ‑60 mV when a HeLa cell was perfused with solutions
indicated by the bars above the current trace.At the timings indicated by numbers in parentheses,ramp
voltage pulses were applied. b. Current‑voltage relationships obtained at (1) and (3) in a. c. Current‑
voltage relationships obtained at (2)and (4)in a. d. Diﬀerence currents between (1)and (3)or between(2)and (4).
38

Shinshu Med J Vol.62

TRPM4 channels in HeLa cells

Fig. 3 Eﬀects of drugs on currents observed under hypertonic conditions in HeLa cells.
A‑C. Membrane currents observed at ‑60 mV when a HeLa cell was perfused with solutions indicatedby bars above the current trace.Vertical spikes in current traces are current responses to ramp voltage
pulses. AML :amiloride;RR :ruthenic acid ;ECO:econazole.
D. Percentages of current density at ‑60 mV in the presence of the indicated drugs normalized to thecontrol. The graph shows the mean and S.E. p＜0.05 vs. control.
No. 1, 201439

Gomi.Nakada.Kashihara et al.

reported that some TRPV channels have me-nels.Thus,it is likelythat PLC is not involved in the

chanosensitivity ; however, RR, an inhibitor ofstimulation of TRPM4 channels by hypertonic stim-

TRPV channels ,did not inhibit I

ulus in HeLa cells.

3B,D).ECO also inhibited I

channels (Fig.

channels as eﬃciently

Discussion

as FFA (Fig. 3C, D).
Identiﬁcation of genes encoding ion channels

Activation of cation channels plays an important

activated by hypertonic solution in HeLa cells

role in cellular RVI . In HeLa cells, an increase in

The major class of molecules known to form non‑the osmolarity of the external solution from 340 toselective cation channels is “transient receptor

450 mOsmol/kg‑H O induced relatively large FFA‑

potential” (TRP) channels . Fig. 4A shows thesensitive ion channel currents whose IV relationshipanalysis of the transcripts of these genes by RT‑

was quasi‑linear with the reversal potential at 0 mV

PCR. HeLa cells clearly expressed the transcripts

under the present experimental conditions(Fig.1A).

of TRPC1,C6,M3,M4,M7,M 8,V1 and V2 but not

The channels showed voltage‑dependent activation

M2 despite the recent report .Among these expres-at depolarized potentials and weak inactivation at

sed TRP channels, TRPC6 is activated by FFA;

hyperpolarized potentials (Fig. 1B).Experiments in;and V1 andwhich the ion composition of the bathing solution

V2 are inhibited by RR . From these results andwas changed indicated that the channels are Ca ‑the properties of I

channels, the candidate genes

impermeable,non‑selective cation channels (Fig.2).

for those encoding I

channels are TRPC1,M4 andThese channels are insensitive to AM L or RR but

M8.Among them,only TRPM4 channels are estab-sensitive to ECO (Fig. 3). Consistent with theselished to be Ca ‑impermeable .We then examined

results, the channel currents were signiﬁcantly

the eﬀect of transduction of a dominant‑negative

inhibited byTRPM 4 DN (Fig.4).The channels were

TRPM 4 gene (TRPM 4 DN) into HeLa cells (Fig.

likely to be activated by an increase in ［Ca ］ but

4B, C). Hypertonic solution induced I

currents in

not PLC (Fig. 5). Thus, TRPM 4 channels form I

mock‑but not TRPM4 DN‑transfected HeLa cells.

channels, which are activated by intracellular Ca

Thus, it is most likely that TRPM 4 encodes I

and are at least in part responsible for the RVI ofchannels.

HeLa cells .

M3 and M 7 by hypotonic stimulus

Intracellular signals activating TRPM 4 chan-Wehner et al. for the ﬁrst time described non‑nels in response to hypertonic solution in HeLa

selective cation currents activated by hypertonic

cells

stimulus in HeLa cells.They showed that the chan-

TRPM4 channels are activated by intracellular

nel currents have a linear IV relationship, are per-Ca

. Fig. 5A shows that hypertonic solution

meable to monovalent cations but very imperme(i.e.P /P ＝0.07),and are sensitive tofailed to activate TRPM4 channels when intracel-able to Ca

lular Ca

buﬀer was changed from 1 mM EGTA to

FFA but not AM L,consistent with our observation.

10 mM BAPTA,indicating that hypertonic stimulus

They also showed that FFA partially inhibited theactivates TRPM4 channels by increasing ［Ca ］.

RVI of HeLa cells.However,they did not show theIn order to examine whether the increase involtage‑dependent activation of the channels, a［Ca ］ might result from activation of mechanosen-salient feature of TRPM4 channels

sitive Gq protein‑coupled receptors

followed byour observation (Fig. 1B).The same group of inves-inositol triphosphate‑dependent Ca

release fromtigators recently identiﬁed that the channel currents

the endoplasmic reticulum, HeLa cells were

were mediated by TRPM2 . TRPM 2 channels are

pretreated with a PLC inhibitor (U73122) or its

Ca ‑permeable, non‑selective cation channels (i.e.

inactive analogue (U73343) (Fig. 5B); however,

P /P ＝0.5‑1.6) .They explained this paradox by

U73122 did not signiﬁcantly inhibit TRPM4 chan-arguing that HeLa cells express the TRPM 2 variant

40,contrary to

Shinshu Med J Vol.62

TRPM4 channels in HeLa cells

Fig. 4 Ion channel subunits mediating currents observed under hypertonic conditions in HeLa cells.
Ａ. RT‑PCR of transient receptor potential channel subunits expressed in HeLa cells.
Ｂ. Current responses at ‑60 mV to hypertonic solution in HeLa cells transfected with empty plasmids
or plasmids containing a dominant‑negative TRPM 4 (TRPM 4 DN)gene.Vertical spikes in current
traces are current responses to ramp voltage pulses.
Ｃ. Current density was increased by hypertonic solution in mock‑and TRPM4 DN‑transfected HeLa
cells. p＜0.05 vs. mock.
No. 1, 201441

Gomi.Nakada.Kashihara et al.

Fig. 5Involvement of intracellular Ca in the induction of membrane currents in response to
hypertonic conditions in HeLa cells.

Ａ. Current responses to hypertonic solution in HeLa cells dialyzed with 1 mM EGTA (top trace)or 10
mM BAPTA (bottom trace). The holding potential was ‑60 mV. Vertical spikes in current traces
are current responses to ramp voltage pulses. The right‑hand graph shows the current density
increased by hypertonic solution under each condition. p＜0.05 vs. EGTA
Ｂ. Current responses to hypertonic solution in HeLa cells in the presence of speciﬁc phospholipase C
inhibitor U73122 (bottom trace)or its inactive analogue U73343 (top trace). The holding potential
was ‑60 mV. Vertical spikes in current traces are current responses to ramp voltage pulses. The
right‑hand graph shows the current density increased by hypertonic solution under each condition.
42

Shinshu Med J Vol.62

TRPM4 channels in HeLa cells

that lacked part of an NUDT9 ‑H domain in theincreases the sensitivity of TRPM4 channels tocytosolic C‑terminus (TRPM 2ΔC) and made up

intracellular Ca

Ca ‑impermeable channels.However,we could notincreases PIP content in some cell types including

identify the transcripts of a TRPM2 gene with RT‑

HeLa cells

PCR although a pair of our primers for TRPM2 wasincrease in PIP content might sensitize TRPM4

to detect TRPM 2ΔC (Fig.4A).It is also noteworthy

channels to basal ［Ca ］ and thereby activatethat some investigators reported discernible Ca

TRPM4 channels.

permeability of TRPM2ΔC channels .On the otherand that hypertonic stimulus. Thus, the hypertonicity‑induced

To summarize, we found that TRPM 4 channels

hand,it is well established that TRPM 4 forms Ca ‑mediate I

impermeable, nonselective cation channels . We

of HeLa cells . Inhibition of I

detected the abundant expression of TRPM4 inwith FFA sensitizes HeLa cells to shrinkage‑in-

HeLa cells (Fig. 4A) and could inhibit I

duced apoptosis . FFA is a clinically used non‑currents

currents which are important for RVI
currents and RVI

steroidal anti‑inﬂammatory drug with an estab-with TRPM 4 DN (Fig. 4B, C)
TRPM4 channels are activated by membrane

lished safety record. Therefore, a clinically impor-. Wetant possibility is that intra‑uterus or ‑vaginal

found that 10 mM BAPTA but not 1 mM EGTA

application of FFA may cause antiproliferative/

inhibited hypertonicity‑induced I

proapoptotic eﬀects

depolarization, intracellular Ca

and PIPcurrents (Fig.

oncervical cancer.

5A). Although Gq protein‑coupled receptors serve

Identiﬁcation of the molecules forming I

as a sensor of membrane stretch , PLC was notin this study may lead to development of more

involved in the activation of I

selective and potent inhibitors of I

currents by hyper-

tonic stimulus (Fig. 5B).It might be that some Cachannelschannels in

cervical cancer than FFA.

permeable channels are activated by hypertonicity

Acknowledgement

before TRPM 4 channels are open. Indeed, it isreported that in HEK293 cells, TRPM 4 channels

We are grateful to Ms.Reiko Sakai for secretar-

open in response to extracellular ATP‑induced

ial assistance.This work was supported byresearchtransmembrane Careleasegrants to M.Y. from Ono Pharmaceutical Co., Ltd.

from the endoplasmic reticulum . Thus, TRPM4

There are no companies, organizations or groups

may be located in the vicinity of some Ca ‑per-having conﬂict of interest regarding this work to bemeating channels. It is also noteworthy that PIP

disclosed.

inﬂux but not Ca

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（2013. 8. 7 received ; 2013. 9.10 accepted）

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