Manu B. Johny

Dynamic switching of calmodulin interactions underlies Ca2+ regulation of CaV1.3 channels

Calmodulin regulation of CaV channels is a prominent Ca2+ feedback mechanism orchestrating vital adjustments of Ca2+ entry. The long-held structural correlate of this regulation has been Ca2+-bound calmodulin complexed alone with an IQ domain on the channel carboxy terminus. Here, however, systematic alanine mutagenesis of the entire carboxyl tail of an L-type CaV1.3 channel casts doubt on this paradigm. To identify the actual molecular states underlying channel regulation, we develop a structure-function approach relating the strength of regulation to the affinity of underlying calmodulin/channel interactions, by a Langmuir relation (iTL analysis). Accordingly, we uncover frank exchange of Ca2+-calmodulin to interfaces beyond the IQ domain, initiating substantial rearrangements of the calmodulin/channel complex. The N-lobe of Ca2+-calmodulin binds an NSCaTE module on the channel amino terminus, while the C-lobe binds an EF-hand region upstream of the IQ domain. This system of structural plasticity furnishes a next-generation blueprint for CaV channel modulation.

Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within CaV1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in CaV1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca2+ channelopathies involving S6 mutations.

Allostery in Ca2+ channel modulation by calcium-binding proteins

Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Out of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). As CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, raising questions as to whether CaBPs can exert appreciable biological actions. Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell fluorescence resonance energy transfer assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.

Following Optogenetic Dimerizers and Quantitative Prospects

Optogenetics describes the use of genetically encoded photosensitive proteins to direct intended biological processes with light in recombinant and native systems. While most of these light-responsive proteins were originally discovered in photosynthetic organisms, the past few decades have been punctuated by experiments that not only commandeer but also engineer and enhance these natural tools to explore a wide variety of physiological questions. In addition, the ability to tune dynamic range and kinetic rates of optogenetic actuators is a challenging question that is heavily explored with computational methods devised to facilitate optimization of these systems. Here, we explain the basic mechanisms of a few popular photodimerizing optogenetic systems, discuss applications, compare optogenetic tools against more traditional chemical methods, and propose a simple quantitative understanding of how actuators exert their influence on targeted processes.

Large Ca2+-Dependent Facilitation of CaV2.1 Channels Induced by Ca2+ Photo-Uncaging

CaV2.1 voltage-gated Ca2+ channels are the predominant trigger of neurotransmitter release in the CNS, so their Ca2+/calmodulin-dependent facilitation (CDF) could impact short-term synaptic plasticity. CDF is believed to arise from a Ca2+-dependent increase in channel open probability, but the precise magnitude of this increase has been uncertain, owing to complex superposition of CDF with channel (de)activation under conventional electrophysiological protocols. Here, we utilize photo-uncaging of Ca2+ with CaV2.1 channels fluxing Li+ currents, so that any resulting CDF is driven solely by light-induced increases in Ca2+. The figures black trace shows that a 1 μM Ca2+ step rapidly triggers an increase in current. No current enhancement is seen in the absence of Ca2+ photo-uncaging (gray trace). Because channel (de)activation is steady the instant before Ca2+ uncaging, the increased current relates exclusively to CDF. Notably, the ∼two-fold boost in current suggests that CDF is far stronger than previously suspected. Moreover, half-maximal CDF was reached by Ca2+ concentrations of ∼0.5 μM, well within the physiological range. Given the fourth-power relation between Ca2+ entry and transmitter release, our results suggest that CaV2.1 channel CDF could play a dominant role in producing short-term synaptic facilitation.View Large Image | View Hi-Res Image | Download PowerPoint Slide