Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings.

Large conductance, Ca2+-activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. The single channel conductance of cell body BK channels, recorded from inside-out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P < 0.01). At low open probability (Po), the reciprocal of the slope in the ln(NPo)-voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mVper e-fold change in NPo, respectively. At 40 mV, the [Ca2+]i producing half-maximal activation was 273 nM, as opposed to > 1.53 μM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as ‘type I’ isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37 % of control), or 10 nM iberiotoxin (NPo, 5 % of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings. Rat magnocellular neurones with cell bodies in the supraoptic and paraventricular nuclei of the hypothalamus send their axonal nerve endings into the neurohypophysis, where they release oxytocin or vasopressin into the bloodstream during highly specific firing patterns. Peptide release from the neurohypophysis is closely controlled by an interplay between the duration and frequency of the action potential burst, and the silence that separates the bursts generated by these neurones (Cazalis et al. 1985). Thus, understanding of peptide release from the hypothalamic-neurohypophysial system requires characterization of the conductances that shape action potentials in both the cell bodies and their axonal endings. The physical separation of cell bodies and nerve endings in this system allows a unique opportunity to elucidate the differences in neuronal excitability, and underlying conductances in different cellular domains. Large conductance Ca2+-activated K+ (BK) channels are ubiquitous in neurones. They typically exhibit high permeability and selectivity for K+, and activation upon increases in [Ca2+]i and/or depolarization (for reviews see: McManus, 1991; Kaczorowski et al. 1996), which make them effective ‘negative feedback’ regulators of depolarization and Ca2+ entry, and subsequent processes, such as neurotransmitter release (Robitaille & Charlton, 1992). They contribute to repolarization of individual action potentials, and/or hyperpolarizations following trains of spikes (Crest & Gola, 1993). Mutations in the Drosophila slowpoke gene, which encodes BK channels (Kaczorowski et al. 1996), cause abnormally broad action potentials (Elkins et al. 1986). Ca2+ entry produces opposite effects on action potential shape in neurohypophysial nerve endings vs. supraoptic cell bodies. Exposure of nerve endings to extracellular solutions containing either zero Ca2+, Ca2+-channel blockers, or the tetraethylammonium ion, slows repolarization and broadens individual spikes (Wang & Lemos, 1995), which suggests the participation of Ca2+-sensitive voltage-gated K+ conductances in spike repolarization. Indeed, BK channels have been characterized at both macroscopic and single channel levels in these nerve endings (Wang et al. 1992). In contrast, exposure of cell bodies to zero Ca2+ or Ca2+-channel blockers speeds repolarization and narrows the spike (Widmer et al. 1998), consistent with a reduced Ca2+ influx, but little, if any, contribution of BK channel activity to spike repolarization. Furthermore, iberiotoxin, a specific blocker of BK channels, fails to modify the shape of the cell body spike (Widmer et al. 1998). Using a slice preparation, it has recently been shown that the slow after-hyperpolarization in supraoptic neurones is blocked by charybdotoxin, but not by iberiotoxin, these data being interpreted as a blockade of an intermediate conductance, Ca2+-activated K+ channel (Greffrath et al. 1998). Together, these results are consistent with either the absence of BK channels in the cell bodies, or the existence of a BK channel phenotype with properties markedly different from those of the nerve ending channel. Differential distribution of BK channel types has been reported among different hair cells in the cochlea using the RT-PCR technique (Navaratnam et al. 1997; Rosenblatt et al. 1997). BK channels from vascular smooth muscle or brain, when incorporated into lipid bilayers, exhibit a variety of phenotypes (isochannels) (Reinhardt et al. 1989; Toro et al. 1991; Sansom & Stockand, 1994). However, the topology and function of distinct BK isochannels regionally segregated in the same neurone have not been reported. In the present paper, we describe for the first time, biophysical and pharmacological characteristics of a BK channel subtype in supraoptic cell bodies, allowing a comparison with the BK channel of the corresponding nerve endings in the neurohypophysis. We also investigate the contribution of each BK channel to single spike repolarization within their particular neuronal domain. Moreover, nerve ending BK channels have been shown to be targets of alcohol action (Dopico et al. 1996), possibly contributing to drug-induced reduction of neuropeptide release from the neurohypophysis. The physical separation of cell bodies and nerve endings in the hypothalamo-neurohypophysial system allow us to examine the regionalization of responses of an ion channel class within a neurone to ethanol. A preliminary set of data has appeared in an abstract form (Dopico et al. 1998b).