Philemon S. Yang

Switching of Ca2+-Dependent Inactivation of CaV1.3 Channels by Calcium Binding Proteins of Auditory Hair Cells

CaV1.3 channels comprise a vital subdivision of L-type Ca2+ channels: CaV1.3 channels mediate neurotransmitter release from auditory inner hair cells (IHCs), pancreatic insulin secretion, and cardiac pacemaking. Fitting with these diverse roles, CaV1.3 channels exhibit striking variability in their inactivation by intracellular Ca2+. IHCs show generally weak-to-absent Ca2+-dependent inactivation (CDI), potentially permitting audition of sustained sounds. In contrast, the strong CDI seen elsewhere likely provides critical negative feedback. Here, we explore this mysterious CDI malleability, particularly its comparative weakness in hair cells. At baseline, heterologously expressed CaV1.3 channels exhibit intense CDI, wherein each lobe of calmodulin (CaM) contributes a distinct inactivation component. Because CaM-like molecules (bearing four recognizable but not necessarily functional Ca2+-binding EF hands) can perturb the Ca2+ response of molecules regulated by CaM, we asked whether such CaM-like entities could influence CDI. We find that CaM-like calcium-binding protein (CaBP) molecules are clearly expressed within the organ of Corti. In particular, the rare subtype CaBP4 is specific to IHCs, and CaBP4 proves capable of eliminating even the potent baseline CDI of CaV1.3. CaBP4 thereby represents a plausible candidate for moderating CDI within IHCs.

Enzyme-inhibitor-like tuning of Ca 2+ channel connectivity with calmodulin

Ca2+ channels and calmodulin (CaM) are two prominent signalling hubs that synergistically affect functions as diverse as cardiac excitability, synaptic plasticity and gene transcription. It is therefore fitting that these hubs are in some sense coordinated, as the opening of CaV1–2 Ca2+ channels are regulated by a single CaM constitutively complexed with channels. The Ca2+-free form of CaM (apoCaM) is already pre-associated with the isoleucine–glutamine (IQ) domain on the channel carboxy terminus, and subsequent Ca2+ binding to this ‘resident’ CaM drives conformational changes that then trigger regulation of channel opening. Another potential avenue for channel–CaM coordination could arise from the absence of Ca2+ regulation in channels lacking a pre-associated CaM. Natural fluctuations in CaM concentrations might then influence the fraction of regulable channels and, thereby, the overall strength of Ca2+ feedback. However, the prevailing view has been that the ultrastrong affinity of channels for apoCaM ensures their saturation with CaM, yielding a significant form of concentration independence between Ca2+ channels and CaM. Here we show that significant exceptions to this autonomy exist, by combining electrophysiology (to characterize channel regulation) with optical fluorescence resonance energy transfer (FRET) sensor determination of free-apoCaM concentration in live cells. This approach translates quantitative CaM biochemistry from the traditional test-tube context into the realm of functioning holochannels within intact cells. From this perspective, we find that long splice forms of CaV1.3 and CaV1.4 channels include a distal carboxy tail that resembles an enzyme competitive inhibitor that retunes channel affinity for apoCaM such that natural CaM variations affect the strength of Ca2+ feedback modulation. Given the ubiquity of these channels, the connection between ambient CaM levels and Ca2+ entry through channels is broadly significant for Ca2+ homeostasis. Strategies such as ours promise key advances for the in situ analysis of signalling molecules resistant to in vitro reconstitution, such as Ca2+ channels.

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.

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.

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Quantifying macromolecular interactions in living cells using FRET two-hybrid assays

Förster resonance energy transfer (FRET) is a versatile method for analyzing protein–protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein–protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures. A step-by-step protocol is given for sample preparation, data acquisition and analysis. The method uses relatively inexpensive and widely available equipment and can be performed with minimal training. Implementation of the imaging setup requires up to 1 week, and sample preparation takes ∼1–3 d. An individual FRET experiment, including control measurements, can be completed within 4–6 h, with data analysis requiring an additional 1–3 h.

Erratum: Enzyme-inhibitor-like tuning of Ca2+ channel connectivity with calmodulin (Nature (968-972) 463 (2010))

This corrects the article DOI: 10.1038/nature08766