Burst activity and ultrafast activation kinetics of CaV1.3 Ca²⁺ channels support presynaptic activity in adult gerbil hair cell ribbon synapses

Abstract

Auditory information transfer to afferent neurons relies on precise triggering of neurotransmitter release at the inner hair cell (IHC) ribbon synapses by Ca²⁺ entry through CaV1.3 Ca²⁺ channels. Despite the crucial role of CaV1.3 Ca²⁺ channels in governing synaptic vesicle fusion, their elementary properties in adult mammals remain unknown. Using near-physiological recording conditions we investigated Ca²⁺ channel activity in adult gerbil IHCs. We found that Ca²⁺ channels are partially active at the IHC resting membrane potential (-60 mV). At -20 mV, the large majority (>70%) of Ca²⁺ channel first openings occurred with an estimated delay of about 50 μs in physiological conditions, with a mean open time of 0.5 ms. Similar to other ribbon synapses, Ca²⁺ channels in IHCs showed a low mean open probability (0.21 at -20 mV), but this increased significantly (up to 0.91) when Ca²⁺ channel activity switched to a bursting modality. We propose that IHC Ca²⁺ channels are sufficiently rapid to transmit fast signals of sound onset and support phase-locking. Short-latency Ca²⁺ channel opening coupled to multivesicular release would ensure precise and reliable signal transmission at the IHC ribbon synapse.

Figure 1. Distribution of Ca_V1.3 and CtBP2/RIBEYE in adult gerbil IHCs

A, basal-coil IHC from an adult (P20) gerbil cochlea immunostained for the Ca_V1.3 Ca²⁺ channel (red) and ribbon marker CtBP2/RIBEYE (green). Colocalization is shown in the merge image in the right column. White dotted lines delineate IHCs. Images represent the maximum intensity projection over all layers of the z-stack. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. B, total number of immunopositive spots for Ca_V1.3 (red bar), total number of CtBP2/RIBEYE (green bar) and colocalized (yellow bar). Number of IHCs analysed for cochlear region is indicated above the bars.

Figure 2. Ca²⁺ channel current in adult gerbil IHCs

A, unitary Ca²⁺ currents recorded from adult gerbil IHCs using a high-K⁺ extracellular solution, and 5 mm Ca²⁺ and 5 μm BayK 8644 in the patch pipette. Transmembrane patch potentials are shown next to the traces. Grey horizontal lines indicate the channel closed state. Arrows and arrowheads show single brief and long-lasting clusters of openings, respectively. B, average current–voltage relation for single Ca²⁺ channel currents recorded in high-K⁺ extracellular solution (2 ≤n≤ 10 patches; 10 IHCs). Mean channel conductance: 14.7 ± 0.2 pS. Shaded area provides an indication for the resting membrane potential of adult IHCs. C, average single Ca²⁺ channel current amplitudes in Na⁺-based extracellular solution plotted on the fit from panel B in order to extrapolate the membrane potential (mean ± SD). Number of IHCs tested is shown. Unless otherwise stated, all recordings in this and the following figures were performed at 37°C.

Figure 3. Ca²⁺ channel currents recorded in Na⁺-based solution

A, representative unitary currents recorded from basal IHCs in a Na⁺-based extracellular solution with 5 mm Ca²⁺ and 5 μm BayK 8644. B, examples of channel openings near −20 mV with mode 1 (brief: arrow) and mode 2 (bursts: arrowhead). Grey horizontal lines in A and B indicate the channel closed state. C, ensemble-averaged Ca²⁺ current near −20 mV. The value of the scale bars in the expanded time course of activation are: 0.3 pA and 10 ms. In A–C the patch transmembrane voltage is indicated as the unknown IHC V_m plus the voltage step delivered to the patch pipette (e.g. V_m+ 20 mV: 20 mV depolarization from V_m). The actual estimated patch transmembrane voltage is shown in parentheses (see Results). D, macroscopic I_Ca at −21 mV recorded from a basal IHC using the same Na⁺-based extracellular solution to that used for single-channel recordings. Scale bars near the expanded time course of activation of the single-channel (C) and whole-cell (D) currents are: 100 pA and 5 ms. Superimposed lines in C and D are single exponential fits (activation is also shown on an expanded timescale). E, first latency distribution was obtained by plotting the natural logarithm of the number of observations ms^-1 (Zampini et al. 2010) as a function of latency.

Figure 4. Single Ca²⁺ channel bursting activity in adult IHCs decreases during a sweep

A, calcium channel P_o at around −20 mV as a function of successive sweeps from basal IHCs. B, time of burst onset obtained at around −20 mV. Note that bursts appear more frequently at the very beginning of the sweep.