Presynaptic mechanisms controlling calcium-triggered transmitter release at the neuromuscular junction

Highlights

• The positioning of Ca²⁺ channels relative to synaptic vesicles determines function.

• The Ca²⁺-release relationship can be explained by an excess of calcium binding sites.

• Action potential shape indirectly influences calcium entry at the synapse.

• Low probability of Ca²⁺ channel opening and Ca²⁺ ion-triggered fusion governs release.

• Active zone heterogeneity likely characterizes neuromuscular junction function.

Calcium-triggered neurotransmission underlies most communication in the nervous system. Yet, despite the conserved and essential nature of this process, the molecular underpinnings of calcium-triggered neurotransmission have been difficult to study directly and our understanding to this date remains incomplete. Here we frame more recent efforts to understand this process with a historical perspective of the study of neurotransmitter release at the neuromuscular junction. We focus on the role of calcium channel distribution and organization relative to synaptic vesicles, as well as the nature of the calcium sensors that trigger release. Importantly, we provide a framework for understanding how the function of neurotransmitter release sites, or active zones, contributes to the function of the synapse as a whole.

Introduction

More than a century has passed since the first studies of calcium-triggered acetylcholine release from the neuromuscular junction (NMJ) were published. Early reports by Locke [1] in 1894 and Overton [2] in 1904 provided some of the first pieces of evidence that calcium ions were required for communication between nerve and muscle cells. These observations were followed by more extensive investigations including a series of studies in the 1930s by T.P. Feng [3]. By measuring muscle contractions following nerve stimulation, Feng studied the effects of stimulus frequency, calcium ion concentration, and various pharmacological agents on neurotransmission at the NMJ. While these investigations provided important evidence for the role of calcium in neurotransmission at the NMJ, it was not until the invention of the microelectrode, and the demonstration that it could be used for recording membrane potential from the frog sartorius muscle [4], that detailed study of calcium-triggered chemical transmitter release at the NMJ became possible with sufficient temporal and spatial resolution to begin to elucidate underlying mechanisms.

In a series of publications in the 1950s and 1960s, Paul Fatt, Jose del Castillo, Ricardo Miledi, and Bernard Katz made a number of seminal discoveries which solidified the importance of the influx and timing of presynaptic calcium ions for chemical transmitter release [5, 6]. Among other observations, they showed that varying the extracellular calcium concentration affected the size of the action potential-evoked endplate potential (EPP), but not the size of the spontaneous miniature endplate potentials (mEPPs) [7, 8]. Katz and colleagues also discovered that calcium ions had to be present extracellularly during action potential invasion at the synapse for transmitter release to occur [9, 10]. Katz and Miledi conducted complementary studies using the squid giant synapse to demonstrate that calcium ions enter the nerve terminal during the action potential to trigger transmitter release [11, 12, 13]. In addition, they also used recordings of action potential-evoked and spontaneous transmitter release from the frog NMJ in low extracellular calcium (a critical experimental variable) to formulate the quantal theory of chemical transmitter release [8]. Katz and coworkers’ groundbreaking work also helped popularize the NMJ as a model system for studying calcium-dependent mechanisms of transmitter release. In recognition of the impact of this work, Bernard Katz shared the Nobel Prize in Physiology or Medicine in 1970.

These foundational studies have spurred a renewed effort over the last two decades to better understand the microscopic underpinnings of synaptic function at the NMJ. Aided by novel experimental techniques and computational approaches, recent studies have led to new insights at the NMJ and also provided a framework for better understanding of early discoveries. Below we highlight and discuss several key aspects of synaptic function, present recent discoveries, and discuss how these advances have helped to shed new light on the underlying microscopic principles of synaptic function. Finally, we identify and propose new avenues for future study.