Sodium channels implement a molecular leaky integrator that detects action potentials and regulates neuronal firing.

Voltage-gated sodium channels play a critical role in cellular excitability, amplifying small membrane depolarizations into action potentials. Interactions with auxiliary subunits and other factors modify the intrinsic kinetic mechanism to result in new molecular and cellular functionality. We show here that sodium channels can implement a molecular leaky integrator, where the input signal is the membrane potential and the output is the occupancy of a long-term inactivated state. Through this mechanism, sodium channels effectively measure the frequency of action potentials and convert it into Na+ current availability. In turn, the Na+ current can control neuronal firing frequency in a negative feedback loop. Consequently, neurons become less sensitive to changes in excitatory input and maintain a lower firing rate. We present these ideas in the context of rat serotonergic raphe neurons, which fire spontaneously at low frequency and provide critical neuromodulation to many autonomous and cognitive brain functions.

Regulation of TRPC5 currents by intracellular ATP: Single channel studies.

TRPC5 channels are nonselective cation channels activated by G-protein-coupled receptors. It was previously found that recombinant TRPC5 currents are inhibited by intracellular ATP, when studied by whole-cell patch-clamp recording. In the present study, we investigated the mechanism of ATP inhibition at the single-channel level using patches from HEK-293 cells transiently transfected with TRPC5 and the M1 muscarinic receptor. In inside-out patches, application of ATP to the intracellular face of the membrane reduced TRPC5 channel activity at both positive and negative potentials without affecting the unitary current amplitude or open dwell time of the channel. The effect of ATP was rapidly reversible. These results suggest that ATP may bind to the channel protein and affect the ability of the channel to open or to remain in an open, nondesensitized state. The activity of TRPC5 channels may be influenced by cellular metabolism via changes in ATP levels.

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.

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.

Properties of I(A) 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.

Inhibition of TRPC5 channels by intracellular ATP.

TRPC5 channels are Ca(2+)-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 Galpha(q), almost abolished TRPC5 currents indicating that Galpha(q) 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 Ca(2+) overload occurring after ischemia when ATP is depleted.