CaV2.1 channels conduct P/Q-type Ca2+currents that are modulated by calmodulin (CaM) and the structurally related Ca2+-binding protein 1 (CaBP1). Visinin-like protein-2 (VILIP-2) is a CaM-related Ca2+-binding protein expressed in the neocortex and hippocampus. Coexpression of CaV2.1 and VILIP-2 in tsA-201 cells resulted in Ca2+channel modulation distinct from CaM and CaBP1. CaV2.1 channels with β2asubunits undergo Ca2+-dependent facilitation and inactivation attributable to association of endogenous Ca2+/CaM. VILIP-2 coexpression does not alter facilitation measured in paired-pulse experiments but slows the rate of inactivation to that seen without Ca2+/CaM binding and reduces inactivation of Ca2+currents during trains of repetitive depolarizations. CaV2.1 channels with β1bsubunits have rapid voltage-dependent inactivation, and VILIP-2 has no effect on the rate of inactivation or facilitation of the Ca2+current. In contrast, when Ba2+replaces Ca2+as the charge carrier, VILIP-2 slows inactivation. The effects of VILIP-2 are prevented by deletion of the CaM-binding domain (CBD) in the C terminus of CaV2.1 channels. However, both the CBD and an upstream IQ-like domain must be deleted to prevent VILIP-2 binding. Our results indicate that VILIP-2 binds to the CBD and IQ-like domains of CaV2.1 channels like CaM but slows inactivation, which enhances facilitation of CaV2.1 channels during extended trains of stimuli. Comparison of VILIP-2 effects with those of CaBP1 indicates striking differences in modulation of both facilitation and inactivation. Differential regulation of CaV2.1 channels by CaM, VILIP-2, CaBP1, and other neurospecific Ca2+-binding proteins is a potentially important determinant of Ca2+entry in neurotransmission.

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a key regulator of synaptic responses in the postsynaptic density, but understanding of its mechanisms of action in the presynaptic neuron is incomplete. Here we show that CaMKII constitutively associates with and modulates voltage-gated calcium (Ca V )2.1 channels that conduct P/Q type Ca 2+ currents and initiate transmitter release. Both exogenous and brain-specific inhibitors of CaMKII accelerate voltage-dependent inactivation, cause a negative shift in the voltage dependence of inactivation, and reduce Ca 2+ -dependent facilitation of Ca V 2.1 channels. The modulatory effects of CaMKII are reduced by a peptide that prevents binding to Ca V 2.1 channels but not by a peptide that blocks catalytic activity, suggesting that binding rather than phosphorylation is responsible for modulation. Our results reveal a signaling complex formed by Ca V 2.1 channels and CaMKII that regulates P/Q-type Ca 2+ current in neurons. We propose an “effector checkpoint” model for the control of Ca 2+ channel fitness for function that depends on association with CaMKII, SNARE proteins, and other effectors of Ca 2+ signals. This regulatory mechanism would be important in presynaptic nerve terminals, where Ca V 2.1 channels initiate synaptic transmission and CaMKII has noncatalytic effects on presynaptic plasticity.

Growth cones generate spontaneous transient elevations of intracellular Ca(2+) that regulate the rate of neurite outgrowth. Here we report that these Ca(2+) waves inhibit neurite extension via the Ca(2+)-dependent phosphatase calcineurin (CN) in Xenopus spinal neurons. Pharmacological blockers of CN (cyclosporin A and deltamethrin) and peptide inhibitors of CN [the Xenopus CN (xCN) autoinhibitory domain and African swine fever virus protein A238L] block the Ca(2+)-dependent reduction of neurite outgrowth in cultured neurons. Time-lapse microscopy of growing neurites demonstrates directly that the reduction in the rate of outgrowth by Ca(2+) transients is blocked by cyclosporin A. In contrast, expression of a constitutively active form of xCN in the absence of waves results in shorter neurite lengths similar to those seen in the presence of waves. The developmental expression pattern of xCN transcripts in vivo coincides temporally with axonal pathfinding by spinal neurons, supporting a role of CN in regulating Ca(2+)-dependent neurite extension in the spinal cord. Ca(2+) wave frequency and Ca(2+)-dependent expression of GABA are not affected by inhibition or activation of CN. However, phosphorylation of the cytoskeletal element GAP-43, which promotes actin polymerization, is reduced by Ca(2+) waves and enhanced by suppression of CN activity. CN ultimately acts on the growth cone actin cytoskeleton, because disrupting actin microfilaments with cytochalasin D or stabilizing them with jasplakinolide negates the effects of suppressing or activating CN. Destabilization or stabilization of microtubules with colcemide or taxol results in Ca(2+)-independent inhibition of neurite outgrowth. The results identify components of the cascade by which Ca(2+) waves act to regulate neurite extension.

Excitability has long been recognized as the basis for rapid signaling in the mature nervous system, but roles of channels and receptors in controlling slower processes of differentiation have been identified only more recently. Voltage-dependent and transmitter-activated channels are often expressed at early stages of development prior to synaptogenesis, and allow influx of Ca(2+). Here we examine the functions of spontaneous transient elevations of intracellular Ca(2+) in embryonic neurons. These Ca(2+) transients abruptly raise levels of Ca(2+) as much as tenfold, for brief periods, repeatedly, and can be highly localized. Like cloudbursts on the developing landscape, Ca(2+) transients modulate growth and stimulate differentiation, in a frequency-dependent manner, probably by changes in phosphorylation or proteolysis of regulatory and structural proteins in local regions. We review the mechanisms by which Ca(2+) transients are generated and their effects in regulating motility via the cytoskeleton and differentiation via transcription.

2ϩ -independent manner. Block of myristoylation abolished these effects, leaving regulation that is similar to endogenous CaM. CaBP1/G2A binds to Ca V 2.1 with reduced stability, but in situ protein cross-linking and immunocytochemical studies revealed that it binds Ca V 2.1 in situ and is localized to the plasma membrane by coexpression with Ca V 2.1, indicating that it binds effectively in intact cells. In contrast to CaBP1, coexpression of VILIP-2 slows inactivation in a Ca 2ϩ -independent manner, but this effect also requires myristoylation. These results suggest a model in which nonmyristoylated CaBP1 and VILIP-2 bind to Ca V 2.1 channels and regulate them like CaM, whereas myristoylation allows differential, Ca 2ϩ -independent regulation by the inactive EF-hands of CaBP1 and VILIP-2, which differ in their positions in the protein structure. Differential, myristoylation-dependent regulation of presynaptic Ca 2ϩ channels by nCaBPs may provide a flexible mechanism for diverse forms of short-term synaptic plasticity.