My lab is interested in the modulation of voltage-dependent and ligand gated conductances in acutely isolated, 5-HT containing, central neurons. The use of predominantly isolated adult brain cells (and lately cultured neuronal cell lines), that can be studied at the whole cell or single channel patch-clamp level, has allowed us to answer several questions not previously investigated in adult central neurons.
We are working on the modulation of calcium current in dorsal raphe serotonergic neurons of the rat brain and have discovered a profound inhibitory modulation of this voltage-activated current by the neurotransmitter serotonin. My recent work has involved characterization of the receptor (5-HT1A), the mechanism (G-protein type), possible second messengers and the calcium channel type that is affected (N, and "N-" channels).
Since coming to SUNY my lab has been interested in signal transduction pathways in the serotonergic cells; particularly how a single receptor is capable of modulating two different effector mechanisms simultaneously. Dorsal raphe neurons are a good model in which to study this because we found that 5-HT opens both K+ channels and modulates Ca2+ channels as a result of activation of the same receptor. Are there two G-proteins, or do different subunits of the same G-protein mediate different effects in the same cell? The patch-clamp technique is a useful one to investigate these questions. We recently have found a differential effect of activation of the cellular enzyme protein kinase C (PKC) on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurons. 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. The functional weight of one pathway may be increased by PKC activation, perhaps at the expense of another (Ca2+ current modulation), thus altering the output of the neuron. In this fashion one branch of a bifurcating signal pathway could be temporarily switched off, perhaps representing a short term form of synaptic plasticity.
In our studies, we provided evidence to support the hypothesis that G-protein alpha and -beta/gamma subunits, in their inactive GDP-bound form, are subject to covalent modification by PKC; thus phosphorylation by PKC may occur in a conformation dependent manner. This effect suggests that the G-protein may be a "programmable messe" that after modification by PKC would interact weakly with Ca2+ channels but normally with K+ channels. We have gathered evidence recently suggesting that the action of PMA in decreasing the effectiveness of Ca current modulation, appears to be exerted on the G-protein in raphe cells rather than on the Ca channel; future work will test this idea using molecular biological approaches coupled with electrophysiology.
Figure 1. 5-HT dramatically increased K+ channel activity in an outside-out patch, when it was added to the bath. A, Single channel I-Vs from the o/o patch to which 5-HT was added. Five conductance levels were observed using both slope and chord conductance. B, Unitary activity is plotted on a compressed time base. C, selected periods of the recorded activity are plotted on an expanded time base, obtained at the locations indicated by the lines.
Penington, N. J., Kelly, J. S., and Fox, A. P. (1993). Unitary properties of potassium channels activated by 5-HT in acutely isolated rat dorsal raphe neurones. J. Physiol. 469, 407-426.
Penington, N. J., and Fox, A. P. (1995). Toxin insensitive Ca current in dorsal raphe neurons. J. Neurosci. 15, 5719-5726.
Chen, Y., and Penington, N. J. (1996). Differential effects of PKC activation on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones J. Physiol. 496.1, 129-137.
Chen, Y., and Penington, N. J. (1997). QEHA, a peptide that binds to G-protein beta/gamma-subunits, reduces the inhibitory effect of 5-HT on the Ca2+ current of rat dorsal raphe neurons. Neurosci. Lett. 224, 87-90.
Chen, Y., and Penington, N. J. (2000). Competition between internal aluminum fluoride and receptor-mediated stimulation of dorsal raphe neuron G-proteins coupled to calcium current inhibition. J. Neurosphysiol. 83, 1273-1282.
Chen, Y., Yao, Y., and Penington, N. J. (2002). Effect of pertussis toxin and n-ethyl-malemide on voltage-dependent and independent calcium current modulation in serotonergic neurons. Neuroscience 111, 207-214.
Wu, X., Kushwaha, N., Albert, P. R., and Penington, N. J. (2002). A critical protein kinase C phosphorylation site on the 5-HT1A receptor controlling coupling to N-type calcium channels. J.Physiology (Lond). 538.1, 41-51.
Yudong Yao, B.S., Graduate Student
Xiaoping Wu, M.D, Postdoctoral Trainee, Research Associate
Reviewer for various scientific journals