Characterization of outward currents in neurons of the avian nucleus magnocellularis. J. Neurophysiol. 80: 2824-2835, 1998. Neurons of the nucleus magnocellularis (NM) preserve the timing of auditory signals through the convergence of a variety of voltage- and ligand-gated ion channels. To understand better how these channels interact, we have characterized the kinetics, voltage sensitivity, and pharmacology of outward currents of NM neurons in brain slices. The reversal potential (Erev) of outward currents varied with potassium concentration as expected for currents carried by potassium. However, Erev was consistently more positive than the Nernst potential for potassium (EK). Deviation of Erev from the calculated EK most likely arose from potassium accumulation in extracellular spaces by potassium conductances active at rest and during depolarizing steps. Three outward potassium currents were studied that varied in voltage and pharmacological sensitivity. A tetraethylammonium (TEA)-sensitive, high-threshold current was activated within 1-5 ms of the onset of depolarization, with a half-maximal activation voltage (V1/2) of -19 mV. It was blocked partially by 4-aminopyridine (4-AP) and was the dominant ionic conductance of NM neurons. A dendrotoxin-I (DTX) and 4-AP-sensitive, low-threshold current had a V1/2 of -58 mV, rapid activation kinetics, and only partial inactivation, with decay time constants between 20 and 100 ms. A rapidly inactivating current was observed that was resistant to TEA and DTX and was blocked by intracellular Cs+. The transient current was inactivated almost completely at the resting potential. The onset of inactivation was fastest at potentials negative to those that caused activation. When intracellular K+ was replaced by Cs+, large inward and outward currents were obtained that corresponded respectively to the above-mentioned DTX- and TEA-sensitive currents. Outward, TEA-sensitive current was carried by Cs+, with a PCs/PK of approximately 0.1. In current-clamped neurons, DTX induced repetitive firing and increased membrane time constant near rest but had little effect on action potential duration. These studies indicate that a low-threshold, DTX-sensitive current plays a key role in making NM neurons highly responsive to the onset and offset of synaptic stimuli.

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Nicotinic acetylcholine receptors (AChRs) located in the postsynaptic membrane on neurons are responsible for mediating fast, excitatory synaptic transmission. If synaptic AChRs are also highly permeable to calcium as reported recently for several kinds of neuronal AChRs, the synaptic receptors could regulate calcium-dependent events in the neurons in concert with normal transmission. Chick ciliary ganglion neurons have two classes of AChRs, one located predominantly in the synaptic membrane and responsible for synaptic signaling through the ganglion and the other located almost exclusively in nonsynaptic membrane and having no known function. The nonsynaptic receptors can readily elevate intracellular calcium concentrations. The experiments reported here indicate that synaptic-type receptors can raise intracellular calcium levels to the same extent as the nonsynaptic receptors and that they do so not only by being permeable to calcium themselves but also by activating voltage-dependent calcium channels (VDCCs). Currents of equivalent amplitude are obtained through the synaptic-type receptors when neurons are bathed in solutions containing either sodium or calcium as the sole extracellular cation. Measuring the effect of ion substitutions on the reversal potential of the receptors and applying the Goldman-Hodgkin-Katz constant field equation indicates the receptors are at least as permeable to calcium as to sodium. When neurons are loaded with the calcium-sensitive dye fluo-3 and challenged with nicotine, both the synaptic-type and nonsynaptic AChRs substantially elevate intracellular calcium levels under physiological conditions, and do so largely by activating VDCCs. Confirmation that synaptic-type AChRs can elevate intracellular calcium levels in the absence of contributions from VDCCs was obtained from voltage-clamp experiments on neurons loaded with fluo-3. The fluorescence signals indicate that the nicotine-induced calcium increases in neurons voltage clamped at rest are nearly as great as those induced in the same neurons when VDCCs are maximally activated by a voltage step. Calcium flux through AChRs may be particularly important for mediating local changes in calcium concentrations near the plasma

The recognition that intracellular free calcium serves as a ubiquitous intracellular signal has motivated efforts to elucidate mechanisms by which cells regulate calcium influx. One route of entry that may offer both spatial and temporal fine resolution for altering calcium levels is that provided by cation-permeable, ligand-gated ion channels. Biophysical measurements as well as calcium imaging techniques demonstrate that neuronal nicotinic acetylcholine receptors as a class have a high relative permeability to calcium; some subtypes equal or exceed all other known receptors in this respect. Activation of nicotinic receptors on neurons can produce substantial increases in intracellular calcium levels by direct passage of calcium through the receptor channel. When multiple classes of nicotinic receptors are expressed by the same neuron, each appears capable of increasing calcium in the cell but may differ with respect to location, temporal response, agonist sensitivity, or regulation in achieving it. As a result, nicotinic receptors must be considered strong candidates for signaling molecules through which neurons regulate a diverse array of cellular events.

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