Uniquantal Release through a Dynamic Fusion Pore Is a Candidate Mechanism of Hair Cell Exocytosis

Abstract

The mechanisms underlying the large amplitudes and heterogeneity of excitatory postsynaptic currents (EPSCs) at inner hair cell (IHC) ribbon synapses are unknown. Based on electrophysiology, electron and superresolution light microscopy, and modeling, we propose that uniquantal exocytosis shaped by a dynamic fusion pore is a candidate neurotransmitter release mechanism in IHCs. Modeling indicated that the extended postsynaptic AMPA receptor clusters enable large uniquantal EPSCs. Recorded multiphasic EPSCs were triggered by similar glutamate amounts as monophasic ones and were consistent with progressive vesicle emptying during pore flickering. The fraction of multiphasic EPSCs decreased in absence of Ca(2+) influx and upon application of the Ca(2+) channel modulator BayK8644. Our experiments and modeling did not support the two most popular multiquantal release interpretations of EPSC heterogeneity: (1) Ca(2+)-synchronized exocytosis of multiple vesicles and (2) compound exocytosis fueled by vesicle-to-vesicle fusion. We propose that IHC synapses efficiently use uniquantal glutamate release for achieving high information transmission rates.

The ubiquitous nature of multivesicular release

“…Unlike conventional synapses that require action potentials to release vesicles, the presynaptic cells at many primary sensory synapses typically operate via graded membrane potential changes that can last several seconds. Here, vesicles are often tethered to a specialized protein structure called a ribbon that is thought to maintain and synchronize the release of hundreds of vesicles per second [86–89, 16 but see, 90]. For example, at retinal bipolar cell terminals a 200 ms depolarization can release ~6000 synaptic vesicles at 50 synaptic ribbon release sites, with each ribbon releasing ~120 vesicles, a number similar to the total number of docked vesicles at the ribbon plus those tethered to the ribbon [91].…”

“…Several hypotheses have been proposed to explain the nearsynchronous fusion of several tens of vesicles, including coordination of release sites and tight coupling of calcium channels to the release machinery [89, 96, 21], fusion of several vesicles to each other prior to exocytosis (compound fusion [97]). Recently, however, fusion pore regulation of UVR events has been proposed as an alternate mechanism to explain multiphasic EPSCs [90]. …”

New insights into cochlear sound encoding

“…Recently, uniquantal release through a dynamic fusion pore has been suggested as an alternative hypothesis for IHC synapses of rodents 76 . The motivation came from considering the spike rates of hundreds of hertz over prolonged periods of time, which, according to the multiquantal hypothesis (on average, six vesicles per EPSC 59 ), would require at least sixfold-higher vesicle release rates, which seem very high considering measured rates of sustained exocytosis of about 700 vesicles per second 27 .…”

Molecularly and structurally distinct synapses mediate reliable encoding and processing of auditory information

“…Moreover, multiphasic (less synchronized) and monophasic (synchronized) events were observed (Glowatzki and Fuchs, 2002). Recently, an alternative release mechanism has been suggested (Chapochnikov et al, 2014) that builds on the assumption that a single vesicle can release sufficient amounts of glutamate to generate the measured large EPSCs. In the framework of this hypothesis, the observed heterogeneity of EPSC amplitudes and shape is explained by post-fusion control via a dynamic fusion pore.…”

“…This spatial characteristic ensures an instant reception of released glutamate upon stimulation. The flickering of a dynamic fusion pore releasing only portions of a single vesicle's content would cause multiphasic EPSCs (Chapochnikov et al, 2014). It is left to speculation whether both mechanisms co-exist at IHC ribbon synapses or are mutually exclusive.…”