The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.     

Putative M-type potassium channels in neuroblastoma-glioma hybrid cells: inhibition by muscarine and bradykinin

Putative M-type K(+)-channels ('M-channels') were recorded in differentiated NG108-15 neuroblastoma x glioma hybrid cells transformed to express m1 muscarinic acetylcholine receptors using cell-attached patch-electrodes. Channels showed multiple conductances, with peaks at 6-9 and 12-15 pS. Averaged currents showed time-dependent activation during 1 s depolarization steps to around -30 mV. Steady-state Po increased in a voltage-dependent manner when the membrane was depolarized between 10 and 60 mV, with a limiting slope of 5.5 mV/e-fold change in Po. Steady-state kinetics were fit by two open and three shut times: depolarization shortened shut times and lengthened open times. Application of muscarine (10 microM) or bradykinin (10 microM) to the membrane outside the patch reversibly reduced steady-state in-patch channel activity to 38.4 +/- 11.7 and 28.8 +/- 6.1% of control values, respectively. Inhibition was accompanied by a lengthening of channel shut times without significant change in open times or distribution of conductance levels. No effect of muscarine or bradykinin on whole-cell or membrane patch delayed rectifier currents was detected. It is concluded that M-channels in NG108-15 cells are qualitatively similar to, but sparser than, those previously reported in rat sympathetic neurones. Their inhibition by extra-patch acetylcholine and bradykinin suggests that a mobile messenger is involved in the transduction process leading from receptor activation to channel closure.     

Intracellular calcium directly inhibits potassium M channels in excised membrane patches from rat sympathetic neurons

Complex effects of altering intracellular [Ca2+] on M-type K+ currents have previously been reported using whole-cell current recording. To study the direct effect of Ca2+ on M-channel activity, we have applied Ca2+ to the inside face of membrane patches excised from rat superior cervical sympathetic ganglion cells. Ca2+ rapidly and reversibly inhibited M-channel activity in 28/44 patches by up to 87%, with a mean IC50 of 100 nM. This effect persisted in the absence of ATP, implying that it was not due to phosphorylation/dephosphorylation. A similar effect was observed in 13/13 cell-attached patches when cells were transiently "Ca(2+)-loaded" by adding 2 mM Ca2+ to a 25 mM K+ solution bathing the extrapatch cell membrane. These observations provide new evidence that Ca2+ can directly inhibit M channels, so supporting the view that Ca2+ might mediate M current inhibition following muscarinic receptor activation.     

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP > GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.     

G protein alpha subunit G alpha z couples neurotransmitter receptors to ion channels in sympathetic neurons

The functional roles subserved by G(alpha)z, a G protein alpha subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that G(alpha)z coupled neurotransmitter receptors to N-type Ca2+ channels when transiently overexpressed in rat sympathetic neurons. The G(alpha)z-mediated inhibition was voltage dependent and PTX insensitive. Recovery from G(alpha)z-mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. G(alpha)z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca2+ channels via PTX-sensitive Go/i proteins. In addition, G(alpha)z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that G(alpha)z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.     

Contribution of ATP-sensitive potassium channels to hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro

To investigate the mechanism of generation of the hypoxia-induced hyperpolarization (hypoxic hyperpolarization) in hippocampal CA1 neurons in rat tissue slices, recordings were made in current-clamp mode and single-electrode voltage-clamp mode. Superfusion with hypoxic medium produced a hyperpolarization and corresponding outward current, which were associated with an increase in membrane conductance. Reoxygenation produced a further hyperpolarization, with corresponding outward current, followed by a recovery to the preexposure level. The amplitude of the posthypoxic hyperpolarization was always greater than that of the hypoxic hyperpolarization. In single-electrode voltage-clamp mode, it was difficult to record reproducible outward currents in response to repeated hypoxic exposure with the use of electrodes with a high tip resistance. The current-clamp technique was therefore chosen to study the pharmacological characteristics of the hypoxic hyperpolarization. In 60-80% of hippocampal CA1 neurons, glibenclamide or tolbutamide (3-100 microM) reduced the amplitude of the hypoxic hyperpolarization in a concentration-dependent manner by up to approximately 70%. The glibenclamide or tolbutamide concentrations producing half-maximal inhibition of the hypoxic hyperpolarization were 6 and 12 microM, respectively. The chord conductance of the membrane potential between -80 and -90 mV in the absence of glibenclamide (30 microM) or tolbutamide (100 microM) was 2-3 times greater than that in the presence of glibenclamide or tolbutamide. In contrast, the reversal potential of the hypoxic hyperpolarization was approximately -83 mV in both the absence and presence of tolbutamide or glibenclamide. In approximately 40% of CA1 neurons, diazoxide (100 microM) or nicorandil (1 mM) mimicked the hypoxic hyperpolarization and pretreatment of these drugs occluded the hypoxic hyperpolarization. When ATP was injected into the impaled neuron, hypoxic exposure could not produce a hyperpolarization. The intracellular injection of the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate lithium salt reduced the amplitude of the hypoxic hyperpolarization. Furthermore, application of dinitrophenol (10 microM) mimicked the hypoxic hyperpolarization, and the dinitrophenol-induced hyperpolarization was inhibited by either pretreatment of tolbutamide or intracellular injection of ATP, indicating that the hypoxic hyperpolarization is highly dependent on intracellular ATP. It is therefore concluded that in the majority of hippocampal CA1 neurons, exposure to hypoxic conditions resulting in a reduction in the intracellular level of ATP leads to activation of ATP-sensitive potassium channels with concomitant hyperpolarization.     

Whole cell recording of sodium, potassium and calcium currents of cultured neonatal rat sympathetic neurons

Na, K and Ca currents and other electrophysiological characteristics of cultured neonatal rat superior cervical sympathetic neurons were studied using whole cell clamp technique. The mean passive and active membrane properties measured are as follows: resting membrane potential, -51 +/- 6 mV; input resistance, 1432 +/- 389 M omega; time constant, 130 +/- 32 ms; amplitude of action potential, 96 +/- 10 mV; overshoot, 42 +/- 6 mV. Na, K and Ca currents were isolated upon pharmacological manipulations. The predominant type of K current was a noninactivating delayed rectifier. Voltage-clamp studies also showed the presence of a high voltage-activated sustained inward Ca current, while low voltage could not elicit any transient Ca current.     

Regulation of galanin in rat sympathetic neurons in vitro

The neuropeptide galanin is induced in sensory and autonomic neurons after peripheral nerve lesion. Leukemia inhibitory factor (LIF) has been suggested to be involved in the up-regulation of galanin. A direct effect of LIF on galanin content in pure sympathetic neuron cultures dissociated from newborn rat superior cervical ganglia was investigated by radioimmunoassay and immunohistochemistry. Galanin increases in sympathetic neurons during a 12 day culture period in the presence of NGF (10 ng/ml). Five days after addition of LIF (10 ng/ml) a 7-fold elevation is observed when compared to control cultures. Furthermore, galanin increases significantly in the presence of non-neuronal cells and in response to potassium-induced depolarization. The proportion of galanin-immunoreactive neurons in mixed cultures is similar to that found in adult rat superior cervical ganglia after transection of the major postganglionic branches. The results corroborate the hypothesis that LIF, presumably released from ganglionic satellite cells, induces galanin in a subpopulation of sympathetic neurons in vivo and in vitro.     

Gating of single N-type calcium channels recorded from bullfrog sympathetic neurons

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the pipet, mean N-channel open probability (Po, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., Po at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (tauo) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (taush1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (taush2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel Po during individual sweeps at +40 mV was often high ( approximately 0.8), mean Po was reduced by null sweeps, low Po gating, inactivation, and slow activation. The variability in mean Po across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low Po gating (Po = 0.2, tauo = 1 ms at +40 mV) could be sustained for approximately 1 min in some patches. The clustering of null sweeps and sweeps with low Po gating is consistent with the idea that they result from different modes of N-channel gating. While Po of the main N-channel gating state is high, the net Po is reduced to a maximum value of close to 0.5 by other gating processes.     

Inhibition of M-type K+ and N-type Ca2+ channels by the human gonadotropin-releasing-hormone receptor heterologously expressed in adult neurons

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.     

Mibefradil inhibition of T-type calcium channels in cerebellar purkinje neurons

The antihypertensive agent mibefradil completely and reversibly inhibited T-type calcium channels in freshly isolated rat cerebellar Purkinje neurons. The potency of mibefradil was increased at less hyperpolarized holding potentials, and the apparent affinity was correlated with the degree of channel inactivation. At 35 degrees, the apparent dissociation constant Kapp was 1 microM at a holding voltage of -110 mV (corresponding to noninactivated channels) and 83 nM at a holding voltage of -70 mV (corresponding to 65% inactivation). The increased affinity was attributable mainly to a decreased off-rate. Mibefradil also inhibited P-type calcium channels in Purkinje neurons, but inhibition was much less potent. At a holding potential of -70 mV, the Kapp for mibefradil inhibition of P-type channels was approximately 200-fold higher than that for inhibition of T-type channels. Mibefradil should be a useful compound for distinguishing T-type channels from high voltage-activated calcium channels in neurons studied in vitro.     

Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons

We have measured the onset and recovery speed of inhibition of N-type Ca2+ channels in adult rat superior cervical ganglion neurons by somatostatin (SS), norepinephrine (NE), and oxotremorine-M (oxo-M, a muscarinic agonist), using the whole cell configuration of the patch-clamp method with 5 mM external Ca2+. With a local perfusion pipette system that changed the solution surrounding the cell within 50 ms, we applied agonists at various times before a brief depolarization from -80 mV that elicited I(Ca). At concentrations that produced maximal inhibition, the onset time constants for membrane-delimited inhibition by SS (0.5 microM), NE (10 microM), and oxo-M (20 microM) were 2.1, 0.7, and 1.0 s, respectively. The time constants for NE inhibition depended only weakly on the concentration, ranging from 1.2 to 0.4 s in the concentration range from 0.5 to 100 microM. Inhibition by oxo-M (20 microM) through a different G-protein pathway that uses a diffusible cytoplasmic messenger had a time constant near 9 s. The recovery rate constant from membrane-delimited inhibition was between 0.09 and 0.18 s(-1), significantly higher than the intrinsic GTPase rate of purified G protein Go, suggesting that Ca2+ channels or other proteins in the plasma membrane act as GTPase activating proteins. We also measured the rate of channel reinhibition after relief by strong depolarizing prepulses, which should reflect the kinetics of final steps in the inhibition process. In the presence of different concentrations of NE, reinhibition was four to seven times faster than the onset of inhibition, indicating that the slowest step of inhibition must precede the binding of G protein to the channel. We propose a kinetic model for the membrane-delimited NE inhibition of Ca2+ channels. It postulates two populations of receptors with different affinities for NE, a single population of G proteins, and a single population of Ca2+ channels. This model closely simulated the time courses of onset and recovery of inhibition and reinhibition, as well as the dose-response curve for inhibition of Ca2+ channels by NE.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.     

Excision-activated chloride channels in cultured postnatal rat hippocampal neurons

The non-enveloped picornaviruses, which are particularly resistant to physicochemical inactivation, include the aetiological agents of poliomyelitis, hepatitis A and E and infectious common cold (rhinovirus). In this work we used human rhinovirus type 5 (RV-5) cultivated in VERO cells to study the photoinactivating effects of several phthalocyanines and naphthobenzoporphyrazines. Free RV-5 was photoinactivated by aluminium trisulphonated naphthobenzoporphyrazine at 5 x 10(-8) M concentration. This photosensitizer was also active on replicating virus when the infected VERO cells were treated with 5 x 10(-6) M concentration followed by a very short illumination period. On the other hand, the ZnPc(3-MeO-Py)4 phthalocyanine, which possesses four positive charges, does not photoinactivate free rhinovirus, but this molecule protects VERO cells against RV-5 infection when added to the cultures before virus inoculation, in the presence or absence of subsequent illumination, and may therefore be considered as an antiviral agent in itself.     

Modulation of multiple potassium currents by metabotropic glutamate receptors in neurons of the hypothalamic supraoptic nucleus

We studied the effects of activation of the metabotropic glutamate receptors on intrinsic currents of magnocellular n urons of the supraoptic nucleus (SON) with whole cell patch-clamp and conventional intracellular recordings in coronal slices (400 micron) of the rat hypothalamus. Trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 10-100 microM), a broad-spectrum metabotropic glutamate receptor agonist, evoked an inward current (18.7 +/- 3.45 pA) or a slow depolarization (7.35 +/- 4.73 mV) and a 10-30% decrease in whole cell conductance in approximately 50% of the magnocellular neurons recorded at resting membrane potential. The decrease in conductance and the inward current were caused largely by the attenuation of a resting potassium conductance because they were reduced by the replacement of intracellular potassium with an equimolar concentration of cesium or by the addition of potassium channel blockers to the extracellular medium. In some cells, trans-ACPD still elicited a small inward current after blockade of potassium currents, which was abolished by the calcium channel blocker, CdCl2. Trans-ACPD also reduced voltage-gated and Ca2+-activated K+ currents in these cells. Trans-ACPD reduced the transient outward current (IA) by 20-70% and/or the IA-mediated delay to spike generation in approximately 60% of magnocellular neurons tested. The cells that showed a reduction of IA generally also showed a 20-60% reduction in a voltage-gated, sustained outward current. Finally, trans-ACPD attenuated the Ca2+-dependent outward current responsible for the afterhyperpolarization (IAHP) in approximately 60% of cells tested. This often revealed an underlying inward current thought to be responsible for the depolarizing afterpotential seen in some magnocellular neurons. (RS)-3,5-dihydroxyphenylglycine, a group I receptor-selective agonist, mimicked the effects of trans-ACPD on the resting and voltage-gated K+ currents. (RS)-alpha-methyl-4-carboxyphenylglycine, a group I/II metabotropic glutamate receptor antagonist, blocked these effects. A group II receptor agonist, 2S,1'S,2'S-2carboxycyclopropylglycine and a group III receptor agonist, (+)-2-amino-4-phosphonobutyric acid, had no effect on the resting or voltage-gated K+ currents, indicating that the reduction of K+ currents was mediated by group I receptors. About 80% of the SON cells that were labeled immunohistochemically for vasopressin responded to metabotropic glutamate receptor activation, whereas only 33% of labeled oxytocin cells responded, suggesting that metabotropic receptors are expressed preferentially in vasopressinergic neurons. These data indicate that activation of the group I metabotropic glutamate receptors leads to an increase in the postsynaptic excitability of magnocellular neurons by blocking resting K+ currents as well as by reducing voltage-gated and Ca2+-activated K+ currents.     

Block of potassium currents in rat isolated sympathetic neurones by tricyclic antidepressants and structurally related compounds

1. The block of K+ currents by the tricyclic antidepressants (TCAs), imipramine and amitriptyline and three structurally related compounds, chlorpromazine, tacrine and carbamazepine was investigated in rat isolated sympathetic neurones by whole-cell voltage-clamp recording. 2. At a concentration of 10 microM, imipramine, amitriptyline and chlorpromazine all blocked the delayed rectifier K+ current (IKv) by about the same extent, 54%, 47% and 53%. Tacrine was less effective (10%) while carbamazepine was ineffective at all concentrations tested. 3. The degree of block by the four effective compounds was relatively independent of the size of the voltage-step. Neither the activation nor the inactivation rates of IKv were altered by the blocking drugs. 4. Concentration-response relationships for imipramine and tacrine showed that imipramine was about 7 fold more potent than tacrine but that the maximum inhibition and the Hill slope were the same for both compounds. 5. Amitriptyline, chlorpromazine and imipramine (at 10 microM) were 2-3 fold more potent at inhibiting the sustained K+ current (mostly IKv) than the transient K+ current (mostly IA). Tacrine, however, was equally effective in blocking both components.     

Modulation of high voltage-activated calcium channels by somatostatin in acutely isolated rat amygdaloid neurons

We investigated actions of somatostatin (Som) on voltagegated calcium channels in acutely isolated rat amygdaloid neurons. Somatostatin caused a dose-dependent inhibition of the high voltage-activated (HVA) Ca2+ current, with little or no effect on the low voltage-activated (LVA) current. Nifedipine (2-10 microM) reduced the peak current by approximately 15% without reducing inhibition of current by Som significantly, ruling out L-type channels as the target of modulation. The modulation appears to involve N- and P/Q-type calcium channels. After pretreatment with omega-conotoxin-GVIA (omega-CgTx) or omega-agatoxin-IVA, the inhibition was reduced but not abolished, whereas the combined application of both toxins nearly abolished the modulation. The Som analog BIM-23060 mimicked the effects of Som, whereas BIM-23058 had no effect, implicating Som type-2 receptors (SSTR-2). The inhibition was voltage-dependent, being minimal for small depolarizations, and was often accompanied by a slowing of the activation time course. Strong depolarizing prepulses partially relieved the inhibition and restored the time course of activation. Intracellular dialysis with GTP gamma S led to spontaneous inhibition and a slowing of the current like that with Som and occluded the effects of the peptide. Dialysis with GDP beta S also diminished the inhibition. A short preincubation with 50 microM of the alkylating agent N-ethylmaleimide (NEM) prevented the action of somatostatin. These results suggest a role for NEM-sensitive G-proteins in the Som inhibition. Application of 8-CPT-cAMP and IBMX did not mimic or prevent the effects of Som.