Differential Ca²⁺‐dependence of transmitter release mediated by P/Q‐ and N‐type calcium channels at neonatal rat neuromuscular junctions

Abstract

N- and P/Q-type voltage dependent calcium channels (VDCCs) mediate transmitter release at neonatal rat neuromuscular junction (NMJ). Thus the neonatal NMJ allows an examination of the coupling of different subtypes of VDCCs to the release process at a single synapse. We studied calcium dependence of transmitter release mediated by each channel by blocking with omega-conotoxin GVIA the N-type channel or with omega-agatoxin IVA the P/Q-type channel while changing the extracellular calcium concentration ([Ca2+]o). Transmitter release mediated by P/Q-type VDCCs showed steeper calcium dependence than N-type mediated release (average slope 3.6 +/- 0.09 vs. 2.6 +/- 0.03, respectively). Loading the nerve terminals with 10 microm BAPTA-AM in the extracellular solution reduced transmitter release and occluded the blocking effect of omega-conotoxin GVIA (blockade -2 +/- 9%) without affecting the action of omega-agatoxin IVA (blockade 85 +/- 4%). Both VDCC blockers were able to reduce the amount of facilitation produced by double-pulse stimulation. In these conditions facilitation was restored by increasing [Ca2+]o. The facilitation index (fi) was also reduced by loading nerve terminals with 10 microm BAPTA-AM (fi = 1.2 +/- 0.1). The control fi was 2.5 +/- 0.1. These results show that P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than were N-type VDCCs at the neonatal neuromuscular junction. This difference could be accounted for by a differential location of these channels at the release site. In addition, our results indicate that space-time overlapping of calcium domains was required for facilitation.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic Ca V 2.1 Ca 2+ channels

“…Neuromuscular transmission in neonatal skeletal muscle is initiated by both Ca V 2.1 and Ca V 2.2 channels, whereas Ca V 2.1 channels exclusively initiate neuromuscular transmission in adult muscle (21,22). We examined whether the IM-AA mutation upregulates Ca V 2.2 channels by recording evoked synaptic transmission from WT and IM-AA NMJs after blocking Ca V 2. , except for measurements of quantal content, which were done in the presence of 2 mM Ca 2+ .…”

The role of synaptic laminins-α4 and -β2 in maturation and maintenance of the neuromuscular synapse

“…This is in agreement with the study by Rosato-Siri et al (2002) which demonstrated ineffective binding of BAPTA, a fast calcium ion chelator to calcium ions that enter through P/Q-type VGCCs. This indicates that calcium ions which enter through this channel are only required to travel a short distance to bind to synaptotagmin at the release sites.…”

“…There are five VGCC subtypes that can be categorised into two groups based on the level of voltage activation properties, with T-type as the low-voltage activated channel and L-, N-, P/Q-, and R-type as the high-voltage activated channels (Nowycky et al, 1985;Fox et al, 1987;Tsien et al, 1988;Zhang et al, 1993). Each of these VGCCs is different in terms of function with P/Qand N-type being involved primarily in neurotransmitter release at both central and peripheral synapses (Uchitel et al, 1992;Rosato-Siri & Uchitel, 1999;Rosato-Siri et al, 2002;Zaitsev et al, 2007), L-type in excitation-contraction coupling (Zhou & January, 1998), and T-type in controlling pacemaker activity of the heart (Le Quang et al, 2013). The exact role of R-type VGCCs however remains unclear but suggested to be involved with finer control of calcium influx (Myoga & Regehr, 2011).…”

“…At the NMJ, N-and P/Q-type VGCCs play essential roles in neurotransmitter release Rosato-Siri & Uchitel, 1999;Rosato-Siri et al, 2002). During the development of the NMJ, neurotransmitter release is mediated by both N-and P/Q-type VGCCs (Katz & Shatz, 1996;Rosato-Siri & Uchitel, 1999;Rosato-Siri et al, 2002).…”

“…During the development of the NMJ, neurotransmitter release is mediated by both N-and P/Q-type VGCCs (Katz & Shatz, 1996;Rosato-Siri & Uchitel, 1999;Rosato-Siri et al, 2002). This was supported by the study which utilised ω-Agatoxin IVA (ω-Aga IVA) and ω-conotoxin GVIA (ω-CTX GVIA) to block P/Q-and N-type VGCCs respectively, in order to investigate calcium channels involved in neurotransmission (Rosato-Siri & Uchitel, 1999).…”