aspects of TRPC channel function. channel of TRPC aspects on other to more reviews recent comprehensive directed are ers read Interested channels. TRPC5 and of TRPC4 activation the in Gproteins of roles heterotrimeric on be the here will focus acteristics: both are potentiated by G -coupled receptors receptors by Gprotein-coupled potentiated are both acteristics: char functional many share channels TRPC5 and TRPC4 The mediate receptor-operated and store-operated Ca store-operated and receptor-operated mediate to known subfamilies TRP one is of three subfamily (TRPC) potential receptor transient or classic canonical The www.landesbioscience.com 2012;©2012LandesBioscience Channels 6:5,333-343;September/October families, that is, G is, that families, of four consist ) (G proteins nucleotide–binding guanine identity. heterotrimeric The 64% homologs, sharing close are TRPC5 and TRPC4 similarities. on sequence based TRPC4/5, and TRPC3/6/7 TRPC2, TRPC1, subgroups, into four divided are channels TRPC mammalian seven The receptors. serotonin and PAR receptors receptors, adrenergic receptors, muscarinic as such byreceptors, G protein-coupled activated are channels http://dx.doi.org/10.4161/chan.21198 05/23/12;Submitted: 06/20/12; Revised: Accepted: 06/20/12 *Correspondence to: Insuk So; Email: [email protected] The roles ofGproteins of intheactivation TRPC4 Hana Kim, Hana and TRPC5 transient receptor potential channels role in cellular signaling. cellular in role G for We roles potential discuss rudimentary. is still channels G of roles the of understanding our However, particular. in factor growth epidermal or receptor G and general in factors growth and receptors Gprotein-coupled of targets best-studied the of Further, one are they events. these controlling play in proteins interacting that their roles the and heteromultimer a or TRPC1 as ahomotetramer including channels these of function and assembly the regarding is known Much neurons. and myocytes gastrointestinal both in excitability electrical of regulators important are channels TRPC5 and TRPC4 1 Department of Physiology; Seoul National University College of Medicine; Seoul, Republic of Korea; Korea; of Republic Seoul, Medicine; of College University National Seoul Physiology; of Department α i/o proteins in channel activation in addition to their known known to their addition in i/o activation channel in proteins REVIEW Technology; Daejeon, Republic of Korea; Korea; of Republic Daejeon, Technology; 1,† JinsungKim, TRPC4 andTRPC5Channels α s, G s, Keywords: Keywords:

α Introduction i, G i, 2,† Jae-Pyo Jeon, Jae-Pyo α q/11 G and TRP channel, TRPC4, TRPC5, G proteins, GPCR, GI tract, endothelial cells endothelial GI tract, GPCR, Gproteins, TRPC5, TRPC4, channel, TRP 2-4 α i/o TRPC4/5 on proteins α q protein coupled coupled q protein 3 α Department of Physiology; Sungkyunkwan University School of Medicine; Suwon, Republic of Korea of Republic Suwon, Medicine; of School University Sungkyunkwan Physiology; of Department 1,† 12/13 Our protein. Jongyun Myeong, Jongyun † These authors contributed equally to this work. this to equally contributed authors These 2+ entry. 1 TRPC TRPC and Insuk So Channels - - 1 Jinhong Wie, and phosphatidylinositol 5kinase. phosphatidylinositol and 3kinase phosphatidylinositol Rac, via receptors factor growth of stimulation after membrane plasma to the delivered rapidly be can channels homomeric TRPC5 systems, expression gous solution. a140 Cs ited with mM pathway may modulate TRPC4-mediated SOC. TRPC4-mediated modulate may pathway and lipids and by extracellular channels of TRPC5 potentiation the include modulations Other Gd and binding domain. (CIRB) ated by micromolar concentrations of the trivalent cations La cations trivalent of the concentrations by micromolar ated the C553the C558 and mutants. used authors the of redox on TRPC5, effect the showing studies C558S of TRPC5. mutants C553A, the from C553S, currents not any record could C558A, nels have similar current-voltage relationships. current-voltage similar have nels couple to G that (GPCRs) (NHERF) and signaling molecules, such as PLC as such molecules, signaling and (NHERF) way. path CaM-MLCK the via membrane plasma to the delivered rapidly be also can channels homomeric TRPC5 that we showed centrations of the trivalent cations La cations trivalent of the centrations con by micromolar potentiated are they that in channels TRP the C terminus of TRPC5 but not via the CaM/Ins(1,4,5) but the not via of TRPC5 Cterminus the at site located aCaM-binding via currents agonist-activated intracellular Mg intracellular folding proteins, such as the Na the as such proteins, folding scaf PDZ-domain (VTTRL). motif PDZ-binding C-terminal phosphorylation by PKC-mediated of GPCR. of cells isolated from the gastrointestinal (GI) tract. (GI) gastrointestinal the from isolated cells muscle smooth of single to those similar are probabilities open of the dependence TRPC4 ing express stably cells HEK from activities channel single similar the We recorded respectively. pS, 66 also and of 42 conductances single-channel and relationship current-voltage doubly rectifying aunique with currents underlie subunits or TRPC5 TRPC4 La cations trivalent of the concentrations by micromolar potentiated

3+ In addition, TRPC4 and TRPC5 channels are unique among among unique are channels TRPC5 and TRPC4 addition, In 1, and Gd and 23 * In addition, CaM itself was reported to accelerate TRPC5 TRPC5 to accelerate reported was itself CaM addition, In 3+ with bath solutions that mainly contain Na but inhib contain mainly solutions that bath with 16,17 6,9 2 Department of Chemistry; Korea Advanced Institute of Science and and Science of Institute Advanced Korea Chemistry; of Department In our experiments, TRPC5 channels are potenti are channels TRPC5 experiments, our In 3+ β ( ; the inhibition of outward TRPC5 currents by currents TRPC5 of outward inhibition ; the 1 even with a bath solution containing mainly Na mainly solution containing abath with even Chansik Hong, Fig. 1 Fig. 2+18 ; and the desensitization of TRPC5 channels channels of TRPC5 desensitization the ; and ). The unitary conductance and voltage voltage and conductance unitary The ). 24 α Both TRPC4 and TRPC5 contain a contain TRPC5 and TRPC4 Both 10 q/11 and/or G q/11 and/or 12 12,13 Additionally, the NO-cGMP-PKG the Additionally, and nitric oxide, nitric and 1 Hyun Jin Kim, Jin Hyun + In our experiments, however, we experiments, our In /H 10 19,20 22 + However, TRPC4 was not was However, TRPC4 exchanger regulatory factor factor regulatory exchanger As previously mentioned, previously As 3+ ( and Gd and Figs. 2 and 3 2and Figs. α i/o and the two chan two the and i/o 3 13 Ju-Hong Jeon Ju-Hong heavy metals heavy 3+ 5-7 independently independently 21 β Homomeric Homomeric In heterolo In 1, been have ). In the the In ). REVIEW P 14,15 333 3 1 + .

R 3+ 11 ------8

©2012 Landes Bioscience. Do not distribute. 334 mM [Cs mM conductance. The plot of NPo was estimated from each potential (bottom panel). (bottom potential each from estimated was NPo of plot The conductance. the in shown are potential each sylation and PIP2 or indirectly via store depletion and exocytosis. and depletion store via indirectly PIP2 or and sylation nitro CaM, , Gi/o proteins, via directly channels TRPC5 and TRPC4 NO,activate and EGF ions, metal lipids, agonist, GPCR the as such signals, chemical Extracellular signals. many by activated are Figure 2. GTP -120 and -60 30, 80, to-140 were mV +80 ( in from potentials mV. ranging holding The voltages TRPC4 expressing stably were Figure 1.

γ S (200 μ S (200 + ] i and 125 and [Cs mM Single-channel currents recorded in the outside-out configuration from HEK293 cells that that cells HEK293 from configuration outside-out the in recorded currents Single-channel Positive regulatory mechanisms of TRPC4 and TRPC5 channels. TRPC4 and TRPC5 channels channels TRPC5 and TRPC4 channels. TRPC5 and TRPC4 of mechanisms regulatory Positive M) was intracellularly applied to activate the TRPC4 channel under the condition of 125 of condition the under channel TRPC4 the toactivate applied intracellularly M) was + ] o and nominally [Ca and nominally β . ( upper panelupper and ) Representative single-channel current traces were recorded at at recorded were traces current single-channel A) Representative 2+ the red line represents the fit to obtain a unitary unitary a obtain to fit the represents line red the ] o -free. ( -free. B ) The corresponding I-V relationships from from relationships I-V corresponding ) The Channels

). A). - (EGF) on TRPC4. (EGF) factor growth of epidermal mechanism activation for the important especially is channels of TRPC4 activation the contributeto tor-operated mechanisms recep or/and store-operated whether about matter The channel. of TRPC5 activation contribute to the mechanisms receptor-operated that suggests dence evi experimental most the hand, other On the TRPC4. particularly channels, of TRPC activation contribute to the can mechanisms receptor-operated and ated Ca ated reduced Ca reduced exhibited mouse knockout TRPC4 the from cells endothelial isolated that ing tion. applica thrombin and ATP tylcholine, and localization. and expression surface control of channel the in implicated been has TRPC4 PDZ ZO-1) (PSD-95, Dlg, of motif cium concentration ([Ca concentration cium cal cytoplasmic free the in increase role of TRPC channels in receptor-oper in channels role of TRPC Ca on extracellular depends completely due to both a release of Ca arelease due to both NHERF. with by interactions influenced to be shown been also has channels TRPC5 of rat activity The TRPC4 and TRPC5. and TRPC4 with to coimmunoprecipitate reported in store-operated Ca store-operated in protein for arole of aTRPC evidence the channel. the of levels membrane plasma the reduced (T-R-L) dramatically TRPC5 mouse in PDZ of the deletion motif experiments, complex oscillatory changes in [Ca in changes oscillatory complex induced EGF that showed studies eral cells. lial Ca component of store-operated essential an as identified was TRPC4 where mouse, null TRPC4 on the studies channels in the . cell the in channels of hTRPC4 stabilization retention and for the required is NHERF and hTRPC4 between interaction the that to suggest authors the led which channel, of the levels membrane plasma the reduced (T-R-L)motif dramatically of hTRPC4 2+ 2+ The addition of EGF causes an an of causes EGF addition The The most compelling experimental experimental compelling most The levels. More recently, however, levels. sev endothe aortic in channels (SOC) 28,29 2+ Clearly, both store-operated store-operated Clearly, both 28 (ROC) entry include the find the include entry (ROC) Interestingly, evidence for a evidence Interestingly, 2+ 20 entry in response to ace response in entry 26 Deletion of the PDZ of the Deletion 2+ 25 entry came from from came entry The C-terminal C-terminal The V olume 6Issue 5 2+ 2+ ] i from the the from ), which which ), 27 In our our In 20,26 2+ ] ------i

©2012 Landes Bioscience. Do not distribute. and TRPC5. and G that We showed recently tract. (GI) gastrointestinal the from isolated cells muscle smooth single in channels cation nonselective activate via a negatively charged amino acid (D252) acid G of the amino charged anegatively via (K715 acids R716) and amino of TRPC4 charged positively icantly inhibited the TRPC4 current. G current. TRPC4 the inhibited icantly of GTP application whereas channel, TRPC4 the vated www.landesbioscience.com G (PTX)-sensitive toxin pertussis The responses. G use and G between identity sequence 94% than respectively. activation of TRPC4 by GTP of TRPC4 activation G G The cyclase. adenylyl inhibit G G of the forms active constitutively we used TRPC5, and of TRPC4 activation the important for pathophysiological effect at in vivo situation. vivo atin effect for pathophysiological important be might on TRPC4 effects inhibition and enhancement the between balance adelicate that suggest effects opposing These spectrin. and NHERF with interaction via of TRPC4 activation and expression surface exocytic (2) and of TRPC4 degradation of (1) the enhancement opposite effects: dual has EGF Thus, mechanism. unknown an by through EGF cells epithelial corneal in current of astore-operated activation MEKK1-AIP4 pathway and decreased the activity of TRPC4. activity the decreased and pathway MEKK1-AIP4 EGFR- via ubiquitination TRPC4 in toled aprofound increase EGF contrary, On the interaction. of spectrin independently G that suggesting current, of form G active constitutively the and the spectrin cytoskeleton was involved in involved in was cytoskeleton spectrin the and hTRPC4 between interaction a direct eton and cytoskel spectrin the with interaction for the were critical 730–758 of hTRPC4 acids amino the addition, factor. In regulatory exchanger in the association of hTRPC4 with the Na the with of hTRPC4 association the in increase by aphosphorylation-dependent panied accom was insertion this of STKs; activity the dependent on amanner in membrane plasma into the of hTRPC4 insertion exocytotic the induced EGF-receptor stimulation addition, In kinase. dominant the to be appearing Fyn with (STKs), kinases tyrosine by Srcated family and activation. and expression surface of hTRPC4 regulation the COS-7 cells. COS-7 of stimulation (EGFR) receptor EGF lowing 959 Tyr-972, and fol were phosphorylated Tyr- (hTRPC4), TRPC4 of human terminus and aCa and reticulum endoplasmic the outer medium. outer the deletion mutant without the spectrin binding domain ( domain binding spectrin the without mutant deletion 758). α α The G The i2 and G and i2 i3 were the most potent activators for TRPC4 and TRPC5, TRPC5, and for TRPC4 potent most activators were the i3 34 α Intracellular GTP Intracellular i/o proteins are important for the activation of TRPC4 of TRPC4 activation for the important are proteins i/o α i family was originally identified by their ability to ability their by identified originally was i family 34 31 32 34,36 α Constitutively active G Two tyrosine residues in the C the in Two residues tyrosine This phosphorylation was medi was phosphorylation This 33 i3, among others, are highly similar, with more with similar, highly are others, among i3, We also expressed the TRPC4 TRPC4 the We expressed also To G which determine 30 TRPC4 was involved in the the involved in was TRPC4 α i subunits (G

γ α S induced a small current, whereas whereas current, asmall S induced i2 can activate the TRPC4 channel channel TRPC4 the activate can i2 α γ S. Constitutively active G i proteins to trigger physiological physiological to trigger i proteins α i proteins, which include G include which i proteins, 2+ influx from influx α i2 induced a larger TRPC4 TRPC4 alarger induced i2 α QL mutants). G α QL mutants). i3 and G and i3 α α isoform is involved in involved in is α isoform i1 G and i2 binds to C-terminal to C-terminal binds i2 + /H - - - - + o mimicked the the αo mimicked

α i3. Hormones Hormones i3. α and TRPC5 channels. On the other hand, PtdIns(4,5) hand, other the On channels. TRPC5 and TRPC4 of pathway inactivation an as PKC and works PKA by channels TRPC5 and TRPC4 of Figure 3. and TRPC5. α i/o proteins proteins i/o i2 protein. protein. i2 γ α α S signif Δ i1 acti i1 i2 and and i2 730– Negative regulatory mechanisms of TRPC4 and TRPC5 channels. Phosphorylation Phosphorylation channels. TRPC5 and TRPC4 of mechanisms regulatory Negative α Channels i1, i1, β 35 - -

the channels or reduced the activation by GTP activation the or reduced channels the None G activity. of the TRPC5 tested the effects of various G of various effects the tested G of activated availability the by altering channels of the regulation the or in channels, GIRK with case the is as channels, of the activation the involved in be are key activators of TRPC4 or TRPC5, G or TRPC5, of TRPC4 activators key are G by all were increased which to TRPC4 contrary of TRPC5 current basal the reduced P G G by activated G by inhibition of changes in PKC and altered interaction with G with interaction PKC in altered and of changes effects the out ruled findings experimental Our Cterminus. the of Thr-972 residue at phosphorylation with of TRPC5 sitization desen to induce by PKC, known phosphorylation channel and enous G enous GTP and G surface expression of TRPC4, modified cellular PtdIns(4,5) cellular modified of TRPC4, expression surface G active constitutively by the of TRPC4/5 inhibition the Hence, mechanism. unknown by an TRPC5 and TRPC4 activate estly G used TRPC4 used study, we previous our In activation. TRPC5 and TRPC4 results imply that intense overstimulation of aG overstimulation intense imply that results mic Ca mic cytoplas or decreased increased including mechanisms, potential We several TRPC5. considered and TRPC4 inhibits pathway

2 α isoforms tested actually reduced the spontaneous current current spontaneous the reduced actually tested α isoforms α α and perhaps increased cytoplasmic Ca cytoplasmic increased perhaps and Activation of the G of the Activation q on channel function. Our results indicated that PtdIns(4,5) that indicated results Our function. q on channel q was completely unexpected in our experiments. our in unexpected completely q was i3 Q205L 2+ α , interference of channel interaction with G with interaction of channel , interference γ was the most effective activator of TRPC5, as all other other all as of TRPC5, activator effective most the was i3. Interestingly, as in GIRK channels, G channels, GIRK in as Interestingly, i3. S-induced currents, likely by competing with endog with by competing likely currents, S-induced isoform to test the effect of PtdIns(4,5) effect the to test β isoform α α q. Moreover, they pointed to an indirect effect of effect q. indirect Moreover, to an pointed they q as the major inhibitors of the current induced induced current of the major inhibitors the q as α α q-PLC pathway has been shown to mod shown been has q-PLC pathway i/o proteins. Although G Although proteins. i/o P 2 α hydrolysis negatively regulates TRPC4 TRPC4 regulates negatively hydrolysis βγ βγ i. To address these questions, we questions, Toi. these address combinations on TRPC4 and and on TRPC4 combinations combinations tested activated activated tested combinations βγ 2+ subunits may also also may subunits are required for required are γ S. 34 α α α α i in channel channel i in i/o subunits subunits i/o i/o proteins proteins i/o α q-activated q-activated 34,36 i, reduced i, reduced These These 335 P P 2 - - - - 2 .

©2012 Landes Bioscience. Do not distribute. 336 brane, thus stabilizing its binding with PtdIns(4,5) with binding its stabilizing thus brane, ited TRPC4 ited Δ the for keeping necessary is actin cortical and proteins ERM NHERF, adaptor the with domain PDZ-binding C-terminal its via interaction whereby TRPC4 gating, of TRPC4 model nistic TRPC4 dent are controversial. One report showed that PtdIns(4,5) that showed One report controversial. are face expression and subsequent increase in Ca in increase subsequent and expression face sur channel of TRPC4 upregulation mediated protein (CREB) element-binding response cAMP SMCs through artery monary pul human in effect of mitogenic its part of ATP exerted dose low- The function. TRPC4 involved in is cAMP that suggested G G that viously We pre reported channel. TRPC5 the inhibits and phorylates phos PKA, molecule, effector downstream furthest the addition, In channels. of TRPC5 translocation membrane plasma the promotes also cascade The channels. TRPC5 potentiates release channels. TRPC4 not activate did alone and TRPC5. and PDZ binding domain ( PDZ domain binding vators. GTP TRPC5. of of Ser796 Ser794 site and phosphorylation atC-terminus via . to contribution development its of pulmonary and responses pathophysiological during remodeling vascular in of TRPC4 role apossible suggesting phosphorylation, CREB sion through expres TRPC4 increased ATP markedly with treatment study 8-Br-cAMP. Co-expression of constitutively active form of form G active of constitutively Co-expression 8-Br-cAMP. and (FSK) forskolin of isoproterenolited (ISO), by treatment Ca of the effect the alleviated we When larger. considerably was inhibition but the observed, The effects of PtdIns(4,5) effects The channel. of isoproterenol on TRPC5 effect the it facilitated cells, HEK293 GTP GTP solution, internal of the constituents on the depending TRPC5 in the activation process of TRPC5. In our previous study, previous our In of TRPC5. process activation the in free Ca free P by PtdIns(4,5) channels TRPC5 and of TRPC4 stimulation When we infused a solution containing both Ins(1,4,5) both asolution containing we infused When by PKA. of TRPC5 regulation negative observed dominantly β intracellular Ca intracellular induces aCa induces the current. G current. PtdIns(4,5) of roles the about controversy the for solving needed are studies 2 2 84AA stretch close to the inner surface of the plasma mem plasma of the surface inner to the close stretch 84AA α . -adrenergic receptors were present in HEK293 cells. When When cells. HEK293 werein present receptors -adrenergic 34,38 2+ Isoproterenol elicited both positive and negative effects on effects negative and positive Isoproterenol both elicited G

s Q227L β response by using a pipette solution representing a high ahigh solution representing a pipette by using response α γ γ 2 In addition, Otsuguro et al. et Otsuguro addition, In 34,36 12/13-Rho-MLC phosphatase pathway might be involved be might pathway phosphatase 12/13-Rho-MLC S, a decrease in TRPC5 current in response to ISO was towas ISO response in current TRPC5 in adecrease S, S and Ins(1,4,5) S and - was co-expressed with TRPC5 in in TRPC5 with co-expressed was receptor -adrenergic 2+ 11 , also inhibited GTP inhibited , also state, ISO inhibited GTP inhibited ISO state,

Under GTP the β α P -adrenergic stimulation followed by the G by the followed stimulation -adrenergic s but not TRPC4 not α but 2 2+ -cAMP-PKA pathway inhibited TRPC5 current current TRPC5 inhibited pathway -cAMP-PKA - anovel mecha suggested also They α inhibition. 40 39 on TRPC4. As in GIRK2 channel, PtdIns(4,5) channel, GIRK2 in As on TRPC4. α γ release mediated by Ins(1,4,5) mediated release GTP 2+ s S is one of the well-established TRPC4/5 acti TRPC4/5 one well-established S is of the negatively regulated GTP regulated negatively was buffered to 5 buffered was γ S-activated TRPC5 current was inhib was current TRPC5 S-activated P Δ 3 TTRL) prevented PtdIns(4,5) TTRL) , even when only endogenous levels of levels endogenous only when , even P γ β 2 S activation system, we would have have we would system, S activation on TRPC4 and TRPC5 channels channels TRPC5 and on TRPC4 -adrenergic stimulation-dependent stimulation-dependent -adrenergic γ S-activated TRPC4 and TRPC5 TRPC5 and TRPC4 S-activated β 37 37 μ whereas others showed the the showed others whereas showed that deletion of the of the deletion that showed γ M. Similarly, Zhang et al. et Zhang Similarly, M. S-activated TRPC5 when when TRPC5 S-activated 34,37,38 γ S-activated TRPC4 TRPC4 S-activated P 2+ 3 R and this Ca this R and influx. In their their In influx. P α 2 P P . Further . Further s cascade 2 2 depen inhib P 3 and and Channels α P 2+ 40 s ------2 ,

current decreased dramatically ( dramatically decreased current 10 EGTA,the solution mM including apipette we used When of TRPC4. activation for the levels calcium intracellular optimal based on its sensitivity to pertussis toxin (PTX). toxin to pertussis on sensitivity its based ally coupledto Ca ally of voltage-dependent Ca I-V curve bell-shaped the and plus ORAI1) (STIM1 of CRAC (I-V) current-voltage curve rectifying inward typical The tion. intracellular Ca intracellular Ca increases beneath the plasma membrane. plasma the beneath increases of Ca release thapsigargin-induced the that showed also needed. are channels TRPC5 and TRPC4 with Gproteins of small interaction the about studies Further Intracellular Ca Intracellular (CaM). to involve not appear does that a manner TRPC5 forms Ca forms TRPC5 that hypothesis the support data These BAPTA. but not with EC an Na with by intracellular activated to be known is channel GIRK2 channels. GIRK activate can Na itself internal whereas G without of TRPC4 activation for the enough α or the plusORAI1 STIM1 by co-expressing Furthermore, tion. depolariza membrane along-lasting supported turn in which ing I-V curve of TRPC5 channels. The Ca The channels. of I-V TRPC5 ing curve doubly rectify typical of the appearance the before ologically effect of Na of effect to the were similar results These recording. patch whole cell for used we usually 0.5 EGTA,which with mM slow compared G even addition, In TRPC5. potentiate does it receptor, whereas of amuscarinic co-expression the without dependent Ca not. rent was GTP whereas inhibitor MLCK tor and inhibi by CaM inhibited was current TRPC5 carbachol-induced subsequent Ca the and transiently channels TRPC5 activated stores internal from TRPC4 channel by adding EGF are needed. are EGF by adding channel TRPC4 from current not any record why we could studies Further channel. lar calcium or Mn calcium lar intracellu increased EGF whereas technique clamp patch with recorded when electrophysiologically channel TRPC5 activate lesser degree when the M1 expressed. the is when receptor degree lesser activity. TRPC5 and TRPC4 on excessive feedback negative produces PKC typical later and feedback of positive a kind is channels TRPC5 and of TRPC4 facilitation calcium initial hand, other On the activity. electrical on excessive feedback negative ducing by pro function physiological important an to serve thought TRPC5 channels with buffering of internal Ca of internal buffering with channels TRPC5 form of G of form by GTP activated current TRPC5 the blocked bath to the inhibitor kinase Rho and

1C and 2+ TRPC5 is dramatically potentiated by intracellular Ca by intracellular potentiated dramatically is TRPC5 G 50 entry through either calcium release-activated or voltage- release-activated calcium either through entry of 30–40 mM. Sodium activation of GIRK channels is is channels of GIRK activation Sodium mM. of 30–40 α i proteins are thought to play a role in EGF signaling to signaling playaroleEGF in thought are i proteins β α 2 subunits of L-type Ca of L-type 2 subunits 12 and G 12 and + 23 on GIRK channels. However, Ca channels. on GIRK 2+ Interestingly, application of both MLCK inhibitor inhibitor MLCK of both application Interestingly, 2+ channels is sufficient for TRPC5 channel activa channel TRPC5 for sufficient is channels 2+ 2+ entry produced a sustained TRPC5 activation, activation, TRPC5 asustained produced entry does not potentiate TRPC4 channel activity activity channel TRPC4 not potentiate does also potentiates TRPC4 channel activity to a activity channel TRPC4 potentiates also 2+ 2+ γ 2+ -activated cation channels that are function are that channels cation -activated S. On the other hand, constitutively active active constitutively hand, other On the S. quenching in HEK cells expressing TRPC4 TRPC4 expressing cells HEK in quenching -selective ion channels through local Ca local through ion channels -selective α 13 did not show any effect on TRPC4. 13 not effect show did any 2+ channels were recorded electrophysi recorded were channels Fig. 4 Fig. 2+ channels, they found that that found they channels, ). The activation was very very was activation The ). γ S-induced TRPC5 cur TRPC5 S-induced 42 In our experiments, experiments, our In α 2+ i/o proteins need need proteins i/o -entry activated activated -entry 2+ 41 2+ itself was not was itself V 43 Gross et al. et Gross α with EGTA EGTA with olume 6Issue 5 EGF could could EGF i/o proteins proteins i/o 2+ from from 2+ in in 2+ 42 36 ------

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com receptor stimulation and results in an influx of Na influx an in results and stimulation receptor to muscarinic response in occurs which activation, ionic channel of Ins(1,4,5) of activation of voltage-dependent Ca of voltage-dependent activation ried by Na ried car primarily current, Under this physiologic ionic conditions, voltage-dependent Ca of activation Under the physiologic conditions, contraction. ates ate currents similar to mI similar currents ate to gener ability for their tested and cells HEK293 in expressed individually been also have channels TRPC GPCRs. membrane of plasma stimulation to the response in activated are that nels muscles. smooth including tissues, various in receptors G-protein-coupled by activated are that channels cationic nonselective for the didates can molecular as attention increasing receiving been have family stimulation. gic to choliner response contractile the underlying mechanism tant (ICCs). of Cajal cells stitial inter the from potential pacemaker the with begins tract (GI) [Ins(1,4,5) inositol-1,4,5-trisphosphate of production the and phoinositides C G PTX-insensitive coupledto is receptor M3 the channels, cation nonselective vate acti and cyclases adenylyl inhibit activated when which proteins, G (PTX)-sensitive toxin to pertussis coupled functionally is receptor M2 the While process. this involved in are receptors muscarinic M2 and M3 that suggested have agonists carinic mus other and to acetylcholine response in contraction muscle rent (mI rent cur cation a muscarinic-activated to as referred current cation of anonselective generation the in results stimulation receptor channels. or receptor-operated store-operated as such mechanisms, different by several mediated be potentially muscle cells. GI smooth in coupling excitation-contraction controls cells, muscle smooth and on ICCs receptors muscarinic via acting line, to that of native mI of native to that similar ion selectivity relative and I–V relationship an exhibits current TRPC5 the that demonstrated comparison a functional from intracellular Ca intracellular from of these events is an increase in the cytosolic [Ca cytosolic the in increase an is events of these Ca an increase in intracellular [Ca intracellular in increase an Intracellular Ca Intracellular costimulation of both the M2 and M3 receptor subtypes. receptor M3 and M2 the of both costimulation membrane potential change induced by mI induced change potential membrane muscles. smooth including tissues, peripheral control of various nervous parasympathetic role the in play a key receptors muscarinic The β (PLC 2+53 Members of the transient receptor potential canonical (TRPC) (TRPC) canonical potential receptor transient of the Members In various gastrointestinal smooth muscles, muscarinic muscarinic muscles, smooth gastrointestinal various In as well), is suggested to be the primary and most impor most and primary the to be suggested is well), as by MuscarinicStimulationintheGITract 7,38,54,55 44-46 Nonselective CationChannel Activated Cat β P ). + ). The spontaneous activity of the gastrointestinal gastrointestinal of the activity spontaneous The 3 , causes depolarization of the cell, resulting in the the in resulting cell, of the depolarization , causes 45,47-53 P 45 ] and diacylglycerol (DAG). This signaling leads to leads signaling (DAG). This diacylglycerol ] and 45,47 3 Pharmacologic studies of gastrointestinal smooth smooth of gastrointestinal studies Pharmacologic to Ins(1,4,5) to TRPCs form Ca form TRPCs 2+ The latter leads to the breakdown of phos breakdown to the leads latter The Intriguingly, the activation of mI activation the Intriguingly, release is triggered primarily by the binding binding by the primarily triggered is release α q/11 proteins that stimulate phospholipase phospholipase q/11 stimulate that proteins Cat 2+ 2+ , stores and the activation of Ca activation the and stores 7,56

channels is critically dependent on the dependent on the critically is channels the possible contribution of TRPC5 of contribution TRPC5 possible the Cat P 46 of the gastric myocyte. Although Although myocyte. gastric of the 3 receptors, whereas Ca whereas receptors, The acetylcho neurotransmitter The 2+ ] as a result of the release of Ca release of the aresult ] as 2+ 2+ channels. -permeable cation chan cation -permeable Cat 44,45 . Thus, the cat the . Thus, 2+ The net effect effect net The + ], which initi which ], (and perhaps perhaps (and 2+ Cat entry can can entry requires requires 2+ entry. entry. α Channels i/o i/o 2+ 48 ------

whose currents contribute > 80% to mI contribute>80% currents whose channel a55-pS forms cation TRPC4 cells, muscle smooth nal intesti in that, showed data Their TRPC6. and TRPC4 both or alone, TRPC6 alone, TRPC4 lacking mice in function cle muscle cells, voltage-activated Ca voltage-activated cells, muscle smooth of intestinal depolarization to the receptors muscarinic Tsvilovskyy et al. et Tsvilovskyy vivo. in motility intestinal small regulating tion, consequently Tsvilovskyy et al. et Tsvilovskyy native ileal smooth muscle cells. muscle smooth ileal native current mI current cation receptor-operated playamajor rolethe may in isoform this that suggested muscle smooth intestinal in currents native and TRPC4 expressed of heterologously properties the in similarities TRPC5 might be a molecular candidate for mI candidate amolecular be might TRPC5 that we suggested SOC, conducts TRPC4 that suggested results some because and currents TRPC4 not detect we could because mI for responsible channels separate two as function TRPC6 and in gastric smooth muscle cells with nested RT-PCR. nested with cells muscle smooth gastric in TRPC5 detect we could experiments, our In tissues. these in not deleted was channel the that showed which data, expression by not supported was contraction muscle smooth to intestinal recorded in TRPC4 knockout mice. knockout TRPC4 in recorded [Ca in increase an and Gproteins PTX-sensitive requires of TRPC4 contribute the remaining < 20%. There appeared to be no to overlap be appeared There < 20%. remaining contribute the and b of (A). bof and a point at obtained bwas aand in shown relationship I-V The shape. rectifying doubly atypical showed TRPC4 of relationship (I-V) voltage low-calcium buffered internal solution (0.5 mM EGTA).mM (0.5 ( solution internal buffered low-calcium the with recorded current tothe contrast in pipette, EGTA patch the in Figure 4. activation of TRPC4 of activation of mV. course time -60 at The recorded traces current Representative

Cat 2+ . Their findings suggest that TRPC4 and TRPC6 couple TRPC6 and TRPC4 that suggest findings . Their ] i . 34,36,37 Current trace in the activation of TRPC4 of activation the in trace Current Cat These properties are similar to those of mI to those similar are properties These . 57 Subsequently, we could show that mI Subsequently, show that we could 59 by G β by 59 presented conclusive evidence that TRPC4 TRPC4 that evidence conclusive presented studied mI studied α i2 was delayed in the presence of 10 of mM presence the in delayed was Cat 47,52 activation and smooth mus smooth and activation 2+ 58 influx and muscle contrac and muscle influx A recent study reported by reported study A recent Importantly, the activation activation the Importantly, Cat and TRPC6 currents currents TRPC6 and by G β by B α ) The current- ) The Cat i2 . ( . A) 7 Cat However, However, 7 Initially, Initially, was not was Cat 337 in in - - -

©2012 Landes Bioscience. Do not distribute. 338 ing membrane potential and increased the frequency of STDs. frequency the increased and potential membrane ing W of muscles the in were reduced neurotransmitters inhibitory and to excitatory responses postjunctional role For of ICCs. example, the generation of mI generation the and channel(s) of cationic activation to the leading M3, and M2 receptors, muscarinic stimulates acetylcholine neurotransmitter The tract. gastrointestinal controlof the nervous sympathetic para role the in play a key receptors muscarinic Therefore, tissue. on the effects nonredundant concurrent but exert independently to function seem channels two the muscle, smooth of intestinal and 5-nitro-2-(3-phenylpropylamino)-benzoic 5-nitro-2-(3-phenylpropylamino)-benzoic and acid Niflumic Ca of influx ensuing The channels. TRPC6. Gi PTX-sensitive involves activation TRPC4 because receptor muscarinic M2 on the mI whereas knockouts, TRPC single the in TRPC6 and by TRPC4 mediated currents the between of or compensation izations (STDs). Carbachol (100 nM–1 Carbachol (STDs). izations depolar transient spontaneous generated intestine small murine the from ICCs conditions, Under or current-clamp stomach. tine intes small of the cells muscle smooth in were notrents observed Sl the critical trigger for the stimulation of voltage-dependent Ca of voltage-dependent stimulation for the trigger critical the Ca tor, which stimulates PLC- tor, stimulates which recep muscarinic M3 to the coupling via activated primarily is the gastric fundus. gastric the which has a smaller contribution to contribution mI asmaller has which TRPC6, hand, other On the pathways. signaling M3 and M2 the of both costimulation requires that channel the to be appears that the Ca the that showed However, also they contraction. muscle smooth regulate thereby, and critically cells muscle smooth gastrointestinal in nels chan receptor-activated muscarinic the constitute TRPC6 and TRPC4 that demonstrated findings These TRPC6. and TRPC4 lacking mice in substantially were reduced of which both ance, clear and motility intestinal by assessing were examined quences physiologic conse Direct effects. these aggravated of TRPC6 deletion Simultaneous terminals. nerve excitatory from release by acetylcholine elicited contractions atropine-sensitive and tions depolariza membrane carbachol-induced diminished greatly displayed myocytes ileal TRPC4-deficient Furthermore, outs. double knock TRPC4/TRPC6 the in eliminated completely facilitation atlower Ca facilitation also consider the roles of ICCs on of GI roles motilities. ICCs the consider also we should tract, controlof gastrointestinal nervous sympathetic (< 20%). TRPC6 and (> 80%) TRPC4 multiple from channels: TRPC4 channel can also account for the dependence of mI dependence for the account also can channel TRPC4 The myocytes. TRPC4-deficient but notthe in cells wild-type the in were observed that changes potential membrane induced mutant animals with defective ICC networks (e.g., ICC networks defective with animals mutant the specific responses to neurotransmitter. Ca to neurotransmitter. responses specific the of basis on the distinguished be might GI muscles in responses and intestinal motility. mI motility. intestinal and / / Sl 2+ To understand the roles of muscarinic receptors in the para the in receptors of roles To muscarinic the understand W

concentration, can explain the oscillations of the carbachol- of the oscillations the explain can concentration, d V ) has made it possible to learn more about the physiological physiological more the about it to learn possible made ) has and Sl and 34,36,37 2+ / -dependent regulation of TRPC4, which includes includes which of TRPC4, regulation -dependent Therefore, TRPC4, the major contributor the to mI TRPC4, Therefore, Sl d , which lack most of the intramuscular ICCs in in ICCs intramuscular of most the lack , which 62,63 Cat The types of cells mediating cholinergic cholinergic mediating of cells types The 2+ , which depolarizes the cells, providing providing cells, the depolarizes , which concentrations but inhibition at higher athigher but inhibition concentrations α and produces DAG. produces β and Cat /o proteins, which is in contrast to contrast in is which proteins, /o is actually composed of currents of currents composed actually is 2+ is essential for contraction for contraction essential is Cat (approximately 20%), 20%), (approximately μ M) depolarized rest depolarized M) 2+ -activated Cl -activated 59,60 61 The use of use The In the case case the In W / W Cat V - and and cur was was Channels Cat Cat 2+ 64 ------,

pacemaker channels. pacemaker and agonists by muscarinic ICCs the in activated current inward Tmem16a by encoded Recently, channels to CCh. responses and STDs proliferation endothelial inhibits function, barrier determines formation tact ated by a vascular endothelium-specific member of the Ca the of member endothelium-specific by avascular ated medi mainly are cells endothelial between contacts Adhesion complexes. signaling of membrane-associated organization lar oxide release and barrier stability, via intracellular Ca intracellular via stability, barrier and release oxide nitric as such functions, endothelial regulate cells Endothelial awhole. as GI tract of the stimulators muscarinic the as channels TRPC6 and TRPC4 the and ICCs the in TMEM16Achannels consider conclusion, we should In cytoskeleton. filament actin cellular to the molecules adaptor catenin-type via linked is that domain acytoplasmic contains cadherins, classical other like which, VE-cadherin, molecules, dependent adhesion activates aCl activates motor primarily from neurons ACh released that suggested data complex. VE-cadherin/catenin of the phosphorylation tyrosine trolling by con permeability layer cell endothelial the modulate VEGF mation and maintenance of tight junctions. of tight maintenance and mation for for the and physically together cells for holding necessary strength adhesive the provides latter the junctions; adherens and tight contains layer cell endothelial quiescent The phenotype. epithelioid quiescent, typical the adhesions, of cell-cell formation or, phenotype upon proliferative adopt afibroblastoid, either can cells endothelial conditions, culture cell on the Depending ing. of the proliferative migratory state of synthetic vascular SMCs. vascular of synthetic state migratory proliferative of the definition the in part to take believed is and upregulated is nels chan of TRPC expression the injury upon vascular For instance, disease. development of the vascular in importance paramount of is of SMCsand response pathophysiological of the part gral inte an to be thought is to synthetic quiescent from (SMC) cell acid (NPPB; both 100 both (NPPB; acid and have repeatedly been suggested to be key determinants of determinants key to be suggested been repeatedly have and cells endothelial in expressed are protein 4(TRPC4) potential enous CCh or by cholinergic neurons were compared. neurons or by cholinergic CCh enous byexog either stimulated muscles of intact responses electrical The neurons. from ACh to the released responses dominant the generates of cell type which to determine possible be it might then stimulation, by muscarinic ICCs and cells muscle smooth in muscles may depend upon the expression of expression upon the depend may muscles of GI regulation muscarinic that suggest data These cells. muscle smooth in conductances cation of non-selective activation the be may due to response this of most because acid by niflumic affected were not strongly CCh to exogenous exposed muscles of whole response the contrast, In blocked. was neurons from to released theACh response of most acid, of niflumic presence

The channels formed by the canonical transient receptor receptor transient canonical by the formed channels The 72 were suggested to be responsible for the enhanced enhanced for the responsible to be were suggested The phenotypic change of vascular smooth muscle muscle smooth of vascular change phenotypic The The PhysiologicalRolesofTRPC4 69 70,71 - and has been recognized to govern the molecu the to govern recognized been has and conductance, which is expressed by ICCs. In the the In by ICCs. expressed is which conductance, GPCR agonists (thrombin and histamine) and and histamine) and (thrombin agonists GPCR in EndothelialCells 65,66 If different conductances are activated activated are conductances different If μ M) hyperpolarized cells and blocked blocked and cells hyperpolarized M) Tmem16a 67,68 Cell-cell con V olume 6Issue 5

in ICCs. in 2+ 64 signal Their Their 2+ 73 61 ------

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com Ca TRPC4 acted as a Ca as acted TRPC4 function. promotion contact-dependent of TRPC4 cell-cell the identified system expression HEK293 the in function of TRPC4 Analysis plexes. com into junctional of TRPC4 recruitment for the evidence ther fur provided fusion proteins of fluorescent localization of cellular proteins junctional the with co-precipitated TRPC4 contacts. cell-cell immature form that ters et al. et Zhang addition, In system. expression aheterologous in current TRPC4 the when recording important is very confluence of cell degree the Thus, data). (unpublished method clamp patch the using were more confluent that cultures cell from were recorded currents TRPC4 Lower of TRPC4. level expression the affected culture cell the of confluence the that We observed also signaling. factor growth of cellular tuning the and phenotype-switching lial endothe in player akey as emerge may TRPC4 Thus, phenotype. to a quiescent aproliferating from transition the during state lial Ca stimulated mediates TRPC4 that revealed experiments knockdown negative dominant and siRNA Both contacts. of cell-cell formation moted by the Epidermal growth factor-induced Ca growth Epidermal cells. barrier-forming quiescent, in membrane plasma the from of TRPC4 retrieval the but initiated cells of proliferating brane mem plasma into the TRPC4 recruited factor growth Epidermal tional channels. of func targeting cellular the and recruitment membrane the determines which PDZ domain, interaction aC-terminal via cytoskeleton actin the with associated tightly is that molecule ing asignal is TRPC4 proliferation. of endothelial adeterminant as TRPC4 rolefor key a confirmed authors these Nonetheless, ing. endothelial Ca endothelial the endothelial phenotype. endothelial the thus, and functions barrier and junctions of cell-cell integrity the governs TRPC4 that demonstrated been repeatedly it has nels), thelial cells increased with the formation of cell-cell contacts. of cell-cell formation the with increased cells thelial Ca NO-activated native to conduct likely are channels TRPC4 TRPC5/ and TRPC5/TRPC1 hetero-multimeric and TRPC5 TRPC5/TRPC4 that EGFR-G that G and EGFR between interaction Considering hypertension. bution to development of pulmonary contri its and responses pathophysiological during remodeling vascular in role apossible of TRPC4 and expression surface nel TRPC4 as an endothelial Ca endothelial an as TRPC4 Ca endothelial in aprominent role for TRPC4 against as nitric oxide release and barrier stability. barrier and release oxide nitric as ments in human macrovascular endothelial cells, endothelial macrovascular human in ments experi knockdown of siRNA results by the questioned recently during pathophysiological responses. pathophysiological during remodeling vascular and phenotype to aquiescent a proliferating suggested to conduct stimulated Ca stimulated to conduct suggested with interaction and formation contact 2+ 2+ TRPC4 surface expression in human microvascular endo microvascular human in expression surface TRPC4 entry in a specific endothelial state during the transition from transition the during state endothelial aspecific in entry influx in endothelial cells. endothelial in influx 40 suggested CREB-mediated upregulation of TRPC4 chan of TRPC4 upregulation CREB-mediated suggested α 2+ i-TRPC4 pathway are important for stimulated for stimulated important are pathway i-TRPC4 26 signaling and of endothelial functions, such such functions, of endothelial and signaling channels have intact NO sensitivity because because NO sensitivity intact have β channels Importantly, in addition to the debated role of debated to the addition in Importantly, β -catenin as a signaling molecule that enables enables that molecule asignaling as -catenin 2+ -entry channel that is regulated by cell-cell by cell-cell regulated is that channel -entry

2+ -entry exclusively in proliferating clus proliferating in exclusively -entry 29,75 β -catenin and VE-cadherin. Analysis Analysis VE-cadherin. and -catenin 2+ 13 channel (as ROC chan or SOC (as channel However, this concept was very very was concept However, this α i proteins, there is possibilities possibilities is there i proteins, 2+ entry in a specific endothe a specific in entry 2+ -entry was strongly pro strongly was -entry β -catenin. TRPC4 was was TRPC4 -catenin. 28,29,74,75 76 which argued argued which In addition, addition, In 2+ signal Channels 77 ------

and conducts I conducts and subunits of four coordination the apore through forms that tein ties to I ties for endothelial cell barrier disruption. barrier cell for endothelial signal as a proximal serves that flux calcium transmembrane the controlled critically and channel TRPC4 of the selectivity cium store depletion, promoted cal the calcium after activated be will TRPC4 that probability the increased Orai1 Furthermore, tion. store deple calcium after TRPC1 with and cells endothelial in TRPC4 with interacts constitutively Orai1 that indicated results Their (FRET). transfer energy resonance by Foerster interactions intermolecular for probed they fractions, purified Using bait. as protein 4.1 using fractions, membrane cell endothelial from nel chan TRPC4 endogenous for the to enrich ameans developed al. et Cioffi store-operated, to be hence storeand depletion cium cal to sense proteins for TRPC required is that channels TRPC of subunit essential an be To may Orai1 whether determine I both activates Thapsigargin entry channels, which are essential for mediating the Ca the for mediating essential are which channels, entry can interact with STIM1 to form functional store-operated Ca store-operated functional to form STIM1 with interact can possesses a reversal potential near +40 mV. +40 near potential areversal possesses dependent disruption of the endothelial barrier via thrombin. via barrier endothelial of the dependent disruption 4.1-bound channel. protein to the contribute subunits TRPC4 and TRPC1 Both store-operated Ca store-operated receptor-operated Ca receptor-operated mediate TRPCs that of demonstrating controversy,matter data Furthermore, I Furthermore, as to whether I to whether as remain Questions calcium. reticulum endoplasmic deplete which of all inositolTPEN, 1,4,5-trisphosphate and by thapsigargin, a calcium-selective store-operated current, I current, store-operated a calcium-selective proliferation, migration and angiogenesis. and migration proliferation, that TRPCs can contribute to both types of Ca types contributeto both can TRPCs that 10 suggested past yhave the from Studies signaling. to GPCR clamp technique or the current amplitude is too small to record to record small too is amplitude current or the technique clamp patch with cells endothelial thin and flat in current TRPC record to it difficult is that It heterodimer. seems or TRPC1/TRPC5 I-Vof TRPC1/TRPC4 curve rectifying outwardly the even cells, endothelial in of I-V TRPC4 curve double rectifying typical to show no report is To there signal. knowledge, us calcium the contributeto may pathways calcium other that consider should we channels, role of of the TRPC to study methods surement mea calcium we use When cells. endothelial in channels SOC of roles the to investigate used was addition and removal calcium with method measurement acalcium Furthermore, not clear. are channel, CRAC the except channels, SOC of the relationships I-V the or EGF. contrast, In histamine) serotonin, thrombin, (acetylcholine, agonists GPCR as such of astimulator presence of the regardless (I-V) current-voltage relationship bly rectifying dou typical show the TRPC1, except channels, TRPC of the calcium current, I current, calcium selective. I SOC

As previously discussed, both the receptor-operated and the the and receptor-operated the both discussed, previously As Although the role for TRPC channels in SOCE is still a still is SOCE in channels role for the TRPC Although is small (approximately 1.5 pA/pF), inwardly rectifying and and rectifying 1.5 inwardly (approximately pA/pF), small is SOC 84-86 , although I although , Orai1 is a 4 transmembrane spanning domain pro domain spanning a4transmembrane is Orai1 SOC SOC CRAC 2+ CRAC controls permeability, whereas I whereas permeability, controls is the same as the calcium-release activated activated calcium-release the as same the is entry mechanisms can be activated in response response in activated be can mechanisms entry 78-80 . 87-91 2+ , which displays similar biophysical proper biophysical similar displays , which CRAC entry pathways are quite conclusive. All All quite conclusive. are pathways entry This channel was suggested to conduct to conduct suggested was channel This Endothelial cells express Orai1. express cells Endothelial is thought to be more highly calcium calcium more to be highly thought is SOC and I and 95 CRAC 86 TRPC1 and TRPC4 TRPC4 and TRPC1 in endothelial cells. cells. endothelial in 80-83 SOC 2+ I , in endothelia; endothelia; , in -entry channels. channels. -entry SOC CRAC is activated activated is regulates regulates 2+ -entry- 86,92,93 339 2+ 94 95 ------

©2012 Landes Bioscience. Do not distribute. 340 turn suggests that TRPC4 might be a ROC in endothelial cells. cells. endothelial aROC be in might TRPC4 that suggests turn in which vasorelaxation, for acetylcholine-induced responsible was TRPC4 cells, Even endothelial in GI tract. the in channel aROC as act and stimulation by muscarinic activated currents cation nonselective for the responsible are channels TRPC4 TRPC5. and of TRPC4 activation for the essential are proteins ments with heterotrimeric G proteins and found that G that found and Gproteins heterotrimeric with ments experi the we conducted GI tissue, native from results previous ( review this in mentioned early pathways multiple by the activated easily was which current, TRPC5 the than current TRPC4 the us to record for much more difficult it was Initially, channel. aSOC as act and channels TRPC4 or TRPC1 bind with ORAI, and STIM channel, CRAC the for candidates new the that possibilities still are There mice. knockout TRPC4 in were impaired ROC channels induced neurotransmitter- that showed however, studies subsequent I-V curve; rectifying inwardly an showed results those because channels or for CRAC SOC essential is TRPC4 that suggested results first The mice. knockout TRPC4 the with began nels chan of TRPC4 mechanism activation the about debate The I-V curve. typical their showing clearly technique clamp patch with cells endothelial in needed are heterodimer or TRPC1/TRPC4 TRPC4 about experiments careful future, the In channels. TRPC4 express which cells cle mus smooth from of I-V TRPC4 curve double rectifying typical clearly showing currents GPCR-induced to record easy rather is it hand, other On the cells. muscle smooth in to those contrary PtdIns(4,5) PLC, protein, Gq the Gi/o protein, the heteromultimer), or alone (M2, receptor M3 GPCR of consists that caveolae at complex asignal as exist may channels TRPC5 and TRPC4 nels. Figure 5.

The schematic diagram for GPCR-mediated regulation of TRPC4 and TRPC5 chan TRPC5 and TRPC4 of regulation GPCR-mediated for diagram schematic The P 2 SESTD1 and RGS. and SESTD1 Summary Fig. 2 Fig. ). Based on the Based ). α Channels i/o i/o - - - in the inside-out patch mode, the roles of roles PtdIns(4,5) the mode, patch inside-out the in cellular signaling. cellular inrole known their to addition in trafficking and assembly nel chan role in of the Gproteins and PKC desensitization the in GIRK, done for was as Finally, concentration. calcium intracellular with simultaneously recorded be can currents TRPC4 whether ( do for TRPC5 they as of TRPC4 activation for the supply calcium ORAI and STIM whether and complex STIM and TRPC4 activate protein can G whether I-V curve, doubly rectifying expected show the will or ORAI STIM with of TRPC4 co-expression the whether and Technology (MEST). Technology and Science of Education, Ministry of the BK21 from project gram J.P.J.,C.H., J.W. pro S.Y.K. and graduate by the were supported (2008-2005948, 2010-0019472 and 2012R1A2A1A01003073). (MEST) government Korean by the funded of Korea Foundation Research National by the and (A100079) of Korea Republic Affairs, Family and Welfare of Health, Ministry project, R&D 21 Health Korea the from by agrant supported was study This Korea. Seoul, of Medicine, College University National Seoul the at Center Imaging Biomedical of roles the the We acknowledge lar calcium itself, the effect of polyamine and Mg and of polyamine effect the itself, calcium lar lular Ca lular of cel aspects one fundamental of underlie the rearrangements molecular and assembly dynamic their wherein compartments lipid raft into specialized Cav1) and Orai1, localized STIM1 are TRPC4, (TRPC1, major proteins regulated; more complexly

98 - site for intracellu binding the further study we should 2+ signaling. We require more data that demonstrates demonstrates We that more data require signaling. - rafts. In aggregate, SOC channels are perhaps perhaps are channels SOC aggregate, In rafts. Cav1 lipid with in interaction its modulating by preferentially channels of TRPC4 activity the regulate functionally may STIM1 Thus, mediated gating of the channels, of the gating mediated STIM1-for essential as identified been have of TRPCs C-terminus the in (DD) acids amino acidic conserved two Importantly, cells. endothelial in channel aSOC as acts TRPC4 that to confirm technique clamp a patch with currents TRPC4 the record also should we methods, measurement calcium used usually channel aSOC is TRPC4 that showed that results experimental the Because signal. calcium contributeto the may ways - path calcium other that consider we should channels, role of of the TRPC to study ods meth measurement calcium we use When olin1 (Cav1)-binding domain in TRPC4. cave C-terminal putative the within localized Acknowledgments Fig. 5 Fig. ). It will also be important to test to test important be also It). will V 2+ olume 6Issue 5 96 on TRPC4 TRPC4 on which are are which P 2 and and α i2 i2 97 - - - - -

©2012 Landes Bioscience. Do not distribute. www.landesbioscience.com u SZ, Zeng B, Daskoulidou N, Chen GL, Atkin 15. ukumar P, Beech DJ.Stimulation of TRPC5 cationic 14. oshida T, Inoue R,Morii T, Takahashi N, Yamamoto 13. u SZ,Sukumar P, Zeng F, LiJ,Jairaman A,English 12. ong C,KimJ,Jeon JP, Wie J,Kwak M,Ha K,etal.Gs 11. 10. trübing C,KrapivinskyG,L,Clapham 9. TM, Kim YC, Sim JH,RheeJC,KimSJ, 8. YM, KimBJ,HJ, Yang DK,ZhuMH,Lee 7. 6. kada T, Shimizu S, Wakamori M,Maeda A,Kurosaki 5. ilius B, Owsianik G. Transient receptor potential 4. ilius B,OwsianikG. The transientreceptor potential 3. u LJ,Sweet TB, ClaphamDE.International Union 2. 1.

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2+ - - - - -

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