Human T lymphocytes express a Ca²⁺-activated K⁺ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca²⁺-binding sites; however, we mapped a Ca²⁺-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (V _m) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (V _m ∼0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological V _m values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca²⁺ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca²⁺ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca²⁺ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. Toxin block of Ca²⁺ entry via CaV2.2 abolished this stimulated release. Thus, the CRMP-2-Ca²⁺ channel interaction represents a novel mechanism for modulation of Ca²⁺ influx into nerve terminals and, hence, of synaptic strength.

Microglial activation following central nervous system damage or disease often culminates in a respiratory burst that is necessary for antimicrobial function, but, paradoxically, can damage bystander cells. We show that several K⁺ channels are expressed and play a role in the respiratory burst of cultured rat microglia. Three pharmacologically separable K⁺ currents had properties of Kv1.3 and the Ca²⁺/calmodulin-gated channels, SK2, SK3, and SK4. mRNA was detected for Kv1.3, Kv1.5, SK2, and/or SK3, and SK4. Protein was detected for Kv1.3, Kv1.5, and SK3 (selective SK2 and SK4 antibodies not available). No Kv1.5-like current was detected, and confocal immunofluorescence showed the protein to be subcellular, in contrast to the robust membrane localization of Kv1.3. To determine whether any of these channels play a role in microglial activation, a respiratory burst was stimulated with phorbol 12-myristate 13-acetate and measured using a single cell, fluorescence-based dihydrorhodamine 123 assay. The respiratory burst was markedly inhibited by blockers of SK2 (apamin) and SK4 channels (clotrimazole and charybdotoxin), and to a lesser extent, by the potent Kv1.3 blocker agitoxin-2.

Global spread of severe acute respiratory syndrome coronavirus 2 continues unabated. Binding of severe acute respiratory syndrome coronavirus 2's spike protein to host angiotensin-converting enzyme 2 triggers viral entry, but other proteins may participate, including the neuropilin-1 receptor (NRP-1). Because both spike protein and vascular endothelial growth factor-A (VEGF-A)—a pronociceptive and angiogenic factor, bind NRP-1, we tested whether spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuron firing was blocked by spike protein and NRP-1 inhibitor EG00229. Pronociceptive behaviors of VEGF-A were similarly blocked through suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A “silencing” of pain through subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.

Collapsin response mediator proteins (CRMPs) mediate signal transduction of neurite outgrowth and axonal guidance during neuronal development. Voltage-gated Ca²⁺ channels and interacting proteins are essential in neuronal signaling and synaptic transmission during this period. We recently identified the presynaptic N-type voltage-gated Ca²⁺ channel (Cav2.2) as a CRMP-2-interacting partner. Here, we investigated the effects of a functional association of CRMP-2 with Cav2.2 in sensory neurons. Cav2.2 colocalized with CRMP-2 at immature synapses and growth cones, in mature synapses and in cell bodies of dorsal root ganglion (DRG) neurons. Co-immunoprecipitation experiments showed that CRMP-2 associates with Cav2.2 from DRG lysates. Overexpression of CRMP-2 fused to enhanced green fluorescent protein (EGFP) in DRG neurons, via nucleofection, resulted in a significant increase in Cav2.2 current density compared with cells expressing EGFP. CRMP-2 manipulation changed the surface levels of Cav2.2. Because CRMP-2 is localized to synaptophysin-positive puncta in dense DRG cultures, we tested whether this CRMP-2-mediated alteration of Ca²⁺ currents culminated in changes in synaptic transmission. Following a brief high-K⁺-induced stimulation, these puncta became loaded with FM4-64 dye. In EGFP and neurons expressing CRMP-2–EGFP, similar densities of FM-loaded puncta were observed. Finally, CRMP-2 overexpression in DRG increased release of the immunoreactive neurotransmitter calcitonin gene-related peptide (iCGRP) by ∼70%, whereas siRNA targeting CRMP-2 significantly reduced release of iCGRP by ∼54% compared with control cultures. These findings support a novel role for CRMP-2 in the regulation of N-type Ca²⁺ channels and in transmitter release.