Functions of Presynaptic Voltage-gated Calcium Channels

Abstract Voltage-gated calcium channels are the principal conduits for depolarization-mediated Ca2+ entry into excitable cells. In this review, the biophysical properties of the relevant members of this family of channels, those that are present in presynaptic terminals, will be discussed in relation to their function in mediating neurotransmitter release. Voltage-gated calcium channels have properties that ensure they are specialized for particular roles, for example, differences in their activation voltage threshold, their various kinetic properties, and their voltage-dependence of inactivation. All these attributes play into the ability of the various voltage-gated calcium channels to participate in different patterns of presynaptic vesicular release. These include synaptic transmission resulting from single action potentials, and longer-term changes mediated by bursts or trains of action potentials, as well as release resulting from graded changes in membrane potential in specialized sensory synapses.


Introduction
Voltage-gated calcium (Ca V ) channels are well understood to function as the route for Ca 2þ entry into cells, particularly excitable cells, in response to depolarization. However, they represent a family of channels with a variety of biophysical properties that are exploited differentially to perform particular functions in presynaptic terminals. These varied roles will be explored in relation to different types of synaptic boutons. It is important to understand how the membrane potential of the presynaptic terminal, which is dictated in part by other channels present, as well as the intracellular free Ca 2þ , affects the dynamics of the Ca V channel activity. Their properties, in addition to the positional anchoring of the particular channels, dictate their ability to trigger and sustain vesicular release.

Molecular properties of Ca V channels
Distinct voltage-dependent Ca 2þ conductances were first characterized by electrophysiological and pharmacological means, involving both whole-cell and single-channel recording. A number of different currents were identified, 1-3 and termed L-type, 4 T-type, or low voltage-activated, 2,4 N-type, 4 P-type, 5 and R-type 6 ( Table 1). Subsequent molecular cloning identified three subfamilies of mammalian Ca V channels: Ca V 1 with four members (all of them giving rise to L-type currents), Ca V 2 with three members (forming P/Q-, N-, and R-type currents), and Ca V 3 with three members, all producing T-type currents ( Table 1).
The pore-forming Ca V a 1 subunits all have very similar structures with 24 transmembrane segments separated into four domains, each with a voltage-sensing and a pore module. 16,17 The domains are joined by intracellular loops, and a long . 15 The table describes the 10 mammalian CaV genes products and collects data on V50, activation from a number of original sources. It is necessary to refer to the papers for details of the auxiliary subunits, splice variants, and other conditions used, which can further affect the biophysical properties of the channels. LVA and HVA refer to the original nomenclature defining two types of calcium current (low-and high-voltage-activated). 2 C-terminal tail. The Ca V 1 and Ca V 2 channel a 1 subunits are each associated with an auxiliary ß and a 2 d subunit. There are four ß and four a 2 d subunits, which have divergent cellular expression patterns, and confer some differing properties on the channels with which they associate (see below). The Ca V 2 channels, particularly Ca V 2.1 and Ca V 2.2 are the main channels involved in presynaptic function.
How the Biophysical Properties of Ca V Channels Can Shape Their Function Ca V channels have a variety of characteristics that will be considered in this review, including voltage-dependent, kinetic and Ca 2þ -dependent properties ( Table 1, Figure 1). The complex interplay between these elements determines the amount and timing of Ca 2þ entry that occurs during depolarization, for example, during an action potential. Voltage-dependent activation dictates the range of voltages over which the channels will activate when depolarized, which, for presynaptic terminals, is key to their excitability. Although Ca V channels were originally divided into low (Ca V 3) and high (Ca V 1 and Ca V 2) voltage-activated channels, it is clear that there is actually a continuum of activation ranges between these channels, when they are compared under more physiological conditions (Table 1). Such comparisons are nevertheless difficult to equate with physiological activation of these channels in neurons, and more specifically in presynaptic terminals. This is in part because the specific mix of channel splice isoforms 10,20 and the associated auxiliary subunits associated with each channel, which can strongly affect their biophysical properties (Table 1), are rarely known. It is also the case that studies of the biophysical properties of Ca V channels necessarily use non-physiological conditions to isolate the calcium currents, together with a variety of divalent cation concentrations (Table 1), which affect membrane charge screening to differing extents, and therefore influence the voltage drop across the membrane experienced by the channels.
Since voltage-dependent inactivation also occurs for most Ca V channels over a range of physiological voltages, which are for the most part more negative than their activation range, the resting potential will determine the proportion of channels available to open. This availability will be different for each channel type; furthermore, in the resting membrane potential range of most neurons, or during small subthreshold depolarizations, only T-type currents will have the ability to exhibit any significant Ca 2þ entry, termed the window current ( Figure 1A, B).
Some Ca V channels exhibit full voltage-dependent inactivation ( Figure 1A, B), whereas for others it is incomplete ( Figure 1C), meaning that a small proportion of the channels remain available for extended periods at depolarized potentials. This is particularly relevant to the functioning of the slowly  19 showing the relative inactivation rate of L-type and T-type Ca 2þ currents recorded from embryonic chick sensory neurons on repolarization to À80 mV in 5 mM Ca 2þ . The time constants of the tail currents, fitted by single exponentials (dotted lines) were $4 ms (T-type) and $0.6 ms (L-type).
inactivating L-type channels, Ca V 1.3 and Ca V 1.4, in specific presynaptic terminals in the inner ear and retina, respectively (see below). In addition to voltage-dependent inactivation, a second Ca 2þ -dependent inactivation process is important for some channels, and this may be triggered by global Ca 2þ levels or local Ca 2þ entry. 21 The activation and inactivation of particular channels, as well as other properties, can be influenced by differential splicing, 10,22-24 by auxiliary subunit composition, 22,25,26 and by Ca 2þ -binding protein interaction. 21,[27][28][29] Although the inactivation processes may be too slow to affect Ca 2þ entry during most presynaptic single action potentials, they can strongly influence Ca 2þ entry over the course of action potential trains or bursts, and at specialized retinal and auditory synapses in which continuous Ca 2þ entry occurs, which is modulated in a graded manner by membrane potential (see, for example, Ohn et al. 30 ).
An important point that is infrequently considered is the deactivation rate of channels in response to repolarization of the membrane potential, since, together with activation rate, this can dictate the amount of Ca 2þ entering a presynaptic terminal, as the extent of Ca 2þ entry, particularly during a brief action potential, will be strongly affected by the rate of Ca V channel closing. T-type channels have a slower deactivation rate, which is also voltage-dependent, being longer at more depolarized potentials, 19 whereas for Ca V 1 and Ca V 2 channels, the deactivation rate is much more rapid ( Figure 1D). Another key feature is the driving force for Ca 2þ entry, dictated both by the Ca 2þ concentration gradient and the membrane potential of the terminal.
Skeletal muscle calcium channels (Ca V 1.1 or a 1 S) are unusual in that they act primarily as voltage sensors via mechanical coupling to open ryanodine receptors on the sarcoplasmic reticulum, a direct process not involving Ca 2þ entry. 31,32 Activation of the Ca V 1.1 ionic conductance is very slow, relative to movement of its voltage sensors, 32 and therefore Ca 2þ entry is negligible during a single action potential. However, there is no clear evidence for significant functional expression of Ca V 1.1 in neuronal tissue or for any presynaptic function.

Multiple Roles of Ca V Auxiliary Subunits
The ß and a 2 d auxiliary subunits of calcium channels increase the transport of Ca V channels to the plasma membrane, and this is particularly relevant to ß subunits, which prevent endoplasmic reticulum-associated proteasomal degradation of the Ca V a 1 subunits. 33,34 Subsequently, there is an additional trafficking effect of a 2 d subunits. 35 The auxiliary subunits also confer a variety of properties on Ca V 1 and Ca V 2 channels; for example, certain splice variants of ß2 (ß2a and ß2e) slow the inactivation of Ca V 1 and Ca V 2 channels and are themselves membrane-associated. [36][37][38] The a 2 d subunits generally increase Ca V channel activation and inactivation rates, 39,40 but also reduce long-closed states. 40 Our work has shown that proteolytic cleavage of the pro-form of a 2 d into mature a 2 d acts as a permissive molecular switch for the function of Ca V 1 and 2 channels. 41 It should also be noted that although a 2 d proteins increase the trafficking of Ca V channels, they may also be able to traffic to the plasma membrane and to presynaptic terminals alone 41 in the absence of calcium channels, 42 and can have additional roles on synapse morphology. [43][44][45] Some Distinct Membrane Properties of Presynaptic Terminals Presynaptic terminals generally have lower membrane excitability than axons, since voltage-gated Na þ channels are often more sparse than at nodes of Ranvier. 46 In the presynaptic calyx of Held, Na þ channels are absent from the calyx terminal region, but concentrated in the final unmyelinated segment of axon (heminode) leading up to the calyx. 47 The concentration of specific voltage-gated K þ channels, particularly inactivating K þ channels, controls presynaptic excitability, [46][47][48][49] such that presynaptic action potentials are generally either brief, 47 or attenuated. 46 Other channels that may be present presynaptically, such as hyperpolarization-activated HCN channels, also have the ability to affect resting membrane potential. 50 Although a recent study has highlighted that rapid Ca 2þ entry can occur through tetrodotoxin-sensitive Na þ channels, which are highly concentrated in the axon initial segment, 51 the sparsity of presynaptic Na þ channels means it is unlikely that this route contributes significantly to presynaptic Ca 2þ entry.
The presynaptic membrane potential has been directly measured in several types of accessible terminals. For example, in the calyx of Held excitatory terminal, it was about À80 mV, and in the same study the resting intracellular Ca 2þ was estimated to be about 50 nM. 52 In hippocampal mossy fiber boutons, the resting membrane potential was between À60 and À85 mV, 48 and in inhibitory Purkinje cell terminals in culture, the membrane potential was À69 mV. 46 At these potentials even Ca V 3 channels, if present, would show little tonic activity ( Figure 1A).

Implications of Different Presynaptic Ca V Channel Compositions for Neurotransmitter Release
From the foregoing discussion, it is clear that the membrane potential of most presynaptic terminals is sufficiently negative that the vast majority of Ca V 2 channels are closed, rather than inactivated in the absence of ongoing activity. Thus, Ca V 2 channels are available to open upon action potential arrival. Ca V 2.1 channels generally activate at similar potentials to Ca V 2.2 in cell lines (Table 1), but activate more rapidly. 13 However in calyx of Held synapses, presynaptic N-type I Ca was found to activate $ 8 mV more depolarized than P/Q type current, 53 and this was also seen in chromaffin cells. 54 The third subtype of Ca V 2 channel (Ca V 2.3) also known as R-type has a somewhat more hyperpolarized membrane potential 14 (Table 1), potentially pointing to differences in function.
For most synapses, Ca V 2.1 (P/Q)-and Ca V 2.2 (N)-type channels are involved in varying proportions in synaptic transmission, depending on the synapse in question and the developmental stage. Broadly, Ca V 2.1 channels become of increasing importance in many synapses as they develop, such that they predominate in some mature neurons, 53,55 and are also more tightly associated with the release machinery 55 (see below). At some synapses, Ca V 2.3 channels, activated by smaller depolarizations, play an important role, rarely as the main channel involved in vesicular release, although this is the case in habenula cholinergic neuron terminals in the interpeduncular nucleus. 56 More often Ca V 2.3 has been found to underlie other processes such as delayed or asynchronous release, for example from small hippocampal boutons, 57 and it also plays a role in long-term potentiation. 58 A key factor to consider is action potential duration, relative to the rate of deactivation of the calcium channels, as much of the Ca 2þ entry mediating synchronous release will occur on the repolarization phase of each brief action potential-mediated presynaptic depolarization, which has the effect of increasing the driving force for Ca 2þ . In contrast, asynchronous release is the term for release resulting from stochastic opening of individual channels near the membrane potential, often after a burst of action potentials, 57,59 resulting in long-duration presynaptic Ca 2þ transients. Although it has been suggested that spontaneous openings of Ca V 2.3 channels may be in part responsible for asynchronous release occurring after action potentials at some synapses, 57 Ca V 2.1 and Ca V 2.2 channels, particularly when associated with the ß2a subunit which reduces their inactivation, may also play a role. 59 For example, at synapses formed by different subtypes of hippocampal GABA-ergic interneuron, Ca V 2.1 is involved in the mainly synchronous release from fast-spiking parvalbumin interneurons, whereas Ca V 2.2 channels predominantly mediate GABA release from cholecystokinin-containing interneurons, of which a much greater fraction is asynchronous release. 60 At some specialized sensory synapses, L-type channels, particularly Ca V 1.3 and Ca V 1.4, are critical for function. These mainly concern the auditory inner hair cells (Ca V 1.3) 61,62 and retinal photoreceptors and bipolar neurons (Ca V 1.4), 11,63,64 in which the presynaptic responses are graded. These particular Ca V 1 channels have properties suited to this function, in that they remain available at depolarized potentials ( Figure 1C).

Concerted Calcium Channel Involvement in Release from Individual Synapses
As described above, both Ca V 2.1 and Ca V 2.2 calcium channels are involved, to varying extents, in vesicular release at most individual central nervous system terminals, as judged by x-agatoxin IVA and x-conotoxin GVIA inhibition, respectively. [65][66][67] However, the relative amount of block by each toxin cannot be used directly to determine the prevalence of these channels, because of the nonlinear, approximately fourth power, relationship between intracellular Ca 2þ levels and neurotransmitter release. [68][69][70][71][72] There are several related forms of Ca 2þ cooperativity that have been described, that between multiple Ca V channels required to release a single vesicle 73 and the number of Ca 2þ ions that must bind cooperatively to Ca 2þ sensors, and the cooperative action of those sensors, to trigger release of a vesicle. 74 Thus, there is generally found to be synergy between the opening of multiple channels to reach the mM levels of Ca 2þ at the Ca 2þ sensors whose occupancy mediates release of each vesicle in an active zone. The numbers of channels involved have been estimated to be very small in some synapses 67,[75][76][77] , to over 60 in immature calyx of Held synapses. 78 In a few cases a single channel has been found to be sufficient, 75,76 although the probability of release will be low. 77 The number of channels present in each active zone is much greater than those that open in response to each action potential, because of the low probability of opening of each channel and the stochastic nature of channel openings, meaning they occur with a variable delay following a depolarizing stimulus, which can also lead to failure of exocytosis.

Anchoring of Calcium Channels in Presynaptic Active Zones is Key to Their Differing Roles in Synaptic Transmission
The proximity of the presynaptic Ca V channels to the vesicular release site is an extremely important factor in determining the properties and speed of neurotransmitter release. In order to study this, knowledge of the relative locations of the channel subtypes, as well as modeling studies are required, in addition to an understanding of the biophysical and biochemical distinctions between Ca V 2.1 and Ca V 2.2 channels. 77,79 There are well-studied differences in the anchoring of the two main Ca V 2 channels in presynaptic active zones. Both Ca V 2.1 and Ca V 2.2 channels are tethered in active zones by the RAB3A-interacting molecule (RIM), 80 and Ca V 2.3 channels may also associate with RIM proteins. 80 Furthermore, RIM-binding protein interacts with Ca V 2.1, Ca V 2.2 and Ca V 1.2 channels, but recruits only the former two channels via interaction with RIM specifically to the active zone. 80 However, Ca V 2.1 is selectively associated with certain Munc13 isoforms potentially leading it to be localized closer to docked vesicles than Ca V 2.2 55 (Figure 2). In contrast to the obvious central phenotype of Ca V 2.1 knockout mice, 82 the lack of marked phenotype in Ca V 2.2 knockout mice suggests that their role is less crucial, and other types of Ca V channel (particularly Ca V 2.1) are able to compensate for the loss of Ca V 2.2 at most synapses. However, Ca V 2.2 channels have a predominant role at primary afferent synapses in the pain pathway, 83,84 and this pathway is indeed disrupted in Ca V 2.2 knockout mice. 85 Thus, both the properties and distribution of Ca V 2.1 channels result in greater activation and Ca 2þ entry for a brief action potential through these channels than for Ca V 2.2. 13 This has been observed, for example, in mossy fiber boutons, where a single terminal was estimated to contain about 2000 channels, and brief presynaptic action potentials activated a presynaptic Ca 2þ current that was found with pharmacological blockers to be dependent on P/Q ($66%), N ($26%), and R ($8%)-type channels. 86 Other proteins have also been found to interact with Ca V 2 channels, 87 and some of these proteins affect the properties of the channels, such as the CRMP-2 interaction with Ca V 2.2. 88 Another presynaptic protein, Syntaxin 1A has been found to interact with part of the II-III linker of Ca V 2.2 channels (synprint site), increasing both slow inactivation and steady-state inactivation, and thus reducing channel availability. 89,90 By contrast, an analogous effect on Ca V 2.1 channels may depend on channel splice variant. 91 In presynaptic terminals, this could affect the relative availability of Ca V 2.1 and Ca V 2.2 channels. However, this synprint site is not essential for presynaptic targeting 92 or neurotransmission. 93

Ca V 2 Channel Modulation Dramatically Affects Their Presynaptic Function
Since Ca V 2 channels are subject to inhibition by several second messenger pathways, this will affect their availability. Thus, the integral of Ca 2þ entry at any synapse depends on a multitude of factors that are unique to each condition and to the pattern of action potentials arriving at the terminal. In particular, G-protein-mediated inhibition is an important property of Ca V 2 channels. This can result from stimulation of many presynaptic G-protein coupled receptors linked to G i/o , such as GABA-B receptors, opioid receptors, and others whose activation leads to the release of Gßc subunits. [94][95][96][97][98][99] This inhibition, which may have a tonic component, shifts the voltage-dependence of Ca V 2 channel activation to more positive potentials, and slows activation kinetics, 100,101 which can be overcome by prior depolarization, including in some cases an action potential train. 102 This macroscopic current slowing is mediated at the singlechannel level by a prolongation of the latency to first opening both of native N-type single-channel currents 103 and of cloned Ca V 2.2 channels, 104,105 with no change in single-channel conductance.
Gßc binding mediates the inhibition, and voltage-dependent Gßc unbinding underlies the slow activation of the Ca V 2 channels, and triggers the depolarization-mediated reversal of inhibition. 96,98 Here it should be noted that Ca V 2.1 channels are less subject to G-protein modulation than Ca V 2.2, since the Gßc off-rate from these channels is more rapid. 98 Given that, as described above, only a few Ca V channels may open in response to a single action potential at individual synapses, and Gßc-mediated inhibition involves slowing of their activation, the effect on synaptic transmission has the potential to be profound, particularly where a high proportion of Ca V 2.2 channels is present, such as primary afferent terminals. 6 T-type Channels Are Partially Inactivated at Resting Membrane Potentials T-type channels are present in certain presynaptic terminals, and they may play an important role in influencing resting Ca 2þ levels, or in providing Ca 2þ for downstream events. Although Ca V 3 channels do not normally supply significant amounts of Ca 2þ for neurotransmitter release resulting from action potentials arriving at the terminal, nevertheless their availability can be affected by the interplay of other channels such as HCN channels and Ca 2þ -activated K þ channels, which affect membrane potential. 50 Functional HCN1 channels are present on particular glutamatergic synaptic terminals, for example onto entorhinal cortical layer III pyramidal neurons, where they depolarize the membrane potential and reduce neurotransmitter release. These effects at least partly result from reduced availability of Ca V 3.2 channels. 50 Furthermore, Ca V 3 channels were also found to play an important part in asynchronous dendrodendritic release of glutamate from olfactory bulb mitral cells. 107 In another study GABA release from interneurons could be promoted by activation of presynaptic nicotinic receptors and subsequent activation of presynaptic Ca V 3.1 channels, together with release of Ca 2þ from ryanodinesensitive intracellular stores. 108 Thus, there is evidence from numerous studies for a variety of presynaptic roles for T-type channels.

A Role for Ca 21 -induced Ca 21 Release in Presynaptic Terminals
Although Ca 2þ -induced Ca 2þ release (CICR) is mainly associated with Ca V 1.2 channel function, for example in cardiac muscle cells, nevertheless smooth endoplasmic reticulum is present in presynaptic terminals, 109 and there is evidence that CICR occurs from this endoplasmic reticulum which can affect neurotransmitter release. 110,111 The channels involved in presynaptic CICR are mainly ryanodine receptors, 112,113 and the initial source of Ca 2þ for CICR could be Ca V channels, particularly T-type or Rtype, which are activated by small depolarizations, 113 or other presynaptic Ca 2þ -permeable channels such as a7 nicotinic receptors. 108,114 It was further suggested that clustering of the endoplasmic reticulum sensor of Ca 2þ depletion, STIM1, may directly inhibit Ca V channels. 110 The importance of CICR in neurotransmitter release is more evident following prolonged activation rather than single action potential-induced responses, 111,113 although single action potentials can also result in CICR. 111,115 The Roles of Mitochondria in Controlling Intracellular Ca 21

in Presynaptic Terminals
Mitochondria are present in about half of all presynaptic terminals, 116 and they can sequester presynaptic Ca 2þ entry resulting from trains of action potentials. 117,118 Presynaptic mitochondria are found to have a low threshold for Ca 2þ uptake, relative to those in other tissues, which is conferred by a brain-specific protein MICU3, allowing mitochondria to take up Ca 2þ directly from the cytoplasm near to sites of Ca 2þ entry through the plasma membrane. 119 Indeed, mitochondria have been visualized to be tethered to presynaptic terminal membranes in the calyx of Held. 120 Furthermore, Ca 2þ is required for optimal ATP levels, and presynaptic mitochondria promote synaptic transmission in active synapses by supplying the essential ATP. Maintenance of the voltage and ionic gradients related to presynaptic function is also a major consumer of ATP, 119 and thus mitochondria fulfill multiple presynaptic roles.

Conclusions
The molecular and biophysical properties of Ca V channels are finely tuned to their roles in presynaptic terminals to mediate neurotransmitter release. Although there are many types and geometries of synapse, the channels in these terminals function in broadly similar ways to mediate Ca 2þ entry that triggers vesicular release. Since the opening of a few channels, or even a single channel, is able to mediate release at discrete small excitatory and inhibitory synapses, it is extremely important to understand the individual and distinct properties of these channels, in order to appreciate how this process of release is constrained by the localization, tethering, properties, and modulation of the channels. Similarly, the different mix of types of channels present, and their relative active zone distribution, is tuned to the functions of individual synapses and to changes during development and synaptic activity.