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 ∼ 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 ∼ 1800 Ca2+ channels and ∼700 Ca2+-activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains ∼90 Ca2+ and ∼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 μM 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.