Measurement of orexin (hypocretin) and substance P effects on constitutively active inward rectifier K(+) channels in brain neurons.

Electrophysiological experiments in our laboratory have led to the discovery that the cholinergic neurons in the nucleus basalis in the rat forebrain possess constitutively active inward rectifier K(+) channels. Unlike cloned inward rectifier K(+) channels, these constitutively active inward rectifier K(+) channels were found to have unique properties, and thus were named "KirNB" (inward rectifier K(+) channels in the nucleus basalis). We found that slow excitatory transmitters, such as orexin (hypocretin) and substance P, suppress the KirNB channel, resulting in neuronal excitation. Furthermore, it was discovered that suppression of KirNB channels by these transmitters is through protein kinase C (PKC). This chapter describes detailed electrophysiological techniques for investigating the effects of orexin and substance P on constitutively active KirNB channels. For this purpose, we also present a method for culturing nucleus basalis cholinergic neurons in which KirNB channels exist. Then, we describe the procedures through which PKC has been determined to mediate inhibition of KirNB channels by orexin and substance P. There are probably many other transmitters which may produce effects on KirNB channels. This chapter will enable researchers to investigate the effects of such transmitters on KirNB channels and their roles in neuronal functions.

Morphological and physiological properties of serotonergic neurons in dissociated cultures from the postnatal rat dorsal raphe nucleus.

We have developed dissociated primary cultures of the dorsal raphe nucleus from postnatal 9-12-day-old rats. The nucleus was dissected out from brain slices, dissociated, and cultured over a glial feeder layer. Serotonin immunocytochemistry revealed that 62% of cultured neurons were serotonergic. There was no significant difference in diameters between serotonergic and non-serotonergic neurons. With the whole-cell patch-clamp method, cultured neurons were tested for responses to 8-hydroxydipropylaminotetraline (8-OH-DPAT, a selective agonist for 5-HT(1A)), and then treated with serotonin immunocytochemistry. Ninety-two percent of neurons responding to 8-OH-DPAT were serotonergic. These results were used to identify serotonergic neurons. In most cases, serotonergic neurons did not show spontaneous firings of action potentials. Constant current depolarizations elicited trains of action potentials that usually did not show marked adaptation. Application of 8-OH-DPAT inhibited action potential firing. The current-voltage relation of the 8-OH-DPAT-induced current indicated an inward rectification with its reversal potential near E(K). Serotonergic neurons were depolarized by phenylephrine, bombesin, and gastrin-releasing peptide. This culture system will serve as a useful tool for elucidating the cellular, physiological, and molecular properties of brain serotonergic neurons.

Interaction of Galphaq and Kir3, G protein-coupled inwardly rectifying potassium channels.

Activation of substance P receptors, which are coupled to Galpha(q), inhibits the Kir3.1/3.2 channels, resulting in neuronal excitation. We have shown previously that this channel inactivation is not caused by reduction of the phosphatidylinositol 4,5-bisphosphate level in membrane. Moreover, Galpha(q) immunoprecipitates with Kir3.2 (J Physiol 564:489-500, 2005), suggesting that Galpha(q) interacts with Kir3.2. Positive immunoprecipitation, however, does not necessarily indicate direct interaction between the two proteins. Here, the glutathione transferase pull-down assay was used to investigate interaction between Galpha(q) and the K(+) channels. We found that Galpha(q) interacted with N termini of Kir3.1, Kir3.2, and Kir3.4. However, Galpha(q) did not interact with the C terminus of any Kir3 or with the C or N terminus of Kir2.1. TRPC6 is regulated by the signal initiated by Galpha(q). Immunoprecipitation, however, showed that Galpha(q) did not interact with TRPC6. Thus, the interaction between Galpha(q) and the Kir3 N terminus is quite specific. This interaction occurred in the presence of GDP or GDP-AlF(-)(4). The Galpha(q) binding could take place somewhere between residues 51 to 90 of Kir3.2; perhaps the segment between 81 to 90 residues is crucial. Gbetagamma, which is known to bind to N terminus of Kir3, did not compete with Galpha(q) for the binding, suggesting that these two binding regions are different. These findings agree with the hypothesis (J Physiol 564:489-500, 2005) that the signal to inactivate the Kir3 channel could be mainly transmitted directly from Galpha(q) to Kir3.

Dominant negative effects of a Gbeta mutant on G-protein coupled inward rectifier K+ channel.

HEK293 cells were transfected with cDNAs for Gbeta1(W332A) [a mutant Gbeta1], Ggamma2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gbeta1gamma2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gbeta1(W332A) suppressed this current. Most likely, Gbeta1(W332A) inhibited the action of the endogenous Gbeta. Such a dominant negative effect of Gbeta1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gbeta1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gbeta from activating the channel.

Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel.

Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP(2)). SP rapidly - with a half-time of approximately 10 s with intracellular GTPgammaS and approximately 14 s with intracellular GTP - inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP(2) than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP(2). A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP(2) than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP(2) theory of channel inhibition. Co-immunoprecipitation reveals that Galpha(q) binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that G(q) activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Galpha(q) to the channel.

Single-cell RT-PCR analysis of GIRK channels expressed in rat locus coeruleus and nucleus basalis neurons.

G protein-coupled inward rectifier potassium channels (GIRK, Kir3) play a crucial role in determining neuronal excitability. Currently, four mammalian GIRK members (GIRK1-4) have been genetically identified. We have been investigating physiological properties of GIRKs in cultured noradrenergic neurons from the locus coeruleus (LC) and cholinergic neurons from the nucleus basalis (NB). Yet, precise information is lacking about which types of GIRK channels are present in these neurons. We performed single-cell RT-PCR on these cultured neurons. In 13 noradrenergic LC neurons, GIRK1, GIRK2, GIRK3, and GIRK4 mRNAs existed in 12, 13, nine, and six neurons, respectively. In six cholinergic NB neurons, GIRK1, GIRK2, GIRK3, and GIRK4 mRNAs existed in six, four, one, and three neurons, respectively. Therefore, GIRK1 and GIRK2 mRNAs are most frequently encountered in both LC and NB neurons.

Dissociated histaminergic neuron cultures from the tuberomammillary nucleus of rats: culture methods and ghrelin effects.

The tuberomammillary nucleus (TMN) in the hypothalamus is the sole source of histamine in the brain. This nucleus, by innervating various brain regions, plays an important role for vital functions such as arousal and appetite. We have developed dissociated primary histaminergic neuron cultures from TMN of postnatal (3 and 10-day-old) rats. More than 50% of our cultured neurons from the TMN were histaminergic as revealed by adenosine deaminase (AD) as well as histamine immunocytochemistry. Among large neurons (diameter, >22 microm), more than 88% were histaminergic. Such large neurons (mean diameter, 26.5 microm) were used for electrophysiology. Using about 2-month-old TMN cultures, we investigated the effects of ghrelin, a recently discovered appetite-stimulating endogenous peptide. In GTPgammaS-loaded neurons, ghrelin (3 microM) suppressed currents that had previously been activated by an inhibitory neuropeptide, nociceptin. The mean current suppression by ghrelin was 471+/-128 pA (S.E.M., n=7). The I-V relationship revealed that the ghrelin-suppressed current was inwardly rectifying with a reversal potential around E(K). These results suggest that ghrelin inhibits G protein-coupled inward rectifier K+ channels (Kir3, GIRK) of TMN neurons and that our TMN cultures are useful for investigating physiological properties of brain histaminergic neurons.

Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3.

G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade. The Gbeta/Galpha interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between Gbeta1 and yeast beta (STE4) suggested that the outer strands of blades 1 and 2 of Gbeta1 could be an interaction area between Gbeta1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between beta-strands, substantially reduced GIRK channel activation, suggesting that these residues are Gbeta/GIRK interaction sites. These mutations did not affect the expression of Gbeta1 or its ability to stimulate PLCbeta2. These residues are surface-accessible and located outside Gbeta/Galpha interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of Gbetagamma with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of Gbeta1 impaired the functional interaction of Gbeta1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of Gbeta1 with Galpha as well as all effectors so far tested.

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome.

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.