Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells

Abstract

Calcium ions serve as intracellular messengers in 2 activities of hair cells: in conjunction with Ca2(+)-activated K+ channels, they produce the electrical resonance that tunes each cell to a specific frequency of stimulation, and they trigger the release of a chemical synaptic transmitter. Our experiments indicate that both of these functions are conducted within a region that extends a few hundred nanometers around each presynaptic active zone. In focal electrical recordings from the plasma membranes of isolated anuran hair cells, we found nearly all of a cell's Ca2+ channels and Ca2(+)-activated K+ channels clumped at a fixed ratio in an average of 20 clusters on the basolateral membrane surface. Because serial-section electron microscopy indicated that each hair cell has approximately 19 afferent synaptic contacts with a similar distribution upon its basolateral surface, we conclude that the channel clusters coincide with synaptic active zones. Ensemble-variance analysis of current fluctuations indicated that each cell has a total of approximately 1800 Ca2+ channels and approximately 700 Ca2(+)-activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains approximately 90 Ca2+ and approximately 40 Ca2(+)-activated K+ channels. Freeze-fracture electron microscopy demonstrated an average of 133 large intramembrane particles in the presynaptic membrane at each active zone, an observation that suggests that the particles are the clustered channels. We used the K+ channel's sensitivity to intracellular Ca2+ to assay the concentration of free Ca2+ in the presynaptic cytoplasm, which we found to vary between 10 microM and 1 mM over the physiological range of membrane potentials. The inferred concentrations agreed with the values predicted for free diffusion of Ca2+ away from Ca2+ channels scattered randomly within a 300-nm-diameter synaptic active zone. The close association among Ca2+ channels, Ca2(+)-activated K+ channels, and synaptic active zones is necessary both for the rapid activation of K+ currents required in electrical resonance and for the transmission at afferent synapses of information about the phases of high-frequency stimuli.

[Ca2+]iElevations Detected by BK Channels during Ca2+Influx and Muscarine-Mediated Release of Ca2+from Intracellular Stores in Rat Chromaffin Cells

“…Therefore, there is increasing interest in using intrinsic Ca 2ϩ -dependent proteins or processes to define the amplitudes and time course of [Ca 2ϩ ] i elevations. In the squid giant axon (Adler et al, 1991) and frog hair cells (Roberts et al, 1990), this approach has supported the theoretical expectation that [Ca 2ϩ ] i elevations during Ca 2ϩ influx are large and rapid. Furthermore, these studies have suggested that particular Ca 2ϩ -dependent processes are coupled closely to sites of Ca 2ϩ influx.…”

Ca2+from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

“…There is evidence for CaK channels colocalized with the Ca 2ϩ channels in the frog NMJ terminal (Robitaille and Charlton, 1992;Robitaille et al, 1993). It is therefore possible that perhaps 30 -50% of the particles in the AZ are CaK channels, similar to the AZs of hair cells (Roberts et al, 1990).…”

A quantitative measurement of the dependence of short-term synaptic enhancement on presynaptic residual calcium

“…These rapid, high transients produce the rapid exocytosis of vesicles containing neurotransmitter substances from presynaptic nerve terminals (Simon and Llinas, 1985;Zucker and Fogelson, 1986;Roberts et al, 1990;Augustine et al, 199 1;Llinas et al, 1992). Dissipation of these transients by diffusion and buffering adds a small Ca*+ load to the longer-lasting spatially uniform [Ca*+],-termed the residual free calcium concentration-of the terminal.…”