The N-type calcium channel is one of the isoforms of high voltage-activated (HVA) calcium channels; it is ubiquitously expressed in various neurons and control neurotransmitter release at presynaptic nerve terminals. From a therapeutic view point, N-type calcium channels play a critical pathophysiological role in nociceptive responses, especially chronic inflammatory and neuropathic pain. Therefore, the N-type calcium channel is an interesting target to investigate regulatory mechanisms of neurotransmission, and also to develop analgesic drugs for neuropathic pain, which represents a significant unmet medical need.
Analogous to other HVA calcium channels, N-type calcium channels appear to be comprised of a pore-forming α1 subunit and auxiliary β and α2δ subunits. Using recombinant channel expression systems with Xenopus oocytes and mammalian cell lines, it has been shown that auxiliary subunits β and α2δ typically increase N-type calcium channel currents through a direct interaction with the α1 subunit. However, unphysiological conditions, such as high concentrations of divalent cations as a charge carrier and hyperpolarized holding potentials (HPs), have often been used to investigate biophysical properties of calcium channels.
Therefore, this study, first re-evaluated the effect of auxiliary subunits on N-type calcium channel function under conditions approaching physiological states using the Xenopus oocyte expression system. Under these conditions, a novel inhibitory effect of the β3 subunit (a dominant β subunit in native neurons) on N-type calcium channels was found. Overexpressed β3 significantly suppressed N- and also R-type, but not L-type, calcium channel currents at "physiological" HPs of -60 mV and -80 mV Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without β3, and that β3 caused a significant negative shift of this inactivation. In the presence or absence of β3, the channel inactivation developed extremely slowly with a minute-order time constant in the "closed-state", demonstrating that β3 inhibits the N-type calcium channel current causing a hyperpolarizing shift of the "ultra-slow" and "closed-state" inactivation.
The β3-induced current inhibition was compared between N-type calcium channel splice variants (central and peripheral nerve splice isoforms), and a significant difference in HP-dependence of the β3 effect was found. Similar distinct HPdependences were observed for P3-induced modulation of "open-state" inactivation kinetics. Presumably, these differences in the HP-dependence of the splice isoforms relate to the difference in resting membrane potential of neurons which express either splice isoform dominantly, providing optimal β3-induced channel regulation. The β3- induced modulation of N-type calcium channel function was dramatically affected by swapping N-terminus 15 amino acid residues with a corresponding part of the β2a subunit, which contains palmitoylation sites. With this chimeric approach, it was clarified that the N-terminus palmitoylation plays a key role in the modulation of the inactivation properties, including the kinetics "open-state" inactivation and the onset of "closed-state" inactivation, whereas location of β subunits in the vicinity of the plasma membrane appears to be more important for β regulation of current amplitude and the shift in "closed-state" inactivation. In addition, it was found that β3-induced current inhibition was α1:β3 ratio-dependent phenomenon. Although there is no plausible evidence, irreversible interaction between α1 and β3 is speculated.
Second this study investigated the influence of auxiliary subunits, especially the α2δ-l subunit which appears to be involved in neuropathic pain, on N-type calcium channel pharmacology using various ω-conotoxins. While the β3 subunit had little influence on the on- and off-rates of ω-conotoxins, α2δ significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. This result may have implications for the antinociceptive properties of ω-conotoxins, given that the α2δ-l subunit is upregulated in certain pain states. With regard to the mechanism of α2δ-l -induced toxin insensitivity of N-type calcium channels, the influence of α2δ-l was not affected by deglycosylation which targeted the heavily glycosylated extracellular large domain of the α2δ-l subunit.
Finally in this project, a comprehensive study of the effects of auxiliary subunits on various HVA calcium channels was performed in the mammalian, HEK cell, expression system to compare each subunit effect systematically. Overall, a previously underestimated role of the α2δ-l subunit was elucidated with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.