Electrophysiological properties of BK channels in Xenopus motor nerve terminals.

Large conductance calcium-activated potassium (BK or Maxi-K) channels are found in many nerve terminal membranes, where they are associated with voltage-gated Ca2+ channels and help modulate release (Mallart, 1985; Lindgren & Moore, 1989; Roberts et al. 1990; Sivaramakrishnan et al. 1991; Robitaille & Charlton, 1992; Wang et al. 1992; Robitaille et al. 1993; Wangemann & Takeuchi, 1993; Bielefeldt & Jackson, 1993; Issa & Hudspeth, 1994; Blundon et al. 1995; Knaus et al. 1996; Katz et al. 1997; Sakaba et al. 1997; Sun et al. 1999; Womack & Khodakhah, 2002). Blockage of BK channels with the peptide toxins charybdotoxin (CTX) or iberiotoxin (IBTX) causes broadening of action potentials and usually increases transmitter release (Morita & Barrett, 1990; Pattillo et al. 1999; Faber & Sah, 2003), suggesting that a prominent role is the truncation of action potentials and reduction in Ca2+ entry and release. In other cases, BK channels may cause more subtle changes in action potential shape that enhance release (Pattillo et al. 1999, 2001; Van Goor et al. 2001). BK channels are also functionally coupled with Ca2+ channels in the bouton-like synapses formed by Xenopus motoneurone neurite varicosities on muscle cells in culture; this preparation is one of the very few in which it is possible to patch clamp both pre- and postsynaptic membranes simultaneously to correlate presynaptic currents with neurotransmitter release during synaptic activity (Yazejian et al. 1997, 2000; Sand et al. 2001; Pattillo et al. 2001). The Xenopus BK channel gene has recently been cloned. It is expressed in five alternatively spliced variants, including one, xSlo59, which is neurone-specific, is not expressed until late in neuronal differentiation, and differs from other variants in being activated at lower voltages (Kukuljan et al. 2003). It is probably this neurone-specific BK channel that is found in the Xenopus motoneurone terminals in culture. For these channels to be useful in modulation of release in fast-releasing synapses, it is necessary that they be closely colocalized with Ca2+ channels, have activation kinetics in the submillisecond range and have a Ca2+ affinity appropriate to concentrations that might occur in or near microdomains at active zones, without saturating. BK channels that have been characterized biophysically tend to vary greatly in their kinetics and Ca2+ and voltage sensitivity (for reviews see McManus, 1991; Vergara et al. 1998; Gribkoff et al. 2001; Sah & Faber, 2002). Those found in non-neuronal membranes, or even in neuronal cell bodies, tend to exhibit activation kinetics too slow and/or Ca2+ affinity too high (Ikemoto et al. 1989; Wang et al. 1992; Bielefeldt & Jackson, 1993; Safronov & Vogel, 1998) to respond to changes in [Ca2+] on the submillisecond time scale between ∼1 μm and >100 μm. In contrast, the few BK currents (IBK) that have been studied in ‘fast’-transmitting nerve terminals (Sakaba et al. 1997; Sun et al. 1999) or terminal-like cells (e.g. hair cells, Art et al. 1995) have shown significantly faster kinetics and often lower Ca2+ affinity. Interestingly, cloned Slo-1 channels expressed in Xenopus oocytes exhibit Ca2+ affinity close to that of nerve terminals (Xia et al. 2002). BK channels in the Xenopus varicosity synapses are activated so rapidly by the combination of depolarization and Ca2+ influx that whole-terminal BK currents can track changes in local Ca2+ concentration during action potentials or Ca2+ tail currents with delays of no more than a few tens or hundreds of microseconds (Yazejian et al. 2000). The reported close functional coupling between Ca2+ channels and BK channels has also led to use of the latter to quantify the [Ca2+] achieved during depolarization in frog and turtle hair cells (Roberts et al. 1990; Art et al. 1995), bipolar cell terminals (Sakaba et al. 1997), and rat chromaffin cells (Prakriya & Lingle, 1999). This method, in principle, has many advantages over using Ca2+-sensitive dyes to measure [Ca2+], since BK channels are endogenous molecules located in the membrane near Ca2+ channels and their level of activation can be studied without introduction of extrinsic Ca2+ binding molecules or washout of endogenous buffers or essential cofactors. To clarify the kinetic basis of BK channel activity during action potential invasion and to validate the usefulness of BK channels in reporting, quantitatively, changes in [Ca2+] near release sites in nerve terminals, we have investigated single channel sensitivity to [Ca2+]i and to voltage, the degree of coupling to Ca2+ channels and the responses of BK channels to transient membrane potential changes at different [Ca2+]. We find that these nerve terminal BK channels exhibit lower Ca2+ affinity and faster activation kinetics than most others studied to date, that the time constant of BK channel activation is a more accurate reflection of [Ca2+] than is channel open probability, and that differences in activation time constant can distinguish [Ca2+] over a range from ∼0.2 μm to >100 μm. The data support the conclusions that BK channels are closely colocalized with Ca2+ channels, that they can be used to accurately measure [Ca2+] during Ca2+ transients, and that they can contribute importantly to shaping the terminal action potential.