Nicholas J. Penington

Whole-cell recordings of inwardly rectifying K+ currents activated by 5-HT1A receptors on dorsal raphe neurones of the adult rat.

1. An inwardly rectifying K+ current activated by serotonin (5‐HT) was recorded from acutely isolated adult dorsal raphe (DR) neurones using the whole‐cell recording mode of the patch clamp technique. 2. The 5‐HT‐induced K+ current (I5‐HT) was only visible at an [K+]0 > 5 mM and it was observed in 69% of the cells. 3. The reversal potential for I5‐HT was close to the potassium equilibrium potential and was shifted by 51 mV per 10‐fold change in [K+]0 indicating that I5‐HT was carried predominantly by K+. The chord conductance of I5‐HT at ‐90 mV was proportional to the external [K+] raised to a fractional power. 4. A dose‐response relationship revealed that I5‐HT was activated with an ED50 of 30 nM. Ba2+ (0.1 mM) blocked I5‐HT completely. Spiperone reversibly antagonized the response to 5‐HT and 8‐OHDPAT (8‐hydroxy‐2‐(di‐n‐propylamino)tetralin) mimicked the response indicating that the receptor activated was of the 5‐HT1A subtype. 5. The response to 5‐HT was largely prevented by in vitro pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism. 6. cAMP and lipoxygenase metabolites, both implicated in the modulation of similar currents in other preparations, were found not to alter the effectiveness of 5‐HT. 7. Glibenclamide and tolbutamide, blockers of the ATP‐regulated K+ channel, did not reduce the effect of 5‐HT in DR neurones. 8. These results show that in acutely isolated adult DR neurones 5‐HT activates an inwardly rectifying K+ current and this involves a PTX‐sensitive G‐protein in the transduction pathway which may interact with the K+ channel directly.

Agonist stimulation of the serotonin1A receptor causes suppression of anoxia-induced apoptosis via mitogen-activated protein kinase in neuronal HN2-5 cells.

Abstract: Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin1A receptor (5‐HT1A‐R), could cause attenuation of the activity of both N‐type Ca2+ channels and N‐methyl‐D‐aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5‐HT1A‐R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca2+ channels. Using a hippocampal neuron‐derived cell line (HN2‐5) that is Ca2+ channel‐deficient, we demonstrate here that an alternative pathway is responsible for 5‐HT1A‐R‐mediated protection of these cells from anoxia‐triggered apoptosis, assessed by deoxynucleotidyl‐transferase‐mediated dUTP nick end‐labeling (TUNEL). The 5‐HT1A‐R agonist‐evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation‐mediated activation of mitogen‐activated protein kinase (MAPK), as evidenced by the elimination of the agonist‐elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3‐kinase (PI‐3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5‐HT1A‐R caused a 60% inhibition of anoxia‐stimulated caspase 3‐like activity in the HN2‐5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5‐HT1A‐R caused an activation of PI‐3Kγ in HN2‐5 cells, our results showed that this PI‐3Kγ activity was not linked to the 5‐HT1A‐R‐promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2‐5 cells, agonist binding to the 5‐HT1A‐R results in MAPK‐mediated inhibition of a caspase 3‐like enzyme and a 60‐70% suppression of anoxia‐induced apoptosis through a Ca2+ channel‐independent pathway.

Differential effects of protein kinase C activation on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones.

1. Activation of the enzyme protein kinase C (PKC) partially uncouples receptors from the inhibition of Ca2+ current. We have studied the effect of PKC activation on 5‐HT1A receptor coupling of Ca2+ currents and 5‐HT‐induced K+ current (IK,5‐HT) in acutely isolated adult rat dorsal raphe neurones. 2. The phorbol ester 4 beta‐phorbol 12‐myristate, 13‐acetate (PMA; 1 microM) did not significantly alter the peak Ca2+ current. A maximal dose of 5‐HT inhibited Ca2+ current on average by 52%; after application of PMA, the inhibition was only 30% and the effect was irreversible for the duration of the experiment. 3. The inactive phorbol ester 4 alpha‐phorbol (1 microM) did not reduce the effectiveness of 5‐HT. When the kinase inhibitor staurosporine (ST; 200 nM) was added, PMA reduced the effect of 5‐HT by only 13.9%. ST partially prevented or reversed the effect of PMA, depending on the order of addition. 4. The voltage‐dependent rate or re‐inhibition by 5‐HT was reduced by PMA, suggesting that fewer activated G‐protein subunits are available to interact with Ca2+ channel after the action of PMA. 5. In contrast, PMA (1 microM) did not have a significant effect on IK,5‐HT. 6. PKC activation has an inhibitory effect on one branch of the 5‐HT1A receptor transduction fork, namely inhibition of Ca2+ influx, but not on the activation of IK,5‐HT.

Unitary properties of potassium channels activated by 5-HT in acutely isolated rat dorsal raphe neurones

1. Single inwardly rectifying K+ channel currents were recorded from acutely isolated adult serotonergic dorsal raphe (DR) neurones using the cell‐attached and outside‐out patch clamp configuration. 2. Four equally spaced conductance levels were observed in both outside‐out and cell‐attached patch recordings with conductance levels averaging 11, 21, 30 and 40 pS. Larger conductance openings (50‐120 pS) were seen less frequently. 3. When using 136 [K+]0 the single channel I‐V relation was linear in the range 0 mV to ‐100 mV in all cases. 4. Transitions between the various conductance levels were observed, as were apparent direct opening and closing to each individual conductance level. Furthermore openings of 11, 21 and 30 pS were observed in almost all the patches. These results suggest that the different‐sized events result from substrates of a single channel rather than several different channels with different conductances. 5. Unitary K+ channel current probability of opening, recorded in cell‐attached patch, was unchanged after 5‐hydroxytryptamine (5‐HT) was added to the bath outside the patch pipette which suggests that no easily diffusible second messenger was involved. 6. The single K+ channel activity, however, was increased on average by 670% following the addition of 5‐HT to the bath when recording channel activity in the outside‐out configuration. Usually all K+ channel subconductance levels increased in activity but the largest increases occurred in the events with 30 and 40 pS conductance. 7. These results suggest that 5‐HT enhances the probability of opening of the resting K+ channel activity, which can open to several levels of conductance, and that no new channel or freely diffusible second messenger is involved in the response.

A critical protein kinase C phosphorylation site on the 5‐HT1A receptor controlling coupling to N‐type calcium channels

The importance of specific protein kinase C (PKC) sites for modulation of the inhibitory coupling of 5‐HT1A receptors to N‐type Ca2+ channels was examined using patch‐clamp techniques in F11 rat dorsal root ganglion × mouse neuroblastoma hybrid cells. The PKC activator phorbol 12‐myristate 13‐acetate (PMA, 10 nm) reduced by 28.6 ± 6.8% 5‐HT‐mediated, but not GTP‐γ‐S‐induced, inhibition of Ca2+ current, whereas a higher concentration of PMA (500 nm) inhibited both the actions of 5‐HT and GTP‐γ‐S. 5‐HT1A receptor expression plasmids with or without mutation of a single PKC site in the second intracellular loop (i2, T149A) or of three PKC sites located in the third intracellular loop (i3, T229A‐S253G‐T343A) were stably transfected into F11 cells. The T149A 5 HT1A receptor inhibited forskolin‐stimulated cyclic AMP levels but was largely uncoupled from Ca2+ channel modulation. In one (i2) clone a response rate to 5‐HT of 31.6% was obtained. The T149A mutant displayed markedly reduced sensitivity to PMA (10 nm) compared to wild‐type 5‐HT1A receptors, with only a 13.4 ± 3% reduction in 5‐HT‐induced channel inhibition; when exposed to 500 nm PMA, reductions in the action of 5‐HT were comparable to those of the wild‐type receptor. By contrast, the i3 mutant displayed comparable sensitivity to the wild‐type 5‐HT1A receptor to either concentration of PMA. PMA at 10 nm exhibited a similar uncoupling effect on the response of the endogenous opiate receptor to the agonist d‐alanine‐5‐leucine‐enkephalin (DADLE) in wild‐type and T149A mutant‐expressing clones. The T149 site of the 5‐HT1A receptor is crucial for receptor uncoupling by sub‐maximal PKC activation while at maximal PKC activation, downstream sites uncouple G proteins from the N‐type Ca2+ channel.

QEHA27, a peptide that binds to G-protein βγ-subunits, reduces the inhibitory effect of 5-HT on the Ca2+ current of rat dorsal raphe neurons

Abstract We studied a 27 amino acid peptide (QEHA 27 ) containing the G β γ binding motif QXXER, found in adenylyl cyclase 2 and several effector proteins of the G β γ -subunit. The patch-clamp technique was utilized to record Ca 2+ current from serotonergic dorsal raphe neurons and to introduce the peptide QEHA 27 into the cell. We investigated whether it antagonized the inhibitory modulation of Ca 2+ current. Compared to the control group, 5-HT was 41% less effective at inhibiting Ca 2+ current with 500 μ M QEHA 27 in the pipette. The QEHA 27 peptide did not simply occlude the response to 5-HT because there was no stimulating effect of QEHA 27 on the G-protein mediated response. This appears to be the first report of an interference between this peptide and the modulation of Ca 2+ currents; it suggests that at least part of the action of the G-protein may be mediated by a β γ -subunit.

Inhibition of TRPC5 Channels by Intracellular ATP

TRPC5 channels are Ca2+-permeable nonselective cation channels activated by G-protein-coupled receptors, although the mechanisms responsible for channel activation and regulation are poorly understood. Carbachol-activated TRPC5 currents were recorded by the whole-cell patch clamp technique from human embryonic kidney 293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. Some published studies of TRPC5 currents have included ATP and/or GTP in the patch pipette, whereas others used an ATP- and GTP-free pipette solution. We initially included these two nucleotides in the patch pipette but found that TRPC5 currents were absent or were very small. Recordings made with an ATP- and GTP-free pipette solution produced large and robust TRPC5 currents. Under these conditions, treatment of cells with Pasteurella multocida toxin, a selective inhibitor of Gαq, almost abolished TRPC5 currents indicating that Gαq is necessary for activation of TRPC5 by the M1 receptor. To study the effect of intracellular ATP on TRPC5 channels, an intracellular perfusion system was used. Perfusion of ADP or control pipette solution had no effect, whereas perfusion of ATP or AMP-PNP, a nonhydrolyzable analog of ATP, significantly inhibited TRPC5 currents. Thus, the effects of ATP have structural specificity and probably involve a direct effect on the channel rather than a phosphorylation-mediated effect. The activity of TRPC5 channels may be linked to cellular metabolism via changes in ATP levels and could be involved in Ca2+ overload occurring after ischemia when ATP is depleted.

Computational modeling of spike generation in serotonergic neurons of the dorsal raphe nucleus

Serotonergic neurons of the dorsal raphe nucleus, with their extensive innervation of limbic and higher brain regions and interactions with the endocrine system have important modulatory or regulatory effects on many cognitive, emotional and physiological processes. They have been strongly implicated in responses to stress and in the occurrence of major depressive disorder and other psychiatric disorders. In order to quantify some of these effects, detailed mathematical models of the activity of such cells are required which describe their complex neurochemistry and neurophysiology. We consider here a single-compartment model of these neurons which is capable of describing many of the known features of spike generation, particularly the slow rhythmic pacemaking activity often observed in these cells in a variety of species. Included in the model are 11 kinds of ion channels: a fast sodium current INa, a delayed rectifier potassium current IKDR, a transient potassium current IA, a slow non-inactivating potassium current IM, a low-threshold calcium current IT, two high threshold calcium currents IL and IN, small and large conductance potassium currents ISK and IBK, a hyperpolarization-activated cation current IH and a leak current ILeak. In Sections 3-8, each current type is considered in detail and parameters estimated from voltage clamp data where possible. Three kinds of model are considered for the BK current and two for the leak current. Intracellular calcium ion concentration Cai is an additional component and calcium dynamics along with buffering and pumping is discussed in Section 9. The remainder of the article contains descriptions of computed solutions which reveal both spontaneous and driven spiking with several parameter sets. Attention is focused on the properties usually associated with these neurons, particularly long duration of action potential, steep upslope on the leading edge of spikes, pacemaker-like spiking, long-lasting afterhyperpolarization and the ramp-like return to threshold after a spike. In some cases the membrane potential trajectories display doublets or have humps or notches as have been reported in some experimental studies. The computed time courses of IA and IT during the interspike interval support the generally held view of a competition between them in influencing the frequency of spiking. Spontaneous activity was facilitated by the presence of IH which has been found in these neurons by some investigators. For reasonable sets of parameters spike frequencies between about 0.6Hz and 1.2Hz are obtained, but frequencies as high as 6Hz could be obtained with special parameter choices. Topics investigated and compared with experiment include shoulders, notches, anodal break phenomena, the effects of noradrenergic input, frequency versus current curves, depolarization block, effects of cell size and the effects of IM. The inhibitory effects of activating 5-HT1A autoreceptors are also investigated. There is a considerable discussion of in vitro versus in vivo firing behavior, with focus on the roles of noradrenergic input, corticotropin-releasing factor and orexinergic inputs. Location of cells within the nucleus is probably a major factor, along with the state of the animal.

Properties of IA in a neuron of the dorsal raphe nucleus

Voltage clamp data were analyzed in order to characterize the properties of the fast potassium transient current I(A) for a presumed serotonergic neuron of the rat dorsal raphe nucleus (DRN). We obtain maximal conductance, time constants of activation and inactivation, and the steady state activation and inactivation functions m(∞) and h(∞), as Boltzmann curves, defined by half-activation potentials and slope factors. I(A) is estimated as g¯(V-V(rev))m(4)h, with g¯=20.5nS. For activation, the half-activation potential is V(a)=-52.5mV with slope factor k(a)=16.5mV, whereas for inactivation the corresponding quantities are -91.5mV and -9.3mV. We discuss the results in terms of the corresponding properties of I(A) in other cell types and their possible relevance to pacemaking activity in cells of the DRN. Methods of identification of serotonergic DRN neurons and the nature of the K(v) channels underlying the A-type current are also discussed.

Role of protein kinase C in agonist-induced desensitization of 5-HT1A receptor coupling to calcium channels in F11 cells

The 5-Hydroxytriptamine 1A receptor (5-HT1A) is expressed both as a pre- and post-synaptic receptor in neurons. The presynaptic receptor preferentially desensitizes compared to post-synaptic receptors, suggesting different underlying mechanisms of agonist-induced desensitization. Using F11 cells as a model of post-synaptic neurons, the present study examined the role of protein kinase C (PKC) and protein kinase A (PKA) in desensitization of the 5-HT1A-receptor by agonist. Desensitization in whole cell experiments was dependent on internal [Ca(2+)] and was blocked by chelation of intracellular Ca(2+). Using the perforated patch technique, desensitization was reduced when Ba(2+) was used as the conducting cation. Selective inhibitors of conventional PKC isoforms prevented 5-HT-induced desensitization, whereas an inhibitor of PKA did not. In cells in which 3 PKC/PKA sites located in the third intracellular loop (i3) of the 5-HT1A receptor were mutated (i3, T229A-S253G-T343A), 5-HT-mediated desensitization was reduced (and abolished in the absence of intracellular Ca(2+)). In cells in which a fourth mutation was added (T149 in the second i2 loop), the cells responded similarly to the triple mutants suggesting that phosphorylation of T149 does not contribute greatly to the desensitization induced by 5-HT-mediated activation of PKC. Thus agonist-induced uncoupling of the 5-HT1A-receptor is PKC-dependent, but requires a different set of phosphorylation sites than phorbol ester-mediated PKC activation, suggesting differential recruitment of PKC. Furthermore, these studies reveal that 5-HT1A-receptor desensitization utilizes a different kinase in F11 cells and serotonergic neurons, which may in part account for their differential sensitivity in vivo.