Ca2+ regulation of glutamate release from inner hair cells of hearing mice

Significance The first synapse of the auditory pathway faithfully encodes time and intensity of sounds. Ca2+ influx into the inner hair cell via voltage-gated Ca2+ channels links the receptor potential to synaptic vesicle (SV) release. Understanding this Ca2+ signaling and the Ca2+ dependence of SV release is fundamental for deciphering sound encoding. Pre- and postsynaptic patch-clamp recordings in cochleae of hearing mice revealed a supralinear dependence of release on [Ca2+] at the SV release site. Yet, release reports the receptor potential in a near-linear manner. This indicates that [Ca2+] at the SV release site is governed by one or few nearby Ca2+ channels. The supralinear Ca2+ dependence of SV release likely reflects the properties of the Ca2+ sensor of SV release.


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Figure S1 Testing for the potential rundown of exocytosis during manipulations of Ca 2+ influx A,B.Related to Fig. 2 and Fig.3: slow perfusion of Zn 2+ (A) or isradipine (B) progressively reduces the whole-IHC Ca 2+ current (ICa; blue) and the concomitant evoked EPSC charge (QEPSC; orange).Slow washout using normal extracellular solution partially restores ICa and neurotransmitter release for both pharmacological manipulations.In B, wash-in of isradipine once again reduces ICa and QEPSC.C. Related to Fig. 4: during tail-current experiments, rundown of exocytosis was probed as changes in QEPSC elicited by 2 or 10 ms voltage steps to -19 mV over time.We recorded these periodic depolarizations after each full set of tail current protocols in 6 pairs.D. Related to Fig. 5: to address potential rundown of exocytosis, QEPSC elicited by 2 ms pulses at voltages eliciting maximal Ca 2+ influx (-19, -21 and -23 mV) was plotted vs. time (n = 14 pairs).

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Figure S2 Estimating the intrinsic Ca 2+ dependence of SV release for each individual pair Ai-Avii.Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) for each individual pair during perfusion of 1 mM Zn 2+ to reduce the effective fusogenic Ca 2+ .The solid line is a least-squares fit of a power function (QEPSC = a(QCa) m ) to each pair data.For pair # l080118_1 (Aiii), the smaller markers represent the data recorded while washing-out Zn 2+ , which was also included in the fitting.B. Scatter plot of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) of all pairs: different markers and shades of gray for the different pairs (n = 7).C. Ca 2+ cooperativity (m) estimated for each individual pair shown in A. P indicates the boutons contacting the pillar side of the IHC; M indicates the boutons contacting the modiolar side of the IHC.Gray bar corresponds to the median.

Figure S3 .
Figure S3.Apparent Ca 2+ dependence of SV release for each individual pair during isradipine application Ai-Avii.Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) for each individual pair during perfusion 0.5 -2 µM of the dihydropyridine isradipine to progressively shift the Ca 2+ channels to a non-conducting state.The solid line is a least-squares fit of a power function (QEPSC = a(QCa) m ) to each pair data.For pairs # l050318_1 and # l100418_1, the smaller markers represent the data recorded while washing-out isradipine, which was also included in the fitting.B. Scatter plot of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) of all pairs: different markers and shades of gray for the different pairs (n = 7).C. Ca 2+ cooperativity (m) estimated for each individual pair shown in A. P indicates the boutons contacting the pillar side of the IHC; M indicates the boutons contacting the modiolar side of the IHC.Gray bar corresponds to the median.

Figure S4 .
Figure S4.Apparent Ca 2+ dependence of SV release during Ca 2+ tail-current experiments Ai-Aviii.Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) for each individual pair during deactivating (tail) currents upon repolarization B. Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) for all pairs: different markers and shades of gray for the different pairs (n = 8).C. Percentage of failure of synaptic transmission for each individual pair shown in A. P indicates the boutons contacting the pillar side of the IHC; M indicates the boutons contacting the modiolar side of the IHC.Circles and squares are the pairs recorded at 2 mM and 3 mM extracellular [Ca 2+ ], respectively.Black bar represents to the median.D. Scatter plots of the normalized EPSC charges (QEPSC) vs. the corresponding normalized Ca 2+ current integrals (QCa) for all pairs: different markers and shades of gray for the different pairs (n = 7).The solid line is a leastsquares fit of a power function (QEPSC = a(QCa) m ) to the normalized population data for QEPSC and QCa yielded mtails of 1.4 (n = 7 pairs).E. Power function fit to the binned normalized data (bin size ~ 0.15;

Figure S5 .
Figure S5.Apparent Ca 2+ dependence of SV release in the range of IHC receptor potentials Ai-Axiv.Scatter plots of the EPSC charges (QEPSC) vs. the corresponding Ca 2+ current integrals (QCa) for each individual pair in response to 2 ms depolarizations to randomized voltages in the hyperpolarized range.The solid line is a least-squares fit of a power function (QEPSC = a(QCa) m ) to each pair data.Pair #