Unconjugated
High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), β (Cavβ), and α(2)δ subunits. The Cavβ subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavβ subunits in primary sensory neurons. In this study, we found that Cavβ(3) and Cavβ(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavβ(3), but not Cavβ(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavβ(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavβ(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavβ(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavβ(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.
Modulation of phosphorylation states of ion channels is a critical step in the development of hyperalgesia during inflammation. Modulatory enhancement of channel activity may increase neuronal excitability and affect downstream targets such as gene transcription. The specificity required for such regulation of ion channels quickly occurs via targeting of protein kinases and phosphatases by the scaffolding A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 has been implicated in inflammatory pain by targeting protein kinase A (PKA) and protein kinase C (PKC) to the transient receptor potential vanilloid 1 (TRPV1) channel in peripheral sensory neurons, thus lowering threshold for activation of the channel by multiple inflammatory reagents. However, the expression pattern of AKAP150 in peripheral sensory neurons is unknown. Here we identify the peripheral neuron subtypes that express AKAP150, the subcellular distribution of AKAP150, and the potential target ion channels in rat dorsal root ganglion (DRG) slices. We found that AKAP150 is expressed predominantly in a subset of small DRG sensory neurons, where it is localized at the plasma membrane of the soma, axon initial segment, and small fibers. Most of these neurons are peripherin positive and produce C fibers, although a small portion produce Aδ fibers. Furthermore, we demonstrate that AKAP79/150 colocalizes with TRPV1 and Ca(V) 1.2 in the soma and axon initial segment. Thus AKAP150 is expressed in small, nociceptive DRG neurons, where it is targeted to membrane regions and where it may play a role in the modulation of ion channel phosphorylation states required for hyperalgesia.
