Modulation of High-Voltage Activated Ca 2+ Channels by Membrane Phosphatidylinositol 4,5-Bisphosphate

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1 Article Modultion of High-Voltge Activted 2+ hnnels y Memrne Phosphtidylinositol 4,5-isphosphte yung-hng Suh, 1, * Krin Lel, 2 nd ertil Hille 1 1 Deprtment of Physiology nd iophysics 2 Deprtment of Phrmcology nd Progrm in Neuroiology nd ehvior The University of Wshington School of Medicine, Settle, WA , USA *orrespondence: cs@uw.edu DOI 1.116/j.neuron SUMMARY Modultion of voltge-gted 2+ chnnels controls ctivities of excitle cells. We show tht high-voltge ctivted 2+ chnnels re regulted y memrne phosphtidylinositol 4,5-isphosphte (PIP 2 ) with different sensitivities. Plsm memrne PIP 2 depletion y rpmycin-induced trnsloction of n inositol lipid 5-phosphtse or y voltge-sensitive 5-phosphtse (VSP) suppresses V 1.2 nd V 1.3 chnnel currents y 35% nd V 2.1 nd V 2.2 currents y 29% nd 55%, respectively. Other V chnnels re less sensitive. Inhiition is not relieved y strong depolrizing prepulses. It chnges the voltge dependence of chnnel gting little. Recovery of currents from inhiition needs intrcellulr hydrolysle ATP, presumly for PIP 2 resynthesis. When PIP 2 is incresed y overexpressing PIP 5-kinse, ctivtion nd inctivtion of V 2.2 current slow nd voltgedependent gting shifts to slightly higher voltges. Thus, endogenous memrne PIP 2 supports highvoltge ctivted L-, N-, nd P/Q-type 2+ chnnels, nd stimuli tht ctivte phospholipse deplete PIP 2 nd reduce those 2+ chnnel currents. INTRODUTION Voltge-gted 2+ ( V ) chnnels medite 2+ influx in response to memrne depolriztion nd regulte mny physiologicl phenomen including neurotrnsmission, secretion, muscle contrction, nd gene expression (tterll et l., 25). The ctivity of V chnnels is dynmiclly regulted y receptor-dependent signls, such s G proteins, protein kinses, clmodulin, solule N-ethylmleimide-sensitive fusion ttchment receptor (SNARE) proteins, nd the second messengers 2+ nd rchidonic cid (tterll, 2; Dolphin, 23; Roerts-rowley et l., 29). Here, we nlyze in detil the regultion of three high-voltge ctivted (HVA) 2+ chnnels ( V 1.2, V 1.3, nd V 2.2) y the plsm memrne phospholipid phosphtidylinositol 4,5-isphosphte (PIP 2 ). Signls from G protein-coupled receptors (GPRs) suppress N-type V 2.2 chnnels through two pthwys in sympthetic neurons (Hille, 1994). The fst pthwy is voltge dependent, memrne delimited, nd insensitive to the intrcellulr 2+ cheltor APTA. The fst suppression is induced y ctivting receptors coupled to the pertussis toxin (PTX)-sensitive G proteins G o nd G i. It cn e relieved y pplying lrge positive pulses (en, 1989; Lipscome et l., 1989; Zmponi nd Snutch, 1998) nd is understood s direct voltge-dependent inding of G protein g suunits to N-type ( V 2.2) nd P/ Q-type ( V 2.1) 2+ chnnels (Herlitze et l., 1996; Iked, 1996; Dolphin, 23). y contrst, the slow pthwy is voltge independent, insensitive to PTX, nd sensitive to APTA. Activtion of G q -coupled receptors initites the slow pthwy (ernheim et l., 1991; Delms et l., 25; Michilidis et l., 27; Roerts-rowley et l., 29). While the fst nd slow pthwys oth reduce the current pprecily, neither fully elimintes it. A phenomenologiclly similr slow pthwy lso produces inhiitory modultion of L-type 2+ chnnels nd M-type (KNQ) K + chnnels y G q -coupled receptors in sympthetic neurons (Mthie et l., 1992) nd in reconstituted systems (Shpiro et l., 2; nnister et l., 22). We hve speculted tht the underlying signling for slow suppression of these 2+ nd K + chnnels might e the sme (ernheim et l., 1991; Mthie et l., 1992; Hille, 1994). Here, we sk if some or ll of the slow suppression of 2+ currents is due to receptor-medited depletion of PIP 2, s is true for slow suppression of KNQ K + current (Suh nd Hille, 22; Zhng et l., 23; rown et l., 27). urrents in the V 2 chnnel fmily cn e modulted y exogenous mnipultion of memrne phosphoinositides (see reviews Delms et l., 25; Michilidis et l., 27). Wu et l. (22) concluded tht depletion of memrne PIP 2 underlies significnt rundown of V 2.1 (P/Q-type) currents seen in inside-out excised ptch experiments. They showed tht ppliction of PIP 2 ntiody to the intrcellulr side of gint memrne ptches ccelertes the V 2.1 current rundown, wheres rief ppliction of PIP 2 or Mg-ATP retrds the rundown. However, unexpectedly, they lso reported tht pplied PIP 2 reduced the currents. The inhiitory effect of PIP 2 ws ctully strong positive shift of chnnel voltge dependence (y 4 ). It ws ntgonized y conditions tht ctivted cyclic AMP-dependent protein kinse. In , , July 29, 21 ª21 Elsevier Inc.

2 susequent studies, Gmper et l. (24) reported tht V 2.2 N-type chnnels re lso regulted y PIP 2. Mcroscopic current rundown ws significntly slowed y the ppliction of PIP 2 to the intrcellulr side of excised memrne ptches from Xenopus oocytes. In sympthetic neurons, the current suppression during muscrinic receptor ctivtion ws ttenuted nd slowed y intrcellulr perfusion of short-chin Di 8 -PIP 2. When the N-type 2+ currents were inhiited y G q -coupled receptor ctivtion, current recovery ws locked y 5 mm wortmnnin to inhiit PI 4-kinse. Thus, they proposed tht depletion of PIP 2 on the plsm memrne is the cuse of the G q receptor-medited slow inhiition of N-type 2+ currents in sympthetic neurons. The PIP 2 hypothesis hs never een tested for L-, R-, or T-type chnnels in ny system. In contrst, others hve ttriuted the slow receptor-medited inhiition of oth N-type nd L-type 2+ chnnels to production of rchidonic cid (Liu nd Rittenhouse, 23; Liu et l., 26; Roerts-rowley et l., 29). Thus, whether PIP 2 depletion is mjor physiologicl signl for slow receptor-medited suppression of N- nd L-type 2+ chnnels remins controversil (Michilidis et l., 27). G q -coupled receptor signls re notoriously difficult to dissect ecuse they produce so mny downstrem second messengers. For exmple, in studies of G q modultion of 2+ chnnels, PIP 2 depletion occurs simultneously with the downstrem production of rchidonic cid, ctivtion of protein kinse, nd elevtion of cytoplsmic free 2+, which re ll elieved to hve significnt effects on the chnnels. In order to test the PIP 2 hypothesis unmiguously, we here use two strtegies to deplete PIP 2 enzymticlly nd rpidly without producing the downstrem products of PL. We tke dvntge of two exogenous polyphosphoinositide 5-phosphtse systems tht cn convert PI(4,5)P 2 directly to PI(4)P in the plsm memrne in living cell systems without ctivtion of receptors. One system uses chemicl dimeriztion, nd the other uses memrne depolriztion to ctivte trnsfected 5-phosphtse enzymes tht convert PIP 2 to PIP. In chemicl dimeriztion, ddition of rpmycin or its nlog irp to the extrcellulr medium induces irreversile trnsloction of trnsfected yest INP54p 5-phosphtse from cytosol to memrne, inititing PIP 2 dephosphoryltion (Suh et l., 26; Vrni et l., 26). The trnsloction nd dephosphoryltion tke 1 2 s. The other system uses trnsfected voltge-sensitive phosphtse (VSP), n integrl plsm memrne protein tht ecomes ctive when its N-terminl voltge-sensor domin detects lrge memrne depolriztion (Murt et l., 25; Hlszovich et l., 29; Okmur et l., 29). We find tht the VSP system is fster, depleting plsm memrne PIP 2 within second of ctivtion, nd the enzyme turns off quickly when the memrne is repolrized. Here, we focus on the PIP 2 hypothesis nd reserve exmintion of other messengers for lter work. We consider N-type 2+ chnnels, which together with P/Q-type chnnels were the suject of the previous studies (Wu et l., 22; Gmper et l., 24; Lechner et l., 25), nd we consider two L-type chnnels whose PIP 2 dependence hs not een studied efore. In ddition, we screen the other sutypes of V chnnels. We find tht PIP 2 depletion ttenutes oth L- nd N-type 2+ chnnel ctivity through voltge-independent pthwys, nd increses the susceptiility of the chnnels to voltgedependent inctivtion (VDI). We lso find tht resynthesis of PIP 2 from PIP is needed for V chnnel recovery. Our experiments show tht y itself PIP 2 depletion does depress HVA 2+ chnnel ctivity in living cells. RESULTS Our gol ws to test the hypothesis tht 2+ chnnels respond to depletion nd resynthesis of PIP 2 in living cells. To estlish conditions for recording 2+ chnnel currents, we expressed V 1.3 (1D) or V 2.2 (1) chnnel suunits together with 3 nd 2d1 ccessory suunits in tsa cells nd recorded the whole-cell currents with 2+ s the chrge crrier. Depolrizing voltge steps from holding potentil of 7 evoked inwrd currents crried y 2+ (Figure 1A). rium ws used to minimize 2+ -dependent inctivtion of the current so tht ny current decy oserved during the test pulses would e due primrily to voltge-dependent inctivtion (VDI). As expected, the V 1.3 currents inctivted little during the 4 ms test pulses, wheres the V 2.2 currents inctivted more. Pek current-voltge (I-V) reltions showed tht V 1.3 currents peked ner 1 (n = 6), wheres V 2.2 currents peked ner +1 (n = 8) (Figure 1). These respective pek voltges were used for ll test pulses in susequent experiments to ssy the function of these chnnels, except where indicted. hemicl Trnsloction of PIP 2 5-Phosphtse to the Plsm Memrne Attenutes V urrents We egin with the chemicl dimeriztion system nd irp to deplete memrne PIP 2. In ddition to the chnnel suunits nd the M 1 muscrinic receptor, two dditionl components needed to e cotrnsfected: the memrne-loclized irpinding protein Lyn 11 -FR (LDR) nd the fluorescent cytoplsmic enzyme construct FP-FKP-INP54p (F-Inp). The F-Inp hs PIP 2 5-phosphtse ctivity tht converts PIP 2 to PI(4)P. We showed previously tht when LDR nd F-Inp re rought together t the memrne y ppliction of irp, PIP 2 is irreversily depleted nd PIP 2 -dependent KNQ K + chnnels turn off (Suh et l., 26). This system is suitle for experimentl designs tht enefit from the irreversile PIP 2 depletion tht follows chemicl dimeriztion. As n initil control, when F-Inp is expressed ut the memrne nchor LDR is omitted ( LDR), irp hd little direct effect on the currents (top pnels in Figures 1 nd 1D), lthough oth chnnels were redily inhiited y M 1 receptor ctivtion with the muscrinic gonist. When LDR ws included (+LDR), there were two chnges. First, currents in 2+ chnnels were decresed irreversily y irp (ottom pnels in Figures 1 nd 1D), decrese tht ws less thn hd een seen with muscrinic receptor ctivtion. The second effect ws ltertion of the susequent response to muscrinic receptor ctivtion. Prior PIP 2 depletion y ctivted INP54p 5-phosphtse eliminted further inhiition of V 1.3 current y muscrinic receptors (Figure 1), quite possily ecuse the irreversile depletion of PIP 2 rogtes muscrinic genertion of ll PIP 2 67, , July 29, 21 ª21 Elsevier Inc. 225

3 A V m 2+ current (I-1, na) +5 8 V V 1.3 irp F-Inp (-LDR) F-Inp (+LDR) V ms Reltive current D 2+ current (I+1, na) V irp V 1.3 V Voltge () F-Inp (-LDR) F-Inp (+LDR) Figure 1. PIP 2 Depletion Depresses V 1.3 nd V 2.2 Voltge-Gted 2+ urrents (A) Fmilies of whole-cell 2+ currents elicited y voltge steps from 8 to +5 in 1 intervls (see pulse protocol) in cells expressing V 1.3 (left) nd V 2.2 (right) chnnels. Holding potentil is 7 nd dshed line is zero current. losed rrowheds indicte pek inwrd 2+ currents triggered y the depolrizing test pulses. Til currents re clipped. () Pek current-voltge (I-V) reltions for V 1.3 nd V 2.2 currents in whole-cell recording normlized to the mximum current. Points re men ± SEM ( V 1.3, n = 6; V 2.2, n = 8). ( nd D) urrent modultion y irp (5 mm) nd (1 mm) in cells coexpressing M 1 muscrinic receptors nd F-Inp lone (top, LDR) or with LDR (ottom, +LDR). urrents were recorded in response to test pulses to 1 ( V 1.3) or +1 ( V 2.2) every 4 s. Dshed line is zero current. LDR, memrne nchor protein. F-Inp, PI 5-phosphtse. (E nd F) Percent inhiition of V 1.3 (E) nd V 2.2 (F) currents y irp nd compred to initil currents in cells expressing F-Inp lone ( LDR) or with LDR (+LDR). The ppliction of irp significntly inhiited V 1.3 currents (*p <.5 compred to irp effect without LDR, n = 3 for LDR; n = 4 for +LDR) nd V 2.2 currents (**p <.1 compred to irp effect without LDR, n = 3 for oth LDR nd +LDR). Dt re men ± SEM. See lso Figure S1. E % inhiition irp -LDR V 1.3 * irp +LDR clevge products, including inositol trisphosphte nd clcium signling (Suh et l., 26). On the other hnd, further muscrinic modultion of V 2.2 current remined intct nd reched full mplitude (Figure 1D). Quite possily tht pthwy does not require PIP 2. It might involve G protein g suunits or other products of phospholipses including PLA 2 (Melliti et l., 21; Roerts-rowley et l., 29). As preliminry conclusion, muscrinic inhiition of oth chnnels proly occurs vi more thn one pthwy, nd ny PIP 2 depletion component ccounts for only prt of the totl effect. It is well known tht inhiition of V 2.2 current y M 2 muscrinic receptor-medited signling (the fst Gg pthwy) is strongly relieved y lrge positive prepulses (Elmslie et l., 199). We redily verified this effect in cells trnsfected with M 2 (G i -coupled) rther thn M 1 (G q -coupled) receptors (dt not shown). n the irp-induced inhiition of V 1.3 nd V 2.2 chnnels lso e relieved y strong depolrizing F % inhiition irp -LDR V 2.2 +LDR prepulses? The cells were given ** +13 /2 ms prepulse followed 5 ms lter y 1 ms test pulse to mesure chnnel function. Figure S1 (ville online) shows tht inhiition ws irp unchnged y the prepulses for V 1.3 chnnels (Figure S1A) nd for V 2.2 chnnels (Figure S1). Hence, unlike inhiition of V 2.2 chnnels vi Gg, positive prepulses do not relieve the suppression tht follows irp-induced PIP 2 depletion. Depletion of Memrne PIP 2 y Activtion of Attenutes V urrents We turn now to depleting PIP 2 with the voltge-sensitive phosphtse from zer fish (; Okmur et l., 29). This tool is suitle for experimentl designs tht enefit from reversile PIP 2 depletion following n ctivting depolriztion. First we chrcterized the ility of to deplete memrne PIP 2 y using two fluorescent PIP 2 indictors nd mesuring fluorescence resonnce energy trnsfer (FRET) etween them (vn der Wl et l., 21; Jensen et l., 29). In resting cells, the PIP 2 -inding fluoroproes FP-tgged PH(PLd1) nd YFP-tgged PH(PLd1) ind to the PIP 2 t the plsm memrne in high enough surfce density to generte FRET etween them (Figure S2A). If PIP 2 is depleted, the proes dissocite from the , , July 29, 21 ª21 Elsevier Inc.

4 A V 1.3 Reltive current (/) % inhiition , Δt.8 na V ontrol + PIPKIγ % 2 τ = 112 ± 7ms Durtion t +12 (Δt, s) memrne nd move to the cytosol, losing their FRET interction (vn der Wl et l., 21). In cells cotrnsfected with, ppliction of depolrizing pulses to +12 ctivted the phosphtse ctivity. Using 44 nm light to excite FP, the fluorescence of FP (FP ) incresed nd tht of YFP (YFP ) decresed ech time the depolriztion ws pplied (Figure S2, top), nd PIP 2 depletion ws signled s the corresponding decrese in the FRET rtio (FRET rtio = YFP /FP )(Figure S2, ottom). During the lrge depolriztion, the PIP 2 depletion developed rpidly (exponentil time constnt t = 15 ± 18 ms, n = 9) (Figure S2) nd in voltge dependent mnner (V 1/2 =61± 5, n = 5) (Figure S2D). According to the FRET ssy, the depletion with is comprle to tht seen with M 1 muscrinic receptor ctivtion (Figure S2E). However, it is much fster nd results in quite different clevge products. As ws nticipted from the PIP 2 depletion, ctivtion of decresed current in HVA 2+ chnnels. Our protocol ws to pply stndrd test pulse (pulse ) to record seline chnnel current, then lrge depolrizing pulse for vrious times to ctivte, followed y second test pulse (pulse ) (Figure 2A). In control cells not expressing, V D Reltive current (/) % inhiition V , Δt Depolriztion durtion (s) Depolriztion durtion (s) (Δ t) ( Δt) ms ontrol 1.5 na PIPKIγ.4 na 3 ms ontrol 1.5 na +1 V ontrol PIPKIγ % 6 4 τ = 143 ± 1 ms Durtion t +12 (Δt, s) PIPKIγ.5 na Figure 2. Inhiition of V urrents y -Medited PIP 2 Depletion (A nd ) Typicl trces of V 1.3 (A) nd V 2.2 () currents efore nd fter ctivtion of y depolriztions to +12. ells without Dr- VSP (ontrol), cells trnsfected with, or cells trnsfected with plus PIPKIg received test pulse to 1 (A) or +1 () for 1 ms nd then were depolrized to +12 for zero or.5 s (s mrked), followed y second test pulse. The currents efore () nd fter () the +12 -depolrizing pulse re superimposed. Dshed line is zero current. ( nd D) Time-dependent inhiition of L-type ( V 1.3, ) nd N-type ( V 2.2, D) currents y ctivtion. Top, cells were depolrized to +12 for vrious times nd the reltive current rtio (/) ws mesured in control (open circle, n = 6 14 for V 1.3 nd n = 6 for V 2.2), -expressing (closed circle, n = 6 11 for V 1.3 nd n = 6 for V 2.2) cells, nd plus PIPKIg expressing cells (n = 5 8). The dely etween susequent test pulses ws 1 min. ottom, percent inhiition of currents y timegrded ctivtion of t +12. See formul in text. The current inhiition y 1 s depolrizing pulse is leled in ech figure. Dt re men ± SEM. See lso Figure S2. current mplitudes nd were lmost the sme without ( s) or with (.5 s).5 s depolriztion to +12 (Figure 2A, left). In contrst, in cells expressing, the.5 s depolrizing pulse significntly ttenuted the 2+ current in pulse (Figure 2A, middle). Agin, in the sme cell there ws no significnt chnge in current without the lrge pulse. To exmine whether this induced inhiition of V 1.3 current is cused y PIP 2 degrdtion, we tested the effect of ctivtion in cells trnsfected with the PIP 5-kinse type-1g (PIPKIg). This enzyme elevtes PIP 2 concentrtion in the plsm memrne (Wenk et l., 21) nd therey diminishes the ility of G q -coupled receptors to suppress KNQ K + currents (Suh nd Hille, 27). As shown in Figures 2A nd 2 right, the inhiition of V 1.3 nd V 2.2 chnnels y ws significntly ttenuted y PIPKIg expression. Figure 2 plots the time dependence of the / current rtio (top) nd of the percent inhiition with nd without (ottom). The V 1.3 chnnels were mximlly inhiited y 33% with n onset time constnt t = 112 ± 7 ms (Figure 2, ottom left). Further, PIPKIg overexpression significntly ttenuted the -induced V 1.3 inhiition (Figure 2, top left). We lso performed experiments with N-type V 2.2 chnnels. The ottom line ws similr: V 2.2 chnnels were inhiited y ctivtion with mximum inhiition of 56% nd onset t = 143 ± 1 ms (Figure 2D, ottom). However, in these experiments there ws much more evidence of chnnel inctivtion induced y the voltge protocols. Even in the sence of, the V 2.2 current in pulse ws reduced 67, , July 29, 21 ª21 Elsevier Inc. 227

5 A V , 1 s ,.9 s V , 1 s ,.9 s E +1-8 V , 1 s +1-15,.4 s ontrol 1 ms 1 ms - + Hyperpolriztion.5 na ontrol - + Hyperpolriztion 1nA 1nA 5 ms ontrol Til Reltive current (/) V 1.3 ontrol 33% 31% - + Hyperpolriztion D Reltive current (/) V %.4.2. ontrol 57% - + Hyperpolriztion F % inhiition of til current V 2.2 ontrol Figure 3. hnnel Inhiition y orrected for VDI (A nd ) Effect of memrne hyperpolriztion on -induced inhiition of V 1.3 (A) nd V 2.2 () currents. The chnges of V 1.3 nd V 2.2 currents y +12 depolrizing pulse were mesured without ( ) nd with (+) hyperpolrizing step ( 15,.9 s) in control nd -expressing cells. Pirs of current trces were recorded from the sme cell with 1 min intervl. ( nd D) Summry of the reltive pek current (/) of V 1.3 () nd V 2.2 (D) in control nd -expressing cells with nd without the hyperpolrizing step. Dt re men ± SEM ( V 1.3, n = 5 6; V 2.2, n = 4). The percent difference etween control nd is leled in ech condition. (E) Effect of on V 2.2 til currents. Til currents were mesured with hyperpolrizing step ( 15,.4 s) in control nd -expressing cells. Pirs of current trces were recorded from the sme cell 1 min prt. pcitive nd lek currents were sutrcted y P/5 procedure. (F) Summry of the til-current inhiition (%) of V 2.2 in control nd -expressing cells. Dt re men ± SEM (control, 6.1 ± 7.2, n = 5;, 57.6 ± 5.2, n = 8). y s much s 31% ± 2% (n = 6) y the preceding.5 s depolriztion to +12 (Figures 2 nd D). Moreover, N current ws even prtilly reduced without ( s) the lrge depolrizing pulse. To compenste for such control inctivtion, we clculted the percent inhiition due to ction y the formul 1 {1 (/) VSP /(/) ontrol } t ech time point in this figure nd in susequent figures. This correction would pply ccurtely if the inctivtion seen without is unchnged y the ction of, n ssumption tht we revisit lter. Hence,, like the irp-dimerizle INP54p system, depletes PIP 2 nd leds to prllel depression of currents in three sutypes of 2+ chnnels. We did find tht, for V 1.3 nd V 2.2 chnnels, the men inhiition y ws 35% lrger thn tht with the irp system. In summry, experiments with two lipid phosphtses re consistent with the hypothesis tht PIP 2 regultes V chnnels. Inhiition of V hnnels y Is Not Simple VDI We hve lredy sid tht much of the current inctivtion developing during test pulses in the presence of rium is voltge dependent, VDI. In Figure 3 we exmined the role of VDI in our mesurements nd its possile dependence on PIP 2. We sk whether some prts of the inhiition y re simply n enhncement of VDI y testing whether the inhiition cn e removed y lrge hyperpolrizing pulse. The.9 s hyperpolrizing voltge step to 15 ended.1 s efore test pulse (see pulse protocols in Figures 3A nd 3). In control cells, V 1.3 currents showed no or very minor VDI from the +12-/1 s depolrizing pulse, i.e., in control cells, currents nd were very similr even without the hyperpolriztion. With the 15- hyperpolrizing voltge step, the minor VDI ws olished (Figure 3A, top right). In cells expressing, V 1.3 current ws strongly reduced compred to, without nd with the hyperpolrizing step (Figures 3A, ottom, nd 3). Thus, V 1.3 chnnels hd little residul VDI from our pulse protocol, nd the inhiitory effect of ctivtion ws not relieved y hyperpolriztions. In control cells expressing V 2.2 chnnels, there ws some residul VDI fter pulse (Figure 3, top left). This mde the pulse currents 3% smller (Figures 3 nd 3D). The reduction ws totlly relieved , , July 29, 21 ª21 Elsevier Inc.

6 A -8 ontrol ontrol +12, 1 s -15,.4 s V pa 1 ms V pa 3 pa 3 pa I+1 (pa) I (pa) V V Figure 4. Screening V Sutypes for Modultion y nd M 1 Muscrinic Receptors (A) Inhiition of V 1.2 nd 2.1 currents y -induced PIP 2 depletion. V currents were mesured during test pulses efore nd fter +12 /1 s depolrizing pulse in control nd -expressing cells. Typicl current trces for ech chnnel type re superimposed. () Inhiition of V 1.2 nd 2.1 currents y M 1 muscrinic receptor stimultion with (1 mm). Insets, Typicl trces efore nd fter ppliction were superimposed. () Inhiition of V currents y -induced PIP 2 depletion (top) nd M 1 muscrinic receptor stimultion with (). ND, not determined. *The V 2.3 current ws enhnced 2.7 ±.5-fold (n = 4) y the ctivtion of M 1 receptors. Dt re men ± SEM. See lso Figure S4. % inhiition of V current V ND ND 1.1 (L) 1.2 (L) 1.3 (L) ND 1.4 (L) 2.1 (P/Q) 2.2 (N) 2.3 (R) 3.1 (T1) y the 15-/.9 s hyperpolriztion. Indeed, the current in pulse ecme lrger thn tht in pulse s if the hyperpolriztion ws lso relieving some resting inctivtion tht hd reduced pulse current (Figure 3, top right). With, the lrge depolrizing pulse strongly depressed current in pulse s efore. Agin when compred to the control cells, the hyperpolrizing step did not relieve ny of the effect of VSP (Figure 3D). V 2.2 chnnels show prominent til currents. Therefore, we lso could test whether the til currents were inhiited y. Figures 3E nd 3F show strong inhiition in -expressing cells. The inhiition of til currents ws similr to tht of inwrd currents during test pulses. Thus for V 1.3 nd V 2.2 chnnels, the depression of current due to ctivtion did not seem to e some kind of enhncement of VDI. Sutype-Specificity of PIP 2 Modultion of V hnnels We screened for chnnel modultion y expressing different 1 suunits with the sme 3 nd 21 chnnel suunits s efore. * (T2) (T3) Figure 4A shows tht the V 1.2 nd V 2.1 chnnel currents were lso significntly inhiited y ctivtion lthough less thn for V 2.2 chnnels. Since the current-voltge reltions for V 1.2 nd V 2.1 peked t +1 nd, respectively (Figure S3A), those voltges were used for the test pulses in these experiments. The V 1.2 chnnels gve currents similr to those with V 1.3 nd showed lmost the sme inhiition y muscrinic ctivtion (62% ± 8% for V 1.2, n = 5; 59% ± 3% for V 1.3, n = 6) (Figures 4 nd 4, ottom). Furthermore, the effects of ctivtion were very similr (Figure 4, top). With V 1.2 chnnels, the lrge depolrizing pulse produced 35% inhiition of current developing with n onset time constnt t = 138 ± 18 ms (n = 6). V 2.1 chnnels re inhiited y oth nd M 1 receptor stimultion (Figure 4). However, the other sutypes of V chnnels, 1.4, 2.3, nd ll V 3 (T-type), were insensitive to ctivtion (Figures 4 nd S3). Interestingly, the four PIP 2 -depletion-sensitive chnnels were lso strongly inhiited y M 1 receptor ctivtion with, nd the other chnnels were not significntly inhiited y. Indeed V 2.3 R-type currents were strongly enhnced (Figure S3). PIP 2 -Dependent Modultion of V 2.2 hnnels PIP 2 dependence of V current modultion ws investigted in more detil with V 2.2 chnnels. When the cells overexpressed the PIP 2 -inding peptide scvenger, PH domin of PLd1, the current density in the trnsfected cells ws significntly decresed compred to control cells expressing only GFP or cells expressing PH domin of Akt protein which inds to PI (3,4)P 2 nd PIP 3 in the plsm memrne (Figure 5A). Next, we tested if PIP 2 elevtion ove its norml level ttenutes the muscrinic suppression of the V 2.2 chnnels. Overexpression of PIPKIg significntly ttenuted the M 1 muscrinic receptorinduced inhiition (Figure 5) s well s the -induced V 2.2 inhiition (Figure 2). The muscrinic inhiition without PIPKIg ws 72% ± 8% (n = 6), nd with PIPKIg it ws reduced to 35% ± 7% (n = 6). 67, , July 29, 21 ª21 Elsevier Inc. 229

7 A tsa cells + M 1 receptor V 2.2 (pa/pf) GFP ontrol GFP-PH PLδ * PH PLδ YFP-PH Akt PH Akt V 2.2 current (pa) ontrol PIPKIγ % inhiition y ontrol * PIPKIγ Reltive N-current SG neurons Voltge () 6 D +12, 1 s -8-15,.4 s ontrol 1 pa/pf 1 pa/pf Reltive current (/) ontrol 28% Figure 5. PIP 2 -Dependent Modultion of V 2.2 N-Type hnnels (A) V 2.2 current density (pa/pf) ws mesured in cells expressing the V 2.2 chnnels plus GFP, GFP-PH-PLd1, or YFP-PH-Akt. The cells were trnsfected with the sme mounts of cdna. Averge memrne cpcitnces for cells re 22 ± 2 pf for GFP (n = 11), 25 ± 4 for PH-PL (n = 11), nd 24 ± 4 for PH-Akt (n = 12). Top, confocl imges of tsa cells expressing ech fluorescent protein. ell dimeters were 2 3 mm. Dt re men ± SEM. () Elevted PIP 2 levels ttenute V 2.2 chnnel inhiition y M 1 receptor stimultion. V 2.2 currents were mesured in control cells nd in cells trnsfected with PIPKIg. Right, Summry of current inhiition y. Dt re men ± SEM. *p <.5, compred to control. () urrent-voltge (I-V) reltions of N-type V current in isolted rt SG neurons expressing (n = 3) with widefield imge of one neuron. Dt re men ± SEM. (D) Inhiition of N-type V currents y ctivtion in SG neurons. N-type V currents were mesured during test pulses efore nd fter +12 /1 s depolrizing pulse in control nd cells expressing. pcitive nd lek currents were sutrcted y P/5 procedure. Right, Summry of N-type current inhiition y ctivtion in SG neurons. Dt re men ± SEM (n = 3 for control, n = 3 for ). Finlly, we tested if PIP 2 depletion could decrese the endogenous V 2.2 N-type current of sympthetic superior cervicl gnglion (SG) neurons. Figure 5 shows the current-voltge reltionship of N-type current in SG neurons expressing. The current peked t 1. When the ws ctivted y +12 /1 s-depolriztion, it inhiited the N-type current y 28% in SG neurons (Figure 5D). Thus memrne PIP 2 is lso importnt for V chnnel ctivity in differentited neurons. Recovery from -Induced Inhiition Requires Intrcellulr ATP nd PIP 2 Resynthesis After the -induced inhiition, current recovered in <1 min. We tested the need for PIP 2 synthesis in chnnel recovery. urrent ws elicited with the voltge protocols shown in Figures 6A nd 6E. ells were given 1 ms test pulse to mesure the initil current (I ), then depolrized to +12 for 1 s to ctivte. Finlly, recovery from inhiition ws mesured y pplying 1-ms test pulses with successively longer dely fter VSP ctivtion strting t.5 s s indicted ove the trces. As efore, in control cells expressing V 1.3 chnnels, the current ws only slightly inhiited y the lrge depolrizing pulse (Figure 6A, top). With, the V 1.3 current ws reduced y the depolriztion nd recovered to the initil level with recovery time constnt t of 5.9 s (Figures 6A, ottom, nd 6). In control cells expressing V 2.2 current, the current ws reduced y VDI nd then recovered (Figure 6E, top). In cells with, the current ws strongly inhiited y the lrge depolriztion nd, fter correcting for control VDI, recovered with time constnt t of 16.1 s (Figures 6E, ottom, nd 6F), slower thn for V 1.3 chnnels. The slower recovery of V 2.2 compred to V 1.3 did not seem to e due to some form of slowly 23 67, , July 29, 21 ª21 Elsevier Inc.

8 A +12, 1 s -8-1 V 1.3 V m () 1. V 1.3 ontrol 1. V PIPKIγ ontrol D 1. V 1.3 (+ AMP-PP) ontrol I (s) ontrol I (s) 3 ms Reltive current (I/I ) % current 36% inhiition τ = 5.9 s (s) Reltive current (I/I ) (s) % current 1 21% inhiition τ = 1.5 s Reltive current (I/I ) % current1 35% inhiition τ = 2.5 s (s) E +12, 1 s -8 V V m () F 1. V 2.2 ontrol G 1. V PIPKIγ ontrol H 1. V 2.2 (+ AMP-PP) ontrol I (s) ontrol I (s) 3 ms Reltive current (I/I ) % current 56% inhiition 1 8 τ = 16.1 s (s) Reltive current (I/I ) % current 34% inhiition 1 8 τ = 12.5 s (s) Reltive current (I/I ) % current 52% inhiition τ = 31.7 s (s) Figure 6. Recovery of V urrents fter -Induced Inhiition (A nd E) urrent trces for V 1.3 (A) nd V 2.2 (E) chnnels in control (top) nd -expressing (ottom) cells efore nd fter +12 /1 s depolrizing pulse. V 1.3 nd V 2.2 currents were mesured t 1 nd +1, respectively, t the indicted times fter the +12- pulse. Dshed lines indicte zero current, nd dotted lines, the initil V current efore the depolriztion step. ( nd F) Time course of recovery of V 1.3 nd V 2.2 currents fter the -induced inhiition in control (open circles) nd -expressing (closed circles) cells. Dt re men ± SEM ( V 1.3, n = 6 for oth control nd ; V 2.2, n = 5 for oth). Inset shows % current reltive to control cells. ( nd G) Time course of V 1.3 nd V 2.2 current recovery in cells trnsfected with PIPKIg ( V 1.3, n = 5 for control nd n = 8 for ; V 2.2, n = 4 for control nd n = 5 for ). Inset shows % current, compring to control cells. Dt re men ± SEM. (D nd H) Time course of V 1.3 nd V 2.2 current recovery with 3 mm AMP-PP insted of ATP in the pipette solution. Dt re men ± SEM ( V 1.3, n = 7 for control nd n = 1 for ; V 2.2, n = 6 for control nd n = 5 for ). Insets show the % current recovery in the presence of AMP-PP. See lso Figure S4. recovering VDI, since it ws not significntly relieved or speeded y 15- hyperpolrizing step (Figure S4A). We next tested the hypothesis tht PIP 2 resynthesis is needed for V current recovery from the -induced inhiition. First we speeded resynthesis. oexpression of the 5-kinse PIPKIg with V 1.3 or V 2.2 chnnels significntly decresed the current inhiition with the +12 /1 s pulse nd speeded the current recovery (Figures 6, 6, 6F, nd 6G). Next we slowed PIP 2 resynthesis. The synthesis of PIP 2 from PI(4)P requires intrcellulr ATP, so we slowed the kinse ctivity y dilyzing the nonhydrolyzle ATP nlog AMP-PP into the cell. The inclusion of 3 mm AMP-PP insted of ATP in the pipette solution did not significntly ffect mximum chnnel inhiition ut strongly slowed the recovery of oth V 1.3 current (t =21s)(Figure 6D) nd V 2.2 current (t =32s) (Figure 6H) nd diminished the mximum recovery (for trces, see Figure S4). As expected, dilyzing with AMP-PP lso strongly slowed nd depressed PIP 2 resynthesis s mesured y FRET with PH-domin proes (Figure S4). With ATP, the -induced FRET rtio chnges recovered lmost completely (94% ± 3%) with time constnt t of 6.4 ±.9 s (n = 11), wheres with 3 mm AMP-PP the recovery fter one lrge depolrizing pulse ws smller (only 58% ± 3%) nd slower with time constnt t = 32 ± 4 s (n = 5), nd there ws no recovery fter second depolrizing pulse s if the lst remining ATP hd een exhusted (Figure S4D). Inclusion of nother nonhydrolyzle ATP nlog AMP-PNP gve similr retrdtion of the FRET recovery (dt not shown). In summry, we find tht chnnel recovery fter PIP 2 depletion is fster when PIP 2 synthesis is speeded nd slower nd incomplete when PIP 2 synthesis is slowed, implying tht PIP 2 resynthesis underlies V chnnel recovery from the VSP-medited inhiition. Simultneous Mesurements of hnnel Modultion nd PIP 2 Degrdtion in the Sme ells A puzzling finding ws tht recovery of V 1.3 chnnels (Figure 6) closely prlleled tht of FRET rtio mesured in seprte experiments (Figure S2), wheres recovery of V 2.2 chnnels ws slower thn tht of FRET rtio. ould it e tht ecuse we studied one set of cells expressing PH domins nd different sets of cells expressing the chnnels, the comprison ws not 67, , July 29, 21 ª21 Elsevier Inc. 231

9 A -8 V , Δt -1-8 V , Δt +1 V , 1 s +2-1 FRET rtio urrent (Δt) s FRET rtio urrent.5 na (Δt) s FRET rtio urrent Reltive response -1. V 1.3 current FRET rtio Time t +12 (s) 1. Reltive response -1. V 2.2 current FRET rtio Time t +12 (s) 1. Reltive response V 1.3 current FRET rtio Voltge () 1 12 D , 1 s -1 V 1.3 V m () E , 1 s +1 V 2.2 V m () F +12, 3 s V 1.3 (+ AMP-PP) urrent FRET rtio I , 1 s s urrent FRET rtio.3.2 I 12, 1 s s Scled recovery V 1.3 current FRET rtio V 1.3 (+ AMP-PP) +12, 5 s Scled recovery V 1.3 current FRET rtio Scled recovery V 2.2 current FRET rtio Scled recovery V 1.3 current FRET rtio Figure 7. Simultneous Mesurement of V urrent Modultion nd PIP 2 Depletion in Single ells All cells coexpress chnnel suunits, PH-domin proes, nd. (A nd ) Single-cell mesurements of FRET rtio signls nd whole-cell current from V 1.3 (A) or V 2.2 () chnnels. Top, time-dependent induction of effect on current nd FRET rtio mesured simultneously in single cells. ottom, superimposed time courses of current inhiition nd FRET rtio decrese, normlized. () Voltge dependence of ction on V 1.3 current nd FRET rtio chnge in single cell. (D nd E) Time course of recovery of FRET rtio signls nd whole-cell current of V 1.3 (E) or V 2.2 (F) chnnels in single-cell experiments. Top, recovery of currents nd FRET rtio from the -induced chnges ws mesured simultneously in single cells. ottom, superimposed recoveries of current nd FRET rtio in single cell. (F) AMP-PP in the pipette solution ttenutes the recovery of V 1.3 current nd FRET rtio. A single cell dilyzed with 3 mm AMP-PP ws given 3 s or 5 s depolrizing pulse nd current nd FRET rtio were mesured simultneously , , July 29, 21 ª21 Elsevier Inc.

10 A α1 Wildtype α1: N 1 α1 himeric α1-1: N 1 Reltive current V 2.2 (α1) V 2.2 (α1-1) Voltge () N-current (I+1, pa) V 2.2 (α1) + M 2 receptor -4-8 V 2.2 (α1-1) + M 2 receptor % inhiition y 1 α1 None M 2 -receptor α1 α1-1 D V 2.2 (α1-1) +12, Δt Depolriztion durtion (s) (Δt) 1 1 ontrol 3 pa 3 ms 15 pa E % inhiition None (-s) 1-s pulse 56% inhiition ontrol F Reltive response (I/I ) V 2.2 (α1-1) +12, 1 s s.2 ontrol (s) % current Figure 8. Modultion y in Gg-Insensitive himeric V 2.2 hnnel (A) Normlized pek current-voltge (I-V) reltions of wild-type V 2.2 (1) nd chimeric V 2.2 (1-1) chnnels in the whole-cell configurtion. urrents were elicited y voltge-steps from 4 to +4, in 5 intervls, from holding potentil of 8. Points re men ± SEM (n = 5 for oth chnnels). () Time course of M 2 muscrinic receptor ction (, 1 mm) on wild-type V 2.2 (1) (top) or V 2.2 (1-1) (ottom) chnnels. The current mplitude ws mesured t +1 every 4 s. () Summry of the muscrinic inhiition of V 2.2 (1) nd V 2.2 (1-1) currents y M 2 Rs. Dt re men ± SEM ( V 2.2 [1] lone, n = 5; V 2.2 [1] with M 2 receptors, n = 4; V 2.2 [1-1] with M 2 receptor, n = 5). (D) Inhiition of chimeric V 2.2 (1-1) currents y. Typicl trces of V 2.2 (1-1) currents efore nd fter ctivtion y depolriztion to +12. ontrol (left) nd -expressing (right) cells received test pulse nd then were depolrized to +12 for zero or 1 s (Dt), followed y the second test pulse (). The currents efore () nd fter () the depolrizing pulse superimposed. (E) Summry of the current inhiition (%) y the +12- depolrizing pulse in control (n = 8) nd -expressing (n = 5) cells. Dt re men ± SEM. (F) Time course of current recovery from -induced inhiition. ells were depolrized to +12 for 1 s, nd the recovery of currents ws mesured. Inset shows the percent current from compring control nd -expressing cells. Dt re men ± SEM (n = 6). vlid? It seemed necessry to cotrnsfect PH domins nd chnnels nd to mesure the current nd FRET rtio simultneously in the sme cell. The following experiments show in simultneous recordings tht the close prllels etween effects on V 1.3 currents nd FRET rtio chnges persist. Figure 7A mesures the onset of the effects with depolriztions of different durtion (Dt). The durtion dependence ws indistinguishle (Figure 7A, ottom), nd the rtio of the time constnts for onset (t current /t FRET) ws 1.2 ±.9 (n = 5). Figure 7 shows the dependence on the voltge of the pulse for VSP ctivtion; the mid-point voltge ws 58 for oth responses. Similrly, the recovery time courses of current nd FRET rtio fter termintion of the depolrizing pulse were the sme (time constnt rtio.91 ±.9, n = 5; Figure 7D); when AMP-PP replced ATP in the pipette, the recoveries remined prllel lthough much slowed (Figure 7F). On the other hnd, in similr simultneous recording experiments with V 2.2 chnnels, differences in time course persisted. The durtion dependence for onset showed quicker loss of current thn of FRET rtio (time constnt rtio.65 ±.4, n = 4; Figure 7), nd the recovery fter showed slower recovery of current (time constnt rtio 3.7 ±.7, n=6;figure 7E). These rtios ought to e interpreted cutiously since it ws not possile to correct for confounding VDI in these experiments. Modultion of V 2.2 urrents y Is Not Gg inding V 2.2 (N-type, 1) chnnel currents cn e suppressed y memrne Gg suunits in voltge-dependent mnner (Dolphin, 23). Might the -medited chnnel modultion e due in prt to enhnced inding of Gg suunits to V , , July 29, 21 ª21 Elsevier Inc. 233

11 A V , 1 s -1-1 ontrol τ of inctivtion (ms) 2 1, V , 1 s , ontrol 25 ms V 1.3 V ontrol 4 2 ontrol.8 na *.8 na D N-current (pa) V V 2.2 ontrol 1 1 ontrol 2 PIPKIγ +1 PIPKIγ Time (ms) 2 25 ms 4 Reltive current E τ of inctivtion (ms) ontrol V 2.2 ontrol PIPKIγ Test pulses () V 2.2 PIPKIγ Voltge () Figure 9. PIP 2 Depletion Affects hnnel Inctivtion nd Activtion (A) Effect of -induced PIP 2 depletion on the rte of inctivtion of V 1.3 (left) nd V 2.2 (right) currents. V currents were mesured during 5 ms test pulses to 1 ( V 1.3) or +1 ( V 2.2) efore () nd fter () +12 /1 s depolrizing pulse in control cells (top) nd in cells expressing (ottom). Green lines ( ) re current trces scled to the pek mplitude of current. ottom, summry of the time constnts for current inctivtion (n = 5, *p <.1, compred to current ). Dt re men ± SEM. () Elevted PIP 2 levels slow V 2.2 chnnel inctivtion. V 2.2 currents during 5 ms test pulses to +1 or +3 were mesured in control cells with no nd in cells trnsfected with PIPKIg. Typicl current trces for ech test voltge re overlid. () Summry of inctivtion time constnts (t) for V 2.2 current during the +1 nd +3 test pulses with different PIP 2 levels in the plsm memrne. Dt re men ± SEM (n = 5 for control, n = 7 for PIPKIg t oth test pulses). (D) Elevted PIP 2 levels slow V 2.2 chnnel ctivtion. Activtion of V 2.2 chnnels during the depolriztion to +1 ws mesured in control cells with no nd in cells cotrnsfected with PIPKIg. urrents showing similr mplitude re superimposed. (E) urrent-voltge (I-V) reltions of V 2.2 chnnels in control (open circle) nd PIPKIg-trnsfected (closed circle) cells. Reltive currents re plotted ginst test potentil. Points re men ± SEM (n = 8 for control; n = 9 for PIPKIg). See lso Figure S5. chnnels when memrne PIP 2 is depleted? As test we took dvntge of the Gg suunit-insensitive chimeric chnnel construct clled 1-1 (Agler et l., 25). In this construct, the N terminus of V 2.2 (N-type, 1 suunit), which includes one of the Gg inding sites, is replced y the N terminus of V 1.2 (L-type, 1 suunit) (Figure 8A). When expressed in tsa cells, these chimeric chnnels ctivted in the sme voltge rnge s wild-type V 2.2 chnnels ut could not e inhiited y stimultion of M 2 (G i -coupled) muscrinic receptors (Figures 8 nd 8). The wild-type V 2.2 chnnels were redily inhiited (78% ± 5%, n = 5). In cells expressing the chimeric chnnels nd, +12-/1 s depolrizing pulse strongly inhiited the current y 56% when corrected for VDI seen in control cells (Figures 8D nd 8E). Following the inhiition, the chimeric chnnels recovered with time constnt t of 15 s (Figure 8F), comprle to the wild-type chnnels (Figure 6F). These experiments give no evidence for enhnced inding of Gg suunits to the chnnel when PIP 2 is depleted. They lso show tht replcing the N terminus of the V 2.2 with tht of V 1.2 suunits does not chnge PIP 2 -medited chnnel modultion. Does PIP 2 Depletion hnge hnnel Gting Properties? A preliminry exmintion reveled only modest effects on chnnel gting s plsm memrne PIP 2 ws depleted or rised. Some speeding of the development of VDI y PIP 2 depletion is shown in Figure 9A, gin using our / pulse protocols ut with longer test pulses. In control cells without, the +12 /1 s depolrizing pulse hd no effect on the time constnt of VDI development during the susequent 5 ms test pulse (Figure 9A, top pnels). For V 1.3 chnnels, the time constnt (t) of inctivtion ws 2 ± 27 s (n = 5) versus 193 ± 22 s (n = 5), in pulses nd, respectively, nd for V 2.2 chnnels, t vlues were 44 ± 1 s versus 42 ± 1 s (n = 5). However, with, the time constnt of inctivtion ws shortened, especilly for V 2.2 chnnels (49 ± 4 s versus 25 ± 2 s for currents nd, n = 5, *p <.1) (Figure 9A, ottom right). We note tht test-pulse depolriztions to +1 produced no chnge in PH-domin FRET signls nd thus do not ctivte (Figure S2D). For V 2.2 chnnels, we lso explored effects of n increse in memrne PIP 2 levels , , July 29, 21 ª21 Elsevier Inc.

12 y overexpressing the enzyme PIPKIg. With elevted PIP 2, the development of VDI ws slowed y 1.7-fold t +1 nd slowed y 2-fold t +3 compred to control (Figures 9 nd 9). The ctivtion of V 2.2 chnnels ws lso slowed with expression of PIPKIg, delying the time to pek current (Figures 9D, S5A, nd S5). Finlly, effects on the voltge dependence of ctivtion were smll. When PIP 2 ws depleted y the comintion of nd AMP-PP, the voltge dependence of ctivtion of V 1.3 chnnels ws not chnged nd tht for V 2.2 chnnels showed sttisticlly insignificnt left shift (Figures S5 nd S5D). On the other hnd, when PIP 2 ws ugmented y PIPKIg, the current-voltge reltion for V 2.2 chnnels ws significntly right shifted y 5 7 (Figure 9E). None of these smll gting chnges is sufficient to ccount for the lrge depression of currents tht we hve descried following PIP 2 depletion. Rther it seems tht with reduced PIP 2, fewer V chnnels re ville to open. DISUSSION Using direct enzymtic methods to modify PIP 2 levels quickly in living cells, we hve developed compelling support for the hypothesis tht the endogenous PIP 2 of cell mintins high ctivity of V 1.2, V 1.3, V 2.1, nd V 2.2 chnnels nd tht physiologicl reductions of PIP 2 immeditely decrese the chnnel ctivity: (1) irreversile depletion of endogenous memrne PIP 2 using irp-induced trnsloction of INP54p 5-phosphtse to the plsm memrne irreversily decresed the whole-cell V currents. (2) Reversile depletion of memrne PIP 2 y the ctivtion of reversily decresed V currents in less thn 1 s, with little chnge in voltge-dependent chnnel gting. (3) Elevting levels of memrne PIP 2 y trnsfecting with PIP 5-kinse significntly lunted the chnnel inhiition y nd ccelerted recovery from inhiition 4-fold. (4) Attenuting the endogenous PIP 5-kinse ctivity using nonhydrolyzle ATP nlogs hd prllel inhiitory effects on mesured PIP 2 resynthesis nd on recovery of chnnels from inhiition. And (5) the -medited PIP 2 depletion nd the chnnel inhiition, s well s susequent PIP 2 resynthesis nd chnnel recovery, developed with similr time courses in single cells. This correspondence ws especilly tight for V 1.3 chnnels. The loss of V 1.3 current trcks the loss of PIP 2 within milliseconds. Together these new oservtions show tht the ctivities of severl HVA V chnnels depend on endogenous memrne PIP 2 in intct cells. This is the first direct evidence tht L-type chnnels prticipte in such regultion. As cvet, we note tht we re reporting tests with the 3 nd 2d1 ccessory suunits nd specific splice vrints of the 1 suunits. Since severl forms of chnnel modultion re known to e ffected y the sutypes of ech suunit, the quntittive conclusions here cn e pplied strictly only to the suunits we ctully tested (e.g., Ringo et l., 27). Memrne PIP 2 Is Modultory ofctor for V hnnel Activity The centrl question tht motivted our study is whether downstrem signl of PL, PIP 2 depletion, is importnt for signling to V chnnels. We hve studied the most prominent V chnnel types of ntive rt SG neurons 1/3/2d1 ( V 2.2e[37]) nd 1D/3/2d1 ( V 1.3e) (Lin et l., 1996; ell et l., 24) in reconstituted systems. Our dt show how memrne PIP 2 turnover modultes these HVA V chnnels in living cell memrnes nd revel similrities nd novel differences etween vrious sutypes of V chnnels in the modultion y PIP 2. The ctivity of these chnnels is significntly decresed y conversion of PIP 2 to PIP nd remins inhiited until the PIP 2 is resynthesized from PIP y endogenous PIP 5-kinses. Thus the nionic phosphoinositide PIP 2 is cofctor required for full chnnel ctivity. Our dt suggest tht the chnnels must e in equilirium with plsm memrne pools of PIP 2 on time scle much shorter thn the 1 ms tht it tkes for to depress their currents. There must e protein-lipid inding interction of low ffinity. The short time intervening etween PIP 2 dephosphoryltion nd the chnnel response rgues ginst indirect ctions such s downregultion of chnnels y endocytosis. The mgnitude of inhiition is greter vi M 1 receptors thn with exogenous 5-phosphtse-dependent PIP 2 depletion. Our experiments with intct cells did not duplicte ll the phenomen reported for excised ptches with V 2.1 nd V 2.2 chnnels (Wu et l., 22; Gmper et l., 24). In the excised ptch experiments, til currents rn down nerly 1% in 2 min, nd lmost ll of the til current could e restored y direct ddition of PIP 2. Further, the ddition of PIP 2 induced rightwrd shift y 4 in the voltge dependence of chnnel ctivtion. Finlly the rightwrd shift ws locked in conditions fvoring phosphoryltion y camp-dependent protein kinse. In the intct-cell experiments of Gmper et l. (24) nd in our experiments, depletion of PIP 2 suppressed current only prtilly nd ny rightwrd shift with excess PIP 2 ws <1. Perhps in whole-cell experiments, some chnnel phosphoryltions re preserved, or possily other cytoplsmic fctors mke the chnnels less drsticlly PIP 2 sensitive. Perhps lso continuing PIP 2 synthesis prevents full depletion of the PIP 2. Indeed, when we did experiments with nonhydrolyzle ATP nlogs, the inhiition y VSP tended to e lrger nd cumultive. Activting removes the 5 phosphte from PIP 2 nd produces trnsient rise of memrne PI(4)P tht then decys s it is converted ck into PIP 2 (Hlszovich et l., 29). Our finding tht chnnel currents fll during the trnsient PIP 2 depletion mens tht PI(4)P is not s effective s PIP 2 in supporting chnnel ctivity. Since currents re inhiited y only 33% 6% for the three chnnels studied, it is still possile tht the elevted PI(4)P or other cidic phospholipids lso support chnnel ctivity ut significntly less well thn resting levels of PIP 2. Direct experiments with other enzymes would e needed to test tht hypothesis. In our experiments, N-type chnnels were inhiited more thn L-type chnnels y PIP 2 depletion, oth y irp-induced trnsloction nd y. Unexpectedly, the mximum inhiition y ws 25% 35% lrger thn the inhiition y irp-induced phosphtse trnsloction. We suggest tht the difference rises from smll sl PIP 2 5-phosphtse ctivity of the trnsloctle INP54p tht prtilly depletes memrne PIP 2 lredy efore rpmycin is dded. According to our kinetic 67, , July 29, 21 ª21 Elsevier Inc. 235

13 models (Suh et l., 24), even 1% resting ctivity t the memrne would lower the PIP 2 level y 2%, enough to reduce the chnnel currents prtilly. This would mke the susequent irp-induced inhiition smller. Thus, we estimte using the results tht out 55% of V 2.2 nd 35% of V 1.2 nd V 1.3 current is lost when endogenous PIP 2 is depleted. y comprison, out 75% of V 2.2 nd 55% 65% of V 1.2 nd V 1.3 current is lost when M 1 muscrinic receptors re ctivted. We propose tht significnt frction ut not the entire muscrinic inhiition is due to PIP 2 depletion. Previously, we nd others showed tht ctivtion of M 1 or M 3 muscrinic receptors significntly depletes memrne PIP 2 (Willrs et l., 1998; Horowitz et l., 25; Winks et l., 25; Jensen et l., 29). The depletion is >>9% s ssyed y trnsloction or loss of FRET from PH-domin proes nd y direct iochemicl methods. In this multiple-pthwy theory of muscrinic inhiition, there would lso e severl components to the postgonist recovery s ech of the underlying messenger systems mke its own recovery. ommonly discussed dditionl cndidte messengers tht do ct on V 1or V 2 fmily chnnels re divlent ions, Gg suunits, rchidonic cid, nd protein kinse (Delms et l., 25; Michilidis et l., 27; Roerts-rowley et l., 29). The lterntive hypothesis, which we regrd s unlikely on kinetic grounds, is tht stronger inhiition y PL simply reflects n inility of our phosphtses tools to deplete PIP 2 s much s PL does. Mechnisms for the PIP 2 Actions on V hnnels Wht is PIP 2 loss doing to chnnels to decrese the net current they crry? We considered two possiilities with negtive results. We considered whether PIP 2 loss mkes chnnels more susceptile to modultion y Gg suunits. This possiility seems unlikely oth ecuse the PIP 2 effect ws unchnged in mutnt chnnels where the Gg inding site ws crippled nd ecuse the inhiition y PIP 2 depletion ws not relieved y lrge positive fcilitting pulses the wy Gg inhiition would e. We lso considered whether PIP 2 loss enhnces VDI enough to ccount for the suppression of 2+ currents. Although PIP 2 depletion did speed development of VDI nd PIP 2 ugmenttion slowed it, the component of inhiition due to PIP 2 depletion ws not reversed y lrge hyperpolrizing conditioning pulses tht were sufficient to remove norml VDI. Our kinetic results with suggest tht V 1.3 chnnel ctivity follows chnges in the memrne PIP 2 level closely in simple liner mnner. They cn e descried y model with low-ffinity, rpid, first order, noncoopertive inding of PIP 2 to V 1.3 chnnels, where ech ound PIP 2 contriutes certin increment to the chnnel ctivity. Our dt would e consistent with model hving only one fcilittory PIP 2 site on V 1.3 chnnels, ut there lso could e severl. The V 2.2 chnnels ehve differently. Their ctivity possily flls fster thn PIP 2 is depleted nd certinly recovers much slower thn PIP 2 is regenerted. Such ehvior could reflect comintion of need for more thn one ound PIP 2 for ctivity, slow reinding of PIP 2 to chnnels, or the involvement of other PIP 2 -sensitive messenger signls. Working with V 2.1 chnnels, Wu et l. (22) considered model with two PIP 2 inding sites, one with fcilittory nd the other with inhiitory effects. Our experiments were done very differently from theirs, nd our dt re insufficient to discuss such detils, ut we did not encounter ny compelling evidence for inhiitory ctions of PIP 2. The structure, numer, nd influences of PIP 2 inding sites on ny ion chnnels re questions for future work, ut s working hypothesis we would consider model with t lest two fcilittory sites on the V 2.2 chnnel complex oth of which need to e occupied to see enhncement y PIP 2. Tht would mke coopertive kinetics in which chnnel ctivity flls fster thn PH domin FRET rtio during inhiition nd rises more slowly thn FRET rtio during recovery. onclusions Slow modultion of 2+ chnnels y M 1 muscrinic receptors nd more generlly y ny G q -coupled receptor uses multiple signling pthwys. We employed strtegy tht keeps the cell intct yet is le to vry memrne PIP 2 quickly with minimum production of other distrcting messges, especilly with novel use of voltge-dependent phosphtse. We compensted for effects of voltge-dependent inctivtion of chnnels on the test current mplitudes. Depending on the chnnel sutype, such focused experiments demonstrte tht 35% 55% of the 2+ chnnel ctivity is supported y PIP 2 s cofctor. The chnnels do not fil with cutely reduced PIP 2 ut they definitely generte lrger currents when PIP 2 is t its norml endogenous level. Activtion of M 1 Rs removes more current thn just the PIP 2 -dependent component, ut the PIP 2 -dependent component ccounts for more thn hlf of the muscrinic modultion. Our erly proposl (ernheim et l., 1991; Mthie et l., 1992; Hille, 1994) tht slow modultion of KNQ chnnels nd L- nd N-type chnnels in sympthetic gnglion cells shre common pthwy is prtly orne out. They do use common pthwy, ut the 2+ -chnnel modultion lso uses dditionl signls. In sum, the PIP 2 hypothesis hs now een proven for four voltge-gted 2+ chnnel sutypes tht re modulted y M 1 muscrinic receptors, nd it hs een shown not to pply to four other V sutypes tht re not modulted y M 1 muscrinic receptors. EXPERIMENTAL PROEDURES ell ulture nd Trnsfection TsA21 cells (lrge-t-ntigen trnsformed HEK293 cells) were mintined in DMEM supplemented with 1% FS nd.2% penicillin/streptomycin nd trnsiently trnsfected using Lipofectmine 2 (Invitrogen) with vrious cdnas (see Supplementl Experimentl Procedures). For 2+ chnnel expression, cells were trnsfected with the 1 suunit of V, 3, nd 2d1 in 1:1:1 molr rtio. When needed,.1 mg of cdna encoding green fluorescent protein (GFP) or tetrmeric red FP (DsRed) ws cotrnsfected with the cdna s mrker for successfully trnsfected cells. The next dy, the cells were plted onto poly-l-lysine-coted coverslip chips, nd fluorescent cells were studied within 1 2 dys in FRET nd electrophysiologicl experiments. ultured SG neurons were prepred s descried (Mochid et l., 23). riefly, gngli were dissected from 7 dy postntl rts, deshethed, nd incuted with collgense (.65 mg/ml; Worthington iochemicl) in L-15 medium (GIO) t 37 for 4 min. Following enzyme tretment, gngli were triturted gently through smll-pore glss pipette, wshed twice y low speed centrifugtion, nd resuspended in DMEM supplemented with 1% fetl clf serum (GIO), 5% horse serum (GIO), 1% penicillin-streptomycin solution (GIO), nd 25 ng/ml nerve growth fctor (2.5 S; Alomone , , July 29, 21 ª21 Elsevier Inc.