A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.

Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.

Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK.

Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain of function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, we investigated potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. We found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-alpha. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis.

FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation.

L-type Ca(2+) channels possess a Ca(2+)-dependent inactivation (CDI) mechanism, affording feedback in diverse neurobiological settings and serving as prototype for unconventional calmodulin (CaM) regulation emerging in many Ca(2+) channels. Crucial to such regulation is the preassociation of Ca(2+)-free CaM (apoCaM) to channels, facilitating rapid triggering of CDI as Ca(2+)/CaM shifts to a channel IQ site (IQ). Progress has been hindered by controversy over the preassociation site, as identified by in vitro assays. Most critical has been the failure to resolve a functional signature of preassociation. Here, we deploy novel FRET assays in live cells to identify a 73 aa channel segment, containing IQ, as the critical preassociation pocket. IQ mutations disrupting preassociation revealed accelerated voltage-dependent inactivation (VDI) as the functional hallmark of channels lacking preassociated CaM. Hence, the alpha(1C) IQ segment is multifunctional-serving as ligand for preassociation and as Ca(2+)/CaM effector site for CDI.

Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.

L-type (CaV1.2) and P/Q-type (CaV2.1) calcium channels possess lobe-specific CaM regulation, where Ca2+ binding to one or the other lobe of CaM triggers regulation, even with inverted polarity of modulation between channels. Other major members of the CaV1-2 channel family, R-type (CaV2.3) and N-type (CaV2.2), have appeared to lack such CaM regulation. We report here that R- and N-type channels undergo Ca(2+)-dependent inactivation, which is mediated by the CaM N-terminal lobe and present only with mild Ca2+ buffering (0.5 mM EGTA) characteristic of many neurons. These features, together with the CaM regulatory profiles of L- and P/Q-type channels, are consistent with a simplifying principle for CaM signal detection in CaV1-2 channels-independent of channel context, the N- and C-terminal lobes of CaM appear invariably specialized for decoding local versus global Ca2+ activity, respectively.

Rapid and reversible block of N-type calcium channels (CaV 2.2) by omega-conotoxin GVIA in the absence of divalent cations.

Omega-conotoxin GVIA (omegaCGVIA) has been reported to be an irreversible blocker of N-type calcium channels (Ca(V) 2.2). However, recent studies have demonstrated that the omegaCGVIA off-rate is correlated with divalent cation concentration, because increasing [Ba2+]o accelerated the recovery from omegaCGVIA block. This predicts that the dissociation of omegaCGVIA from N-channels will be negligible in the absence of divalent cations. Surprisingly, we find that omegaCGVIA block is rapidly reversible in divalent cation-free (0 Ba2+) external solutions in which current was carried by MA+. The recovery followed a single-exponential time course with tau = 31 sec. Isochronic measurements showed that, at 2 min after the removal of toxin, current returned to 86% of control in 0 Ba2+ compared with 19% in 3 mm Ba2+. The off-rate of omegaCGVIA from N-channels was dependent on [Ba2+]o, because, at an intermediate concentration (3 microm Ba2+), N-current recovered with tau = 64 sec, significantly slower than that in 0 Ba2+ but faster than in 3 mm Ba2+. Recovery from omegaCGVIA block was also observed when Cs+ or Na+ carried the current in divalent cation-free conditions. The off-rate was sensitive to [Ba2+]o only during washout, because current recovered slowly in the presence of 3 mm Ba2+, even after it was blocked in 0 Ba2+. Assuming that the toxin is a pore blocker, our findings are consistent with a model in which Ba2+ interacts at a site on the extracellular surface of the channel to regulate omegaCGVIA dissociation from N-channels.

Voltage control of Ca²âº permeation through N-type calcium (Ca(V)2.2) channels.

Voltage-gated calcium (Ca(V)) channels deliver Ca(2+) to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca(2+) over other cations is thought to involve multiple Ca(2+)-binding sites within the pore. Although the Ca(2+) affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca(V)2.2) channels to investigate the effect of voltage on Ca(2+) flux. We found that the EC50 for Ca(2+) permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca(2+) ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca(V)2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca(2+)-Ba(2+) anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca(2+)-Ba(2+) anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca(2+) permeation through Ca(V)2.2 channels may require at least four Ca(2+)-binding sites. Finally, our results suggest that the high affinity of Ca(2+) for the channel helps to enhance Ca(2+) influx at depolarized voltages relative to other ions (e.g., Ba(2+) or Na(+)), whereas the absence of voltage effects at negative potentials prevents Ca(2+) from becoming a channel blocker. Both effects are needed to maximize Ca(2+) influx over the voltages spanned by action potentials.