Journal of Physiology (1994), 474.1

Muscarinic modulation by a G-protein a-subunit of delayedrectifier K+ current in rat ventromedial hypothalamic neurones

Jarlath M. H. ffrench-Mullen*, Carlos R. Plata-Salam'ant, Noel J. Buckley:and Petra Danks*

*Department of Pharmacology, Zeneca Pharmaceuticals Group, Zeneca Inc.,Wilmington, DE 19897, USA, tSchool of Life and Health Sciences, University of

Delaware, Newark, DE 19716, USA and tNational Institute for Medical Research,The Ridgeway, Mill Hill, London NW7 1AA

1. Rat cultured ventromedial hypothalamic (VMH) neurones obtained from embryonichypothalamus were used to study the muscarinic (carbachol) modulation of voltage-gatedK+ currents with the whole-cell patch-clamp technique.

2. Carbachol produced a potent and concentration-dependent (100 fM to 100 /M) decrease ofthe outward delayed rectifier K+ current (IK) with an IC50 of 44 pM and a Hill coefficientof 0 4. The carbachol-induced depression of IK was reduced by pirenzepine (1-10 /UM) andatropine (1 ,uM). Carbachol had no effect on the transient outward K+ current (IA).

3. Intracellular dialysis with guanosine 5'-O-(2-thiodiophosphate) (GDP-,-S, 500 ,UM)significantly diminished the carbachol-induced depression of IK, suggesting GTP-bindingprotein (G-protein) involvement. Pre-incubation of VMH neurones with pertussis toxin(200-400 ng ml') or cholera toxin (1 jug ml') for 24-48 h had no effect on the carbachol-induced depression of IK. This suggested that the Ga., Gai, and Ga, G-protein a-subunitswere not involved in mediating the carbachol-induced depression of IK in VMH neurones.

4. Treatment (24-48 h) of VMH neurones with antisense phosphothio-oligodeoxynucleotidesto the Gall G-protein subunit (10 ,UM) significantly diminished the carbachol-induced depressionof IK. Treatment with 10 /UM of either Ga,, sense or antisense to Gaq had no effect.

5. These results demonstrate a novel and potent muscarinic depression of IK in VMNneurones, and that this depression is specifically mediated by the Ga,, G-protein subunit.This action of the Ga1, subunit also provides the first demonstration of an associationbetween the Ga,, G-protein subclass (a member of the Gq class) and a neuronal membranecurrent.

The cholinergic innervation and receptors of themammalian ventromedial hypothalamus (VMH) areinvolved in the regulation of autonomic, neuroendocrine(Grijalva & Novin, 1990) and behavioural (Richmond &Clemens, 1988) functions.The muscarinic actions of acetylcholine are mediated by

several muscarinic receptor subtypes, members of the super-family of GTP-binding protein (G-protein)-coupled receptors.Heterotrimeric G-proteins transduce signals from membranereceptors to enzymes (such as phospholipase C), or ionchannels (such as K+ channels) (Simon, Strathmann &Gautam, 1991; Hepler & Gilman, 1992). G-proteininvolvement can be identified by intracellular dialysis withguanosine 5'-O-(2-thiodiophosphate) (GDP-,-S), a non-hydrolysable analogue of GDP that competitively inhibitsthe binding of GTP to G-proteins. Mammalian G-protein a-subunits are classified into four classes (G$, Gj,Gqand G12) based upon amino acid sequence similarity

(Simon et al. 1991; Hepler & Gilman, 1992). Pertussis toxin(PTX) inactivates all of the Gai and Gao subunits (membersof the Gi class) by ADP ribosylation of a cysteine residue,and cholera toxin irreversibly activates the Ga. and GaIofsubunits (members of the G8 class) by ADP ribosylation of anarginine residue (Hepler & Gilman, 1992). Recent studieshave used antisense oligonucleotides to identify G-proteinsubunit subclasses (e.g. Kleuss, Scherubl, Hescheler, Schultz& Wittig, 1993).

In mammalian neurones, a variety of voltage-gated K+currents are modulated by muscarinic (carbachol)stimulation. Carbachol enhances IA in striatal neurones(Atkins, Surmeier & Kitai, 1990), but inhibits it inhippocampal neurones (Nakajima, Nakajima, Leonard &Yamaguchi, 1986). To date, the only report of a muscarinicmodulation of IK was a potentiation of the current inhippocampal neurones via a G-protein-linked intracellularCa2'-dependent process (Zhang, Weiner & Carlen, 1992);

MS 2J642, pp. 21-26 21

J. M. H. ffrench-Mullen and others

however, neither the muscarinic receptor subtype nor theG-protein a-subunit(s) involved were identified.We here report on a novel carbachol modulation of IK'

and identify the involvement of the Ga.l subunit (a memberof the Gq class) in the modulation of a neuronal membranecurrent.

METHODSPrimary cell culture

Pregnant Sprague-Dawley rats (Hilltop Laboratory Animals,Scotsdale, PA,USA) were placed in a sealed chamber into which100% C02 gas was introduced to kill the animals. Death wasconfirmed by a lack of respiration and cardiac arrest. This wasfollowed by rapid removal of the fetuses (embryonic agesE15-E17) which were then quickly decapitated. The heads weredissected in phosphate-buffered saline solution (Gibco LifeTechnologies, Grand Island, NY, USA) supplemented with6 mg ml' glucose (Sigma, USA). The VMH was obtained fromthe ventral half of the mid-region of the hypothalamus aspreviously described (Ono et al. 1990). Cultures were prepared ina manner similar to that described previously (Fiszman, Zuddas,Masana, Barker & Di Porzio, 1991). Cells were obtained usingenzymatic digestion (20 IU ml' papain and 0 005% DNase) andtrituration. Cells were plated in Dulbecco's modified Eaglemedium and nutrient mixture F-12 (1:1, Gibco) supplementedwith 10% fetal bovine serum (Hyclone Laboratories, Logan,UT,USA), 5% heat-inactivated horse serum (HyClone) and3 mg ml- glucose (Sigma). Cultures were incubated at 37 °C in awater-saturated 95% air-5% CO2 atmosphere. The medium waschanged at 5-6 days and cells were used from the fifth to thetwelfth days following plating.

Whole-cell patch-clamp recordingsRecordings were made at 22-25 °C as previously described(ffrench-Mullen & Rogawski, 1989; Harrison, Radke, Talukder &ffrench-Mullen, 1993). The extracellular medium contained (mM):120 NaCl, 3 KCI, 2-3 MgCl2, 0-2 CaCl2, 6 D-Glucose, 10 Hepes/NaOH, pH 7-4 and had an osmolality of 325 mosmol (kg H2O)f.Tetrodotoxin (2 /M) was added to block Na+ channels. CaCl2 waspresent at only 0-2 mm in order to minimize inward Ca2"currents and Ca2+-activated K+ currents (Harrison et al. 1993).Patch pipettes contained (mM): 140 potassium gluconate, 5K2ATP, 2 MgCl2, 0-1 CaCl2, 5 K4-BAPTA, 5 Hepes/KOH, pH 7-2and an osmolality of 315 mosmol (kg H20)'. Seal resistances were> 5 Gil and pipette resistances were 4-6 MQ. Whole-cellcurrents were recorded with an Axopatch-ID amplifier (AxonInstruments, Foster City, CA,USA).Command potential sequences were delivered to the amplifier

and data were simultaneously acquired under computer control.Evoked currents were filtered at 5-10 kHz (-3 dB, 8-pole low-pass Bessel filter) (Frequency Devices, Haverhill, MA, USA),digitally sampled at 500 jus per point (50 ,us per point for tailcurrent measurements). Capacitative and leakage currents weredigitally subtracted from all records on-line with the pCLAMP5.51 (Axon Instruments) software package. Holding potential inall neurones was -40 mV; voltage steps were elicited every 20 sand all traces are the average of three steps.

Test substance applicationA rapid superfusion system consisting of a side-by-side array ofsix 200 #um i.d. capillary tubes was positioned within 400 /uM of

flow was computer controlled via solenoid valves (BME Systems,Baltimore, MD, USA).

Data analysisCurrent measurements of IK were made 10 ms prior to the end ofthe voltage step at +40 mV unless noted; all measurementswere made 30 s after achieving a steady-state level. Percentagedepression was determined according to the formula100 X (1 -Itest sub/Icontrol), where Icontroi is the leak-subtracted

current amplitude prior to the test substance application andItest sub is the current amplitude in the presence of carbacholand/or test substance. Concentration-effect data were fittedwith a non-linear least-squares program (NFIT, IslandProducts, Galveston, TX, USA) according to the logisticalequation: B= 100/1 + (IC50/[test substance])"H where Bis block,[test substance] is the concentration of the test substance, IC50 isthe concentration resulting in 50% depression, and nH is a

parameter that describes the steepness of the curve and has thesame meaning as the Hill coefficient. The reversal potential for IKwas -88+2 mV (n = 5) using tail current analysis (theoretical

value was -87 4) and conductance values, g, at each potentialwere calculated from the measured IK as g = I/( V-EK). Steady-state activation smooth curves were best-fitted to theBoltzmann equation, g /gmax = {1 + exp[(V%- V)/k]}', wheregmax is the maximum conductance, V% is the voltage for half-maximal activation and k is a slope factor describing the steep-ness of the activation curve. Statistical analyses used the two-tailed paired or unpaired Student's t test, the Mann-Whitneytest, or analysis of variance depending on the characteristics ofthe data; results were significant only for P < 0.05. Data are

expressed as means + S.E.M.

MaterialsAll chemicals were obtained from Sigma Chemical Co. (St Louis,MO, USA) except where noted. Oligodeoxynucleotides (18-mers)were thiolated at all positions:

sense-Gal, 605, GTAGGCTACCTGCCCACC,antisense-Ga.z 605, GGTGGGCAGGTAGCCTAC,antisense-Gaz 499, GTAGCCATCTGTGGCGAT,antisense-Gaq 485, CTACACGGTCCAAGTCAT,

and were synthesized by the Johns Hopkins University CoreFacility. Numbers refer to the first base of the sequence to which theprimer corresponds relative to the adenine of the initiating ATG.

RESULTSElectrical and pharmacologicalcharacterization

Whole-cell voltage-clamp studies were conducted toexamine the effects of the non-hydrolysable muscarinicagonist carbachol on IK and IA in cultured VMH neurones.

Carbachol produced a potent and concentration-dependentdecrease in the amplitude of IK which occurred within 20 s

(60-90 s to achieve steady state) and typically reversed to85% of control with wash-out (Fig. 1A).

In contrast, carbachol had no effect on IA in VMHneurones. From the same neurone in Fig. 1A, activation ofboth IA (peak) and IK (late) is simultaneously shown inFig. lB. The same carbachol concentrations depressed IK'

the cell under study. Solutions were applied by gravity feed and

22 J. Phy-siol. 474.1

but had no effect on IA. This is best illustrated by the

Modulation ofK+ current by Ga,, C-protein

A

Control

,--Wash1 pM carbachol

-10 nlm1 /nMM

+40 mVL 1I 0-40

D12

10

en8

00c 6

8

2

0

B

50 Ms

C

ControlWash1 pm carbachol

10 nM1 pM

M.j ~~+40

200 pA 4 0 14200 pA-90 50 ms

E F

300 pA50 ms-40 -20 0 20 40 60

Membrane potential (mV)

23

nControl1 pM carbachol 200 pA10 nlmI

Control

usm PZP + carbachol1 paM50 pM carbachol

250 pA50 ms

Figure 1. Effect of carbachol on K+ currentsA, carbachol reversibly depresses IK evoked by a voltage step (180 ms) to +40 mV from a holdingpotential (Vh) of -40 mV. B, both IA and IK are elicited simultaneously by a pre-pulse (180 ms) to-90 mV from -40 mV( Vh) followed by a step to +40 mV unless noted. Measurements for this, andsubsequent figures were made at +40 mV unless noted. Note the lack of carbachol effect on the peak(IA) versus the late (IK) phase of the step. A and B are from the same neurone; voltage protocolsindicated below current traces. C, isolation of IA (difference currents of A and B) illustrating thecomplete lack of effect of carbachol on this current. D, effect of carbachol on voltage dependence ofactivation of IK. Activation curves were determined from voltage steps delivered in 10 mV incrementsover the range of -40 to +60 mV; current, and hence conductance (see Methods), was measured 10 msfrom the end of the voltage step delivered from a holding potential of -40 mV. Smooth curves were

fitted by the Boltzmann equation (see Methods). Each data point is the mean + S.E.M. of 4-5 neurones.

The difference in values between control and carbachol-treated groups was significant (P< 0 02) usinganalysis of variance. E, IK was depressed 35, 65 and 88% with 100 /UM, 1 and 5 mm TEA, respectively;IK returned to 85 % of control with wash-out. F, carbachol induced a 28 % depression of 'K which was

reduced to 20 and 17 % by 1 and 10/uM pirenzepine (PZP) ; control values were restored after wash-out(not shown).

isolated IA values (obtained from the difference currents ofFig. IA and B) in Fig. IC. In all the cells where IA was

examined, carbachol had no effect (P > 0'7; 736 + 70,

736 + 749 740 +369 732 + 779 735 + 77, 742 + 70 and778+64 pA for control, 1, 100 pM, 1, 10, 100 nm and 1uM

carbachol, respectively; n = 8).The effect of carbachol on the conductance-voltage

relationship of IK is shown in Fig. ID. The activation of IKwas examined and smooth curves were fitted to a

Boltzmann equation to determine the effect of carbachol onthe voltage dependence of activation. The half-maximalactivation voltages (VI) were 22'6, 22-8, 22'9, 22'9 and22-8 mV; the values of the slope of the activation (k) were12-5, 13'5, 13'7, 15-8 and 16 mV for control, 1 and 100 pM,

10 nM and 1 ,uM carbachol, respectively. Carbachol,therefore produced no apparent change in either the V% or

k. The conductance-voltage curves in Fig. ID suggestedthat the carbachol-induced depression of IK occurred in a

voltage-dependent manner. Comparison of the percentageof depression versus test potential (Vt) gave a range at Vt-20 mV of 33 + 5, 63 + 5, 69 + 5 and 76 + 5 % depressioncompared with 10 + 5, 26 + 4, 29 + 4 and 36 + 4% depressionat+60 mV for 1 and 100 pM, 10 nm and 1uM carbachol,respectively (n = 4-5 neurones per data point). Thus, at themore negative test potentials there was a greater depressionof IK than at the more depolarized test potentials.We next clarified the pharmacological verification of IK

and its depression by carbachol. IK is sensitive totetraethylammonium (TEA) (Storm, 1990). TEA produceda reversible and concentration-dependent inhibition of IK(Fig. 1E). Overall, TEA produced 17+1, 27+2, 43+3,61 + 3, 71 + 3, 82 + 6 and 90 + 4 % inhibition with 0'01, 1,

J. Phy&iol. 474.1

J. M. H. ffrench-Mullen and others

100, 500, 1000 uM, 5 and 10 mm TEA, respectively (n = 3);the effect was reversed to 70 + 5 % of control values withwash-out following 100 mm TEA. IA is inhibited by4-aminopyridine (4-AP) (Storm, 1990). In these cells, IA wasreversibly blocked by 5 mm 4-AP (n = 3; not shown);however, the carbachol-induced depression of IK wasunaffected by the presence of 4-AP (n =3; not shown).Although carbachol is a mixed cholinergic agonist, itsactions were mediated by muscarinic receptors. Themuscarinic antagonist pirenzepine (PZP) reduced thecarbachol-induced inhibition by 50%; control values wererestored after wash-out (Fig. IF). With PZP, the 50PMcarbachol-induced depression (23 + 1 %) was reduced to 14 +1and 11 + 1% by 1 and 10/UM PZP, thus giving a 39 and 52 %reduction of the carbachol-induced depression (n = 5).Atropine (1 ,UM) also diminished the carbachol (50 pM)-induced depression from 21 + 3 to 8 + 2 % (n = 3; notshown). Carbachol (10 /uM) had no effect on the fast inwardNa+ current which was sensitive to tetrodotoxin (1,uM)(n = 3; not shown). The non-inactivating voltage-gated K+current, the M-current, was not detected in these neurones(n=6).The concentration-effect curve for carbachol-induced

depression of IK is illustrated in Fig. 2. Carbachol potentlydepressed IK, as the current reduction began at 100 fM andattained a plateau (36% depression) between 1 and 10/M withan IC50 of 44 pM and a Hill coefficient (nH) of0 4.

To test for G-protein involvement, we internally dialysedneurones with GDP-/.-S through the recording electrode.GDP-/J-S (500 /SM; n = 4-5 per point) significantly reducedthe carbachol-induced depression of IK (P < 0 02) for 100 fM,10 nM, 1 and 10/SM carbachol (Fig. 2), suggesting theinvolvement of a G-protein modulating the carbachol-induced depression of IK. Preincubation of VMH neuroneswith PTX (200-400 ng ml'; n = 5-10 per point) for 24-48 hhad no effect on the carbachol-induced depression of IK(Fig. 2). Preincubation with cholera toxin (1 jug ml') for 24 h,also had no significant effect (P > 0 4), (8 + 1, 17 + 2, 25 + 3,29 + 2 and 35 + 2% depression of IK at 1 pM, 100 pM, 10 nM,1/M and 10 SM, carbachol, respectively; n = 4 per point).

C

0-

-0

a

a1)a)

100

9040 v

/

30 -

20 -

10

0

G-protein identification

The data obtained with PTX and cholera toxin indicatedthat the Gao, Gai and Ga. G-protein a-subunits were notinvolved in mediating the carbachol-induced depression ofIK. Many PTX-insensitive actions of G-protein-coupledreceptors are believed to be mediated by Ga,,/Ga,, subunitsof the Gq class ( Simon et at. 1991; Hepler & Gilman, 1992).For example, reconstitution studies (Taylor, Smith &Exton, 1990), transient transfections (Wu, Lee Rhee &Simon, 1992), and antibody studies (Aragay, Katz & Simon,1992), have indicated the involvement of Gaq/Gan1 subunitsin stimulation of phospholipase C-fl,1 activity. Other PTX-insensitive Ga-subunits of the Gq class (Ga14, Ga15 and Ga16)are unlikely to be involved in signal transduction in neuraltissue since their expression is tissue specific to epithelialand haematopoietic cells (Simon et al. 1991; Hepler &Gilman, 1992,). Reconstitution studies and transienttransfections do not recapitulate in vivo interactions, andfunctional studies employing antibodies do not distinguishbetween Gaq and Gall since the C-terminal is sharedbetween these two a- subunits of the G. class. Antisenseoligodeoxynucleotides do not present this limitation sincefunctional blockade can be achieved by oligonucleotidesconstructed against the domain of the transcript, includingthe untranslated regions.We constructed specific antisense phosphothio-oligo-

deoxynucleotides to determine whether the G-proteina-subunits Gaq and/or Gall were involved in mediating thecarbachol-induced depression of IK. VMH neurones weretreated with 10,UM antisense or sense oligodeoxynucleotidesadded to the culture medium for 24 and 48 h prior torecording. For the 48 h treatment, fresh antisense wasadded at 24 h.

Inhibition of the carbachol effect by Gall 605 antisense isillustrated in Fig. 3A and B, where, for example, there was 1and 2% depression at 24 and 48 h for 1 pM carbachol. Incontrast, the 1 pM carbachol-induced depression of IK was15% in the presence of the Gan1 605 sense (Fig. 3C), and 11%in the presence of the Gaq 485 antisense (Fig. 3D).

Figure 2. Concentration-effect curves for the carbachol-induced depression of IKConcentration-effect curves for the carbachol-induceddepression of IK on VMH neurones in drug-free solution (0),pertussis toxin-treated (U) and internally dialysed GDP-,B-S(500 ,/M; A) neurones. Each point is the mean + S.E.M. of5-10 neurones.

-14 -12 -10 -8Carbachol (M)

-6 -4

24 J. Physiol. 474.1

Modulation ofK+ current by Ca,, C-protein

Figure 3. Effects ofoligonucleotide treatmentEffects of phosphothio-oligodeoxynucleotide treatment oncarbachol-induced depression of IKat +40 mV. Cultures were pre-incubated before recording with10 FM antisense or sensephosphothiolated oligodeoxy-nucleotides. A, Gait 605 antisense(24 h); B, Ga I 605 antisense (48 h);C, Gall 605 sense (24 h)corresponding to the inversesequence of Gall 605 antisense; D,Gaq 485 antisense (48 h).

AControl

1 pM carbachol10 nM

+40 mV-40

C

B

Control1 pM carbachol10 nm

D ControlD 4 1 ~~~~~~pmcarbachol4 10 nM

250pA50 ms

A summary of the oligodeoxynucleotide effects is shown inFig. 4. The addition of the Gaq 485 antisense had nosignificant effect on the carbachol-induced response at 24 h(P > 0 7; n = 3; 12 + 2, 28 + 3 and 34 + 3% depression for1 pM, 10 M and 1/M), and also 48 h (P > 0 7; n = 5; 13 + 3,18 + 6, 24 + 4, 27 + 2 and 32 + 4% depression for 1 pM,100 pM, 10 nM, 1 and 10/UM, carbachol, respectively). Incontrast, incubation for 24 h with the Ga.1 605 and Ga. 499antisense oligodeoxynucleotides significantly diminished(P < 0 02) the carbachol-induced depression of IK (Figs 3 and4). A 48 h incubation with the Ga,, 605 antisense had a similareffect (P <0f02; n=4; 3±1, 9±1, 17+1 and 22+1%depression of IK for 1 pM, 100 pM, 10 nm and 1/SM carbachol,respectively). The sense oligodeoxynucleotide Gal, 605 had nosignificant effect (P > 0'5) when compared with control(Figs 3 and 4). Comparison of the two antisense oligo-deoxynucleotides with the sense oligodeoxynucleotide alsoshowed a significant reduction of the carbachol-induceddepression of IK (with Ga11 605 antisense, P < 0-02, and withGa,U 499 antisense, P < 0 03 for 1 pM, 100 pM, 10 nM and 1/ Mcarbachol, respectively). The specificity of Gau1 antisense on

the carbachol-induced depression of IK is suggested because (1)the Gal, sense and the Gaq antisense had no effect, and (2) lowconcentrations of Ga11 antisense had no significant effect. Forexample, the carbachol-induced depression of IK was 16 + 5,27 + 3 and 32 + 6% depression for 1 pM, 10 nM and 10 /SMcarbachol, respectively, in cells treated with 5/uM Gall 605antisense for 24 h (n = 6). Furthermore, the stability ofantisense-treated neurones was confirmed by comparing theamplitude and the time to peak of steady-state IK(at +40 mV) prior to carbachol application in control (non-treated) versus antisense-treated neurones. Currentamplitudes in non-treated (see Fig. 1) compare well withantisense-treated (see Fig. 3) neurones. For example, theamplitude of peak IK (mean + S.D.) in non-treated(1910+269pA, n=28) and Gal, 605 and 499 antisense-treated (1889 + 376 pA, n =13 and 1895 + 258 pA, n = 13,respectively) neurones prior to carbachol application showedno significant difference. The time to peak of IK was bestfitted by the sum of two exponential functions, with timeconstants fast (Tf) and slow (T8), and there was no significantdifference (P > 0'7) between non-treated versus oligodeoxy-

50

Figure 4. Summary of the effects of phosphothio-oligodeoxynucleotide treatments on the carbachol-induced depression of IKControl values were taken from Fig. 2; number of neuronesexamined is in each column; 'a' refers to antisense and 's'refers to sense. There was no significant difference in theinhibition between control and Gall 605s. Both the Gal1 499aand the Gall 605a significantly (*) reduced the carbacholinhibition compared to control (P < 0-02). Furthermore,there was a significant difference (t) in the carbacholdepression between the Gax1605s and the Gall 605a or 499a(P < 0 03) indicating specificity of action of the antisense.

40

Z 30

0co

u)

O 20

0

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0

Carbachol

25J. Physiol. 474.1

26

J. M. H. ffrench-Mullen and othersJ. Physiol. 474.1

nucleotide-treated neurones. The rf values were 1P27 + 0-06,1P28 + 012, 1P21 + 017, 1P29 +011, 1 2+ 009, 1P21 + 015 and126 + 011 ms, and T. values were 8-63 + 0 4, 8 79 + 0 5,8'83 + 03, 8-64 + 0-6, 87 + 04, 8-74 + 03, and 8-52 + 0-5 msfor control (n = 25), Gan1 605 antisense at 24 h (n = 8), Ga,n605 antisense at 48 h (n = 4), Gall 605 sense (n = 8), Gall 499antisense (n = 7), Gaq 485 antisense at 24 h (n = 3) and Gaq485 antisense at 48 h (n = 5).

DISCUSSIONThese findings demonstrate that carbachol potently (pico-molar range) depresses a fraction of the total IK in VMHneurones, and that this depression is specifically mediatedby the Gall G-protein subunit. Depending upon the brainregion, carbachol has opposite effects on both IK and IA. Forexample, IA is potentiated by carbachol in striatal neurones(Atkins et al. 1990), but inhibited in hippocampal neurones(Nakajima et at. 1986). However, in VMH neurones,carbachol had no effect on IA. Carbachol enhances IK inhippocampal neurones (Zhang et at. 1992), while in VMHneurones, we show a novel inhibitory effect. This fractionaldepression of the total IK would shift the cell into aprolonged excitable state. This could prolong depolarizationand increase neurotransmitter release which, in turn, maymodulate the VMH output to influence autonomic,neuroendocrine (Grijalva & Novin, 1990) and behavioural(Richmond & Clemens, 1988) responses.

The identity of various heterotrimeric G-proteins inneuronal muscarinic actions remains unknown. In sym-pathetic ganglion neurones, the depression of IM appears toinvolve PTX-insensitive G-proteins (Brown, Marrion &Smart, 1989). In dorsal lateral geniculate neurones,muscarinic increase of the outward K+ current was mediatedvia a PTX-sensitive G-protein while the muscarinicactivation of a non-voltage-dependent K+ current (which wasdistinct from IM and IAHP) was unaffected by PTX(McCormick, 1992). These results imply the involvement ofPTX-insensitive G-protein a-subunits mediating themuscarinic modulation of various K+ currents. Theidentification of a Gall subunit modulating the muscarinicdepression of IK in VMH neurones suggests that this G-protein a-subunit might be involved in the muscarinicmodulation of other K+ currents. However, it is presentlyunknown whether the Gall subunit is directly coupled to theion channel, or whether intracellular messengers areinvolved. The action of the Gall subunit in the muscarinicmodulation of IK in mammalian neurones also provides thefirst demonstration of an association between a member ofthe G. class of G-protein a-subunits and a neuronal ioniccurrent.

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Received 9 August 1993; accepted 8 September 1993.