Single-channel properties of BK-type calcium-activated potassium channels at a cholinergic presynaptic nerve terminal.

Substantial evidence suggests that most, if not all, nerve terminals that secrete neurotransmitters by action potential-dependent mechanisms exhibit a calcium-activated potassium (KCa) channel. Such terminals include presynaptic nerve terminals at fast-transmitting synapses (Bartschat & Blaustein, 1985; Farley & Rudy, 1988; Anderson et al. 1988; Lindgren & Moore, 1989; Schneider et al. 1989; Astrand & Stjarne, 1991; Sivaramakrishnan et al. 1991; Robitaille & Charlton, 1992; Wangemann & Takeuchi, 1993; Blundon et al. 1995; Rahamimoff et al. 1995; Katz et al. 1997; Sakaba et al. 1997; Yazejian et al. 1997), hormone-secreting nerve terminals (Bielefeldt et al. 1992; Wang & Lemos, 1992; Wang et al. 1992; Bielefeldt & Jackson, 1993) and sense-organ cells (such as hair cells, Edgington & Stewart, 1981; Roberts et al. 1990, 1991; Issa & Hudspeth, 1994; Art et al. 1995) which share many properties with fast-transmitting nerve terminals. It is not clear which specific types of KCa channel are present at most of these sites but, where tested by specific staining or direct recording, the high-conductance, BK-type has been repeatedly observed (Smith et al. 1986; Castle & Strong, 1986; Roberts et al. 1991; Robitaille & Charlton, 1992; Bielefeldt & Jackson, 1993; Wangemann & Takeuchi, 1993; Issa & Hudspeth, 1994; Art et al. 1995; Knaus et al. 1996). Other studies have presented more indirect evidence for the presence of this channel in the nerve terminal on the basis of effects of peptide blockers, primarily charybdotoxin (CTX) or iberiotoxin (IbTX), or intracellular buffers on transmitter release (Kumamoto & Kuba, 1985; Sivaramakrishnan et al. 1991; Robitaille & Charlton, 1992; Stretton et al. 1992; Takeuchi & Wangemann, 1993; Robitaille et al. 1993; Yazejian et al. 1997). Despite the evident broad distribution of BK channels in nerve terminals little is known about their biophysical properties and to what extent these may differ from their relatives in the cell soma. This question has become of particular interest recently with the discovery that the BK channel can be expressed in many functionally different isoforms (Butler et al. 1993; Wei et al. 1998). In addition, the channel can associate with a β subunit (Vergara et al. 1998) that has marked effects on its biophysical and pharmacological properties. Thus, because of this diversity a full understanding of the role of this channel in the nerve terminal requires direct recording of its properties in situ. Furthermore, the complex activation characteristics of the BK channel require analysis at the single-channel level since it is gated by both a ligand and a physical factor, i.e. calcium and voltage. There have been only two nerve terminals at which the single-channel properties of BK channels have been examined (Wang et al. 1992; Wangemann & Takeuchi, 1993; Bielefeldt & Jackson, 1993) due, presumably, to the general small size and inaccessibility of these structures. Few nerve terminals are of sufficiently large size to permit direct patch-clamp recording, and in the cases where this is possible obtaining sufficient data requires considerable experimental effort. This study presents the first characterization of single BK channels at the presynaptic nerve terminal of a neuron-to-neuron synapse. We used the calyx-type synapse of the chick ciliary ganglion (CCG) to record and characterize single presynaptic high-conductance KCa channels. This experimental preparation was the first in which an intact vertebrate presynaptic terminal could be voltage clamped to record whole-terminal currents (Stanley, 1989) or clamped in the cell-attached configuration (Stanley, 1991) to record single-channel activity. We have recently presented evidence for the presence of KCa channels on this terminal by direct recording of an outwardly rectifying K+ current that was sensitive to CTX and IbTX (Tozer et al. 1998). In this study we first used the patch-clamp technique in the outside-out and inside-out configurations to test for block with standard K+ channel blockers. We next used the same techniques to examine the voltage and calcium sensitivity of single BK channels. Finally, we used the cell-attached configuration to test whether presynaptic BK channels could be activated by calcium influx through the same membrane patch.