A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory

Abstract

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca²⁺ signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.

Dream + Wonder

It is 2:00 AM on a typical Tuesday night in Baltimore, Maryland. While many are enjoying a peaceful night of sleep, the lights are still on in Room 713 of the Richard Starr Ross Research Building at the Johns Hopkins University School of Medicine. One can distinctly hear the drumming of the hard drive on an antiquated computer recording digitized electric currents as it has faithfully done so since 1988. The nearby oscilloscope vividly displays a neon green trace – the picoampere flickers of a single Ca²⁺ ion channel gating open and shut. What ensues is a moment of silence, then anticipation, and suddenly a distinct joyous laughter erupts. It is as though the clouds that had shrouded a mystery of nature had parted and the blue sky beyond became apparent – Dr. David T. Yue had a deep insight on the mechanism by which Ca²⁺ channels functioned. He would soon walk over to the blackboard with you and derive a mathematically precise explanation of the observed phenomenon. To his students, trainees, and colleagues at the Calcium Signals Laboratory, such moments of unbridled joy and wonder were a constant inspiration to march forward in the “pursuit of truth, ” and a glimmer of hope that they may yet be able to complete their training.

David T. Yue graduated magna cum laude from Harvard University in 1979 with a Bachelor of Arts in Biochemistry. At Harvard, he was influenced by many prominent scientists including Edward Purcell, who taught him the beauty and elegance of Maxwell's equations. He became captivated by physics and the power of quantitative sciences to explain nature and its many mysteries. For his senior thesis, he worked with J.W. Hastings, a pioneer in bioluminescence. Perhaps as a result of these early influences, he found himself in doubt as he transitioned to become an M.D.-Ph.D student at Johns Hopkins University School of Medicine. “I'm not quite sure I'll ever find the right field. I'll always want to be a bit mathematical, a bit physical – and have the same relevancy of a biologist, ” he wrote to his best friend from high school at the end of his first year of medical school. He continues, “What I hope to do after acquainting myself with the field of battle is to develop a biophysics angle on the situation – a tall order, a definite maybe, and probably a naïve dream. But I'm banking on it.” Herein began his ultimate quest to find “a molecular problem with physics + math ∼ biophysics.”

The first leg of this journey was in the laboratory of Kiichi Sagawa, an eminent cardiovascular physiologist and a Professor of Biomedical Engineering at Johns Hopkins University. The Sagawa laboratory was a scientific environment that encouraged independent thinking. David with his close friends Dan Burkhoff and Joe Alexander soon conjured up their own research projects. They began characterizing force-interval relationships in the heart in order to understand alterations in the strength of cardiac contraction as a result of brief changes in stimulation patterns.^1-3 Through elegant mathematical formulation of these initial macroscopic studies it became apparent that there may be a common underlying feedback loop that related to fundamental properties of excitation-contraction coupling and the dynamics of Ca²⁺ ions in a single myocyte. This negative feedback loop seemed to suggest that the Ca²⁺ influx through the Ca²⁺ channel in the cell depended on the cytosolic Ca²⁺ in the cell.³ The biophysicist in him became ecstatic and translating the tissue-level phenomena and mathematical concepts into the ground molecular reality became his next mission. The vertically integrated approach to interpret systems level physiology through a molecular lens became a recurring theme throughout his career.

With encouragement from his mentor Kiichi Sagawa to pursue his molecular dream, David moonlighted in Gil Wier's Laboratory a few blocks across town at the University of Maryland, Baltimore to measure steady-state [Ca²⁺]-tension relations in intact cardiac muscle – a fundamental parameter in cardiac excitation-contraction coupling.^4-6 This was the early days of intracellular Ca²⁺ measurements before the popular Ca²⁺-sensing dyes from the Roger Tsien laboratory⁷ became widely available and the possibility of genetically engineered Ca²⁺-sensing proteins was more like science fiction. Instead, Yue with Eduardo Marban and Gil Wier utilized pressure injection of aequorin, a Ca²⁺-sensitive bioluminescent protein derived from jelly fish, into a superficial layer of papillary muscle, and measuring changes in luminescence using a photomultiplier tube (Fig. 1).⁵ Aequorin possessing a relatively low affinity for Ca²⁺ and fast on-kinetics was particularly apt for such state-of-the-art measurements.⁸ By integrating mathematical modeling and meticulous luminescence measurements, Yue and colleagues demonstrated an unusually steep relationship between tension and intracellular [Ca²⁺] concentrations (Fig. 1C)⁵ – a result fundamental to our current understanding of cardiac excitation-contraction coupling.⁹

Now fully orbed into molecular level physiology, David Yue faced yet another predicament – a challenge that nearly all of us encounter at some juncture in our careers. He felt conflicted about what direction to steer his life toward even though he had sent in his residency applications. He loved research and teaching, but knew it was hard to make it in science. The Director of Biomedical Engineering, Dr. Richard Johns, had smiled when David asked for a faculty position while still a student, telling him to try again after residency. Nonetheless, after much introspection, David decided that he needed to trust in his growing faith and move in the direction of his dreams, against the advice of his parents and his advisors. To our great benefit, he withdrew his residency applications and instead dedicated himself to quantitative biophysical research. When Dr. Johns saw that David had dedicated himself solely to research, he offered him the faculty position in Biomedical Engineering that David had requested only a few months earlier. David accepted, deferring for a year so that he could join the Marban lab as a postdoc in order to bootstrap his research without faculty obligations.

In the late 1980's, single channel recording of ion channels had become an increasingly popular methodology to obtain high-resolution biophysical understanding of these molecules-nature's version of a transistor.¹⁰ Richard Aldrich, David Corey, and Charles Stevens had utilized such single-channel recordings from Na channels in neuroblastoma cell lines to revise the canonical Hodgkin-Huxley model of channel gating (ACS model)¹¹ – a study that deeply influenced David's thinking. David thus began his rendezvous with ion channels as a post-doctoral fellow in the Marban laboratory, identifying a novel cardiac potassium channel¹² as well as studying the permeation^{13, 14} and gating of voltage-gated sodium and calcium channels.^{15, 16} These studies invariably incorporated mathematical models in the form of discreet and continuous-time Markov processes to explain the biophysics of these channels. Over the next quarter century, David would devote significant time and effort toward incorporating this mode of thinking into a popular graduate-level course entitled “Ion Channels of Excitable Membranes.”

We reminisce in this review an unapologetically biased account of nearly 3 decades of research by David T. Yue, and his Calcium Signals Laboratory that blends atomic structure, single-channel biophysics, cellular physiology, and systems neuroscience to understand the innumerable consequences that a single ion, Ca²⁺, has on human physiology, pathophysiology, and life as we know it.

The Importance of Ca²⁺

Calcium constitutes a vital signaling system critical for normal physiological function. Beyond its importance in making stronger bones, the biological roles of Ca²⁺ remained largely unknown until late 19th century when British physiologist Sidney Ringer characterized Ca²⁺ as an essential component required for the contractility of frog ventricles.¹⁷ More than a century of research since has confirmed the role of Ca²⁺ as ubiquitous second messenger critical for a vast number of signal transduction pathways. For David Yue, the precise regulation of these Ca²⁺ signals in a cell was akin to the feedback control of electrical circuits (Fig. 2). Thus as cardiac, immune, motor and neuronal systems each rely on Ca²⁺ signaling; understanding the detailed components of this biological circuit would have enormous physiological relevance. Here we mention just a few key roles for Ca²⁺, illustrative of its importance across multiple systems.

Figure 1.

David Yue and his early work in the role of Ca²⁺ in excitation-contraction coupling. (A, B) Stress and Ca²⁺ transient measurements in cardiac muscle. (C) The relationship between intracellular Ca²⁺ and relative stress in cardiac muscle. Reproduced from Yue et al., 1986. © Rockefeller University Press. Reproduced by permission of Rockefeller University Press. Permission to reuse must be obtained from the rightsholder.

Figure 2.

Ca²⁺ signals as an electrical circuit. View of cellular Ca²⁺ signaling as a feedback control pathway in an electrical circuit (modified from a slide created by David T. Yue, unpublished).

The importance of Ca²⁺ in the heart is clear and thus constitutes the first system upon which David Yue focused his attention.^{12, 15, 16, 18, 19} Upon depolarization, Ca²⁺ enters the cardiac myocyte through voltage-gated L-type Ca²⁺ channels in the cardiac dyad. This Ca²⁺ influx triggers Ca²⁺ induced Ca²⁺ release from the sarcoplasmic reticulum (SR) via Ca²⁺ binding to the ryanodine receptor.²⁰ This release of Ca²⁺ from intracellular stores is in turn responsible for cardiac contraction via Ca²⁺ binding to troponin C.²¹ Such dependence of cardiac excitation-contraction coupling on Ca²⁺ illustrates the critical nature of Ca²⁺ signaling, but it does not represent the full set of pathways dependent on Ca²⁺ within the heart. Ca²⁺ entry through L-type channels is also a major factor in setting the action potential duration of the heart, ²² making physiological regulation of these channels a necessity¹⁶ as well as an important substrate for pathology.^{22, 23} Additionally, Ca²⁺ activates a number of kinases and phosphatases including CaMKII and calcineurin, both of which may play an important role in cardiac hypertrophy and heart failure.^{24, 25}

Excitation-contraction coupling is not only a vital component of cardiac function, but is a fundamental mechanism of muscle contraction. In skeletal muscle, the role of the voltage gated calcium channel in contraction begins at the neuromuscular junction, where Ca²⁺ entry at the presynaptic terminal is required for neurotransmitter release and subsequent depolarization of the muscle fiber.²⁶ The subsequent opening of L-type Ca²⁺ channels directly couples to ryanodine receptors, inducing a release of Ca²⁺ from the SR leading to muscle contraction.²⁷

Lastly, Ca²⁺ signaling is a major player in neuronal function. Synaptic transmission is dependent on Ca²⁺ entry through voltage gated Ca²⁺ channels in the presynaptiC-terminal. Ca²⁺ entry through P/Q, N, and R-type channels mediate synaptic vesicle fusion, placing neurotransmitter release under the control of Ca²⁺ signaling.²⁸ Furthermore, Ca²⁺ oscillations underlie the rhythmic activity of pacemaker neurons found in various neuronal networks, driving diverse processes from circadian rhythms to precise control of motor movement.^{29, 30} Finally, Ca²⁺ is a critical requirement of both short and long term forms of synaptic plasticity, ^{31, 32} making Ca²⁺ signaling an important mediator for learning and memory.

With such diverse and widespread roles, Ca²⁺ signaling is clearly a critical component of a wide variety of biological functions and a requirement of life itself. It is no wonder that David Yue, a scientist filled with passion and wonder, devoted his career to studying the precise regulation of the Ca²⁺ signals within cells. A deeper biophysical understanding of voltage gated Ca²⁺ channels and their regulatory mechanisms informs both normal and pathological states of the immune, cardiac, neuronal, and motor systems. The generation of cloned Ca_V channels in the 1980's³³ fostered the growth of the calcium channel field. Thus when David Yue began his lab in 1987, a myriad of rich biophysical questions about Ca²⁺ and its entry through voltage gated calcium channels lay before him. The wonder of these biological transistors¹⁰ captured his attention and motivated the next decades of his research.

Ca²⁺ Regulatory Mechanisms

Among all the mysteries of voltage-gated Ca²⁺ channels (Ca_V), David Yue was most fascinated by one peculiar phenomenon – Ca²⁺-dependent inactivation (CDI). First described by Brehm and Eckert in 1978 in Paramecium, ³⁴ Ca_V channels were found to inactivate in response to elevated intracellular Ca²⁺ concentration. The discovery of this feedback inhibition led to the hypothesis that CDI played a prominent role in the homeostatic control of Ca²⁺ levels within the cell. Further investigations revealed that CDI was not restricted to Paramecium, but extended to a myriad of systems including mammalian cardiac and neuronal cell types.³⁵ While this phenomenon was replicated by several groups using whole cell patch clamp recordings, ^{35, 36, 37} the existence of CDI at the single channel level remained elusive.^{38, 39} Driven by the desire to reconcile whole cell and single channel results, David Yue and John Imredy, set out to record single Ca_V channel currents in 1990 in search of CDI on the molecular level.⁴⁰

Before the Ca²⁺ dependence of CDI could be proven, single channel records needed to be obtained in the absence of Ca²⁺. Ba²⁺ was therefore used as a charge carrier since it fluxes well through Ca_V channels without eliciting Ca²⁺ dependent processes. Single channel records of Ba²⁺ currents through the cardiac L-type calcium channel demonstrated robust openings in response to a voltage step (Fig. 3A, left). Ensemble average currents displayed only a modest decline in current during the step, indicative of a voltage dependent inactivation (VDI) process (Fig. 3A, bottom left). However, when Ca²⁺ was used as the charge carrier in these same L-type channels, a significant decrease in channel gating was evident (Fig. 3A, right). Channel openings were not only reduced, but the ensemble average current displayed a marked decay in Ca²⁺ current during the voltage step indicating robust CDI at the single channel level (Fig. 3A, bottom right). This change in channel gating was later shown to be the result of a true shift in channel gating modes, whereby Ca²⁺ entry through the channel promotes a transition from a high open probability mode of gating (mode 1) to a low probability mode (mode Ca).⁴¹

Figure 3.

Ca_V regulatory mechanisms. (A) Single channel recordings of LTCCs unequivocally demonstrate CDI, a reduction in open probability as reflected in the ensemble average in the bottom row of the right column (Ca²⁺ as a charge carrier) as opposed to the left column (Ba²⁺ as a charge carrier). Reproduced from Yue, Backx and Imredy, 1990.⁴⁰ © AAAS. Reproduced by permission of AAAS. Permission to reuse must be obtained from the rightsholder. (B, C) Two forms of calmodulation in Ca_V2.1. The N-lobe of CaM orchestrates CDI (B) while the C-lobe coordinates CDF (C). Reproduced from DeMaria et al., 2001.

At the whole cell level, this CDI manifests simply as the faster decay of the Ca²⁺ current when recorded with Ca²⁺ as the charge carrier, as compared to Ba²⁺, as demonstrated in recombinant Ca_V 2.1 channels⁴² (Fig. 3B). The extent of CDI can be quantified by plotting the fraction of peak current remaining at the end of the test pulse as a function of voltage (Fig. 3B, right). The U-shape of this curve provides a hallmark of Ca²⁺ dependent regulation.^{35, 43} Remarkably, these same channels also exhibit a second type of Ca²⁺ dependent regulation. When shorter voltage pulses, which initiate a negligible amount of CDI, are used to probe the channel, a facilitation of the Ca²⁺ current can be seen (Fig. 3C). This can be quantified by applying a pre-pulse which allows the channel to prefacilitate prior to the test pulse. This duel regulation of the Ca_V2.1 channel reveals a growing theme of Ca²⁺ dependent regulation of Ca_V channels whereby Ca²⁺ entry into a cell can induce disparate modes of channel regulation.

Identifying the sensor for CDI became a subject of intense study within the field. Multiple theories were proposed including Ca²⁺ induced channel (de)phosphoylation, ⁴⁴ direct binding of Ca²⁺ to the channel α subunit, ^{43, 45} or calmodulin⁴⁶ induced channel regulation.⁴⁷ Chimeric channel analyses and site-directed mutagenesis identified critical components of CDI within the carboxyl-tail of Ca_V1.2, ^{43, 48} but identifying the key component of CDI would require a new tool. In 1998, the Adelman lab determined that CaM was the Ca²⁺ sensor for small conductance potassium channels⁴⁹ by utilizing a form of CaM in which all 4 EF-hand domains were mutated (CaM₁₂₃₄). This novel tool helped the Yue lab to definitively identify CaM as the Ca²⁺ sensor for CDI in the L-type Ca²⁺ channel, ⁵⁰ a result which extends to Ca_V2.1 channels as well.^{51, 52} In Ca_V2.1channels, both CDI and CDF were completely eliminated by the overexpression of CaM₁₂₃₄ in HEK cells (Fig. 4A), validating CaM as the Ca²⁺ sensor and indicating a pre-association of the channel with apoCaM.⁴² Further, expression of mutant CaMs in which only the C lobe is able to bind Ca²⁺ (CaM₁₂) specifically abolishes CDI in Ca_V2.1, while a mutant CaM with N lobe Ca²⁺ binding eliminates Ca²⁺-dependent facilitation (CDF) (Fig. 4A). Thus the dual regulation of CaM arises as distinct regulatory mechanisms triggered independently by the 2 lobes of CaM.^{42, 52-56} This functional bipartition of Ca²⁺ regulation by CaM (calmodulation) appears to be a general rule across channel subtypes (Fig. 4C).^{42, 50, 53, 54, 57-61}

Figure 4.

CaM is the Ca²⁺ sensor for Ca_V channels. (A) Simple yet elegant tools, mutations of Ca²⁺-binding residues in one or both lobes of CaM, used to dissect Ca²⁺ regulation by each lobe of CaM. Reproduced from DeMaria etal., 2001.⁴² (B) Usage of various Ca²⁺ buffers differentiates global and local Ca²⁺ signals. Modified from Dick etal, 2008.⁶⁴ (C) Summary of calmodulation in various ion channels.

In addition to bifurcating the Ca²⁺ response, CaM has a remarkable capability to discriminate the spatial origin of a Ca²⁺ signal. Such spatial selectivity is revealed by varying the intracellular Ca²⁺ buffering conditions. Strong buffering restricts Ca²⁺ fluctuations to the nanodomain at the mouth of the Ca_V channel^{62, 63} (Fig. 4B). This local Ca²⁺ signal is sufficient to allow C-lobe calmodulation, while the N-lobe often requires a global elevation in Ca²⁺, observed only in the presence of low intracellular Ca²⁺ buffering.^{42, 50, 53, 59, 64, 65} Thus C-lobe calmodulation permits signaling within a single CaM/channel complex without interference from neighboring Ca²⁺ sources, while N-lobe calmodulation allows coordination of Ca²⁺ regulation across distances within a cell. Such spatial selectivity rationalizes the early challenges in studying CDI in single channels where only a local Ca²⁺ signal exists. Indeed, Ca_V2.1 channels, described as having no CDI at the single channel level (an N-lobe mediated effect), have been shown to exhibit robust C-lobe triggered CDF at the single channel level.⁶⁶

The general principle of the C-lobe as a local Ca²⁺ sensor and the N-lobe as a global Ca²⁺ sensor remained unquestioned until the Yue lab focused more closely on the CDI of Ca_V1.2/1.3. Remarkably, both the N and C-lobe of CaM functioned as local Ca²⁺ sensors within each of these channels.^{64, 65} This switch in the spatial selectivity of the N-lobe of CaM was shown to be the result of an additional CaM binding site within the amino-terminus of Ca_V1.2/1.3 channels called NSCaTE (N-terminal spatial Ca²⁺-transforming element). The presence of NSCaTE tuned the spatial selectivity of CaM, augmenting the repertoire of Ca²⁺ decoding available to channels. Further, the modularity of NSCaTE allowed for careful deduction of the mechanisms underlying the spatial selectivity of CaM lobes leading to a unified mechanism for Ca²⁺/CaM decoding across the Ca_V channel family (Fig. 4C).

Calmodulation of Ca_V channels has emerged as prominent regulatory scheme with extraordinary modes of behavior. But such CaM mediated regulation is not limited to Ca_V channels. Both the SK and KCNQ potassium channels associate with apoCaM and are Ca²⁺ regulated^{49, 67-69} Cyclic nucleotide-gated channels, ^{70, 71} NMDA receptors^{72, 73} and transient receptor potential (TRP) channels add to this growing list of channels proposed to undergo CaM regulation.^74-78 Finally, the Ca²⁺ dependence of voltage gated sodium channels has long been proposed, but the mechanism and extent of such modulation has been controversial.^79-85 Nonetheless, robust CDI was confirmed in Na_V1.4 channels by the rapid delivery of Ca²⁺ to the channels either via photo-uncaging or influx though nearby engineered Ca²⁺ channels.⁸⁶ Furthermore, CaM was shown to mediate this Na_V CDI, primarily though Ca²⁺ binding to its N-lobe. Importantly, the structural similarities between the carboxyl tail of Ca_V and Na_V channels proved to provide functional overlap, unifying the mechanism of calmodulation across the 2 channel families. Thus the principles deduced for CaM regulation of Ca_V channels is paralleled in the Na_V channel family, and may well generalize to a multitude of channels, many yet to be discovered.

Structural Perception Changes the Meaning of Calcium Channel Regulation

David loved to describe voltage-gated Ca²⁺ channels as “biological transistors.” However, these “biological transistors” differ from silicon transistors in one key manner: while the components and details of silicon transistors are known to their human designers, the inner-workings of voltage-gated Ca²⁺ channels are shrouded in mystery. As such, unveiling the molecular machineries which govern Ca²⁺ regulation of voltage-gated Ca²⁺ channels was of intense interest to David, and a large body of his work was dedicated to deducing the mechanism by which parts of the channel interact to induce Ca²⁺ regulation. Utilizing biochemical, optical, and crystallographic means, David contributed significantly to the structural understanding of the voltage-gated Ca²⁺ channels.

Calcium channels are multi-subunit complexes with 3 main components: a pore-forming α₁ subunit, a cytoplasmic β subunit, and a membrane anchored α_2δ subunit. These subunits assemble to form the molecular machinery for Ca²⁺ entry into the cell while CaM serves as the actual Ca²⁺ sensor for feedback regulation. The pore forming α₁ subunit is central for understanding the gating mechanisms of the channel. In particular, the carboxyl terminus of the α₁ subunit was found to harbor a number of critical elements required for Ca²⁺ regulation of Ca_V channels. Two vestigial EF hand motifs were shown to be important components of CDI and CDF via analyses of mutant, chimeric, and alternatively-spliced channels.^{43, 48, 87} Further, the carboxyl-terminus also contains an IQ domain, which has long been a recognized apoCaM binding motif, ⁸⁸ a result confirmed for the IQ domain of Ca_V channels.^89-92 Additionally, the amino-terminus of a subset of Ca_V channels contains the Ca²⁺/CaM binding site, NSCaTE, which is capable of modulating the inactivation profile of Ca_V channels.^{64, 65}

How do these key channel segments interact with CaM so as to produce Ca²⁺ regulation of the channel? To answer this, we begin by considering the typical resting state of the channels. In 2001, the Yue lab developed a novel quantitative FRET 2-hybrid method known as 3³ FRET and used it to demonstrate that in the Ca²⁺ free state, apoCaM pre-associates with Ca_V channels.^{89, 90} The C-lobe of apoCaM has been shown to bind to the channel IQ domain, while the N-lobe is now known to bind to the channel vestigial EF-hand segments (Fig. 5A).^{90, 91, 93, 94} Although definitive crystal structures of apoCaM/channel interactions remain elusive, ab-initio modeling based on functional data provides a structural basis for a mechanistic model (Fig. 5). Traditionally, the structural basis for Ca²⁺ regulation of Ca²⁺ channels was explained by the IQ-centric model. In this model, pre-association of apoCaM on the channel carboxyl-terminus positions CaM as a resident Ca²⁺ sensor and primes the channel for Ca²⁺ regulation. As cytosolic Ca²⁺ rises, the pre-associated CaM binds Ca²⁺ and repositions on the channel inducing CDI or CDF. Although the channels featured a different gating mode corresponding to CDI or CDF, CaM was thought to remain in contact with the IQ region. This IQ centric model was supported by the high affinity of Ca²⁺/CaM with the IQ domain^{42, 50, 54, 61, 91-96} as well as atomic structures of Ca²⁺/CaM in complex with the IQ domain.^97-101 However when the Yue lab presented their crystal structures of Ca²⁺/CaM in complex with the IQ domain of Ca_V2.1 and Ca_V2.3, they noted that the structure was not fully reconciled with the functional data, suggesting that Ca²⁺ /CaM may in fact depart from the IQ region during Ca²⁺ regulation.⁹⁹ This contrary hypothesis was eventually confirmed using exhaustive alanine scanning and a novel analysis utilizing individually transformed Langmuir (iTL) relations.^{93, 94} In this model, the Ca²⁺ bound C-lobe of CaM forms a tri-partite complex with the IQ domain and the vestigial EF-hands within the CI region of the channel (Fig. 5, state I).⁹³ While departing from a pure IQ centric model, this tri-partite arrangement is in agreement with early experiments indicating the importance of the IQ domain and vestigial EF-hand segments.^{43, 50, 102} The Ca²⁺ bound N-lobe of CaM also appears to depart from the IQ domain. For channels containing an NSCaTE motif on the N-terminus, it is likely that the N-lobe of Ca²⁺ CaM interacts with this domain, perhaps bridging the amino and carboxyl termini of the channel (Fig. 5).^{64, 65, 93}

Figure 5.

Structural elements of Ca_V channels. Top: homology model demonstrating dynamic switching of CaM interaction with Ca_V1.3. Bottom: conceptual framework representing the 3 states of CaM regulation of the channel. Modified from Ben Johny et al.⁹³ and Adams et al.¹⁰⁹

Thus a coherent story of the structural rearrangements underlying Ca²⁺ regulation has emerged, but the Yue lab continued to delve deeper into the mystery, revealing yet another unexpected result. Previously, the high affinity of apoCaM for the channel and the ability of overexpressed mutant CaM to change the Ca²⁺ regulatory profile of the channel led to the conclusion that at rest, all channels must contain a preassociated apoCaM.^{89, 91} In 2010, however, the Yue lab provided evidence that this may not, in fact, be the case. By combining patch clamp and optical recordings with quantitative biochemical analysis, Yue and colleagues determined that some variants of Ca_V1.3/1.4 possess a module (ICDI – inhibitor of CDI) within their carboxyl tail which acts as a competitive inhibitor for apoCaM, thus introducing a channel state in which apoCaM is no longer preassociated (Fig. 5, state E) and therefore cannot undergo CDI.^{57, 93}, Moreover, RNA-editing of Ca_V1.3 channels in the brain naturally modulates apoCaM binding affinity, ^{57, 58, 94, 103, 104} tuning the amount of CDI supported by the channel variant.^{94, 105-108} As increased CaM levels restore these CaM-less channels to configuration A by mass action, natural fluctuations in the levels of CaM may determine the distribution of channels among the E and A states, thus conferring physiological variations in CDI.^{94, 103, 108, 109} Finally, apoCaM itself has been found to have a profound effect on Ca_V channels. Recent studies using single channel recordings and chemical dimerization methods demonstrate that configuration E represents a gating mode with a low open probability (P_O).¹⁰⁹ Hence a single mechanistic scheme is proposed to underlie natural variations in channel P_O and CDI: 1) channels in the E state lack a CaM and have low P_O, 2) channels transition to the A state upon binding of apoCaM and exhibit a high P_O and 3) with the addition of Ca²⁺, structural rearrangement of Ca²⁺ /CaM drives the channel into a low P_O state producing CDI (Fig. 5, bottom).

There is significant rearrangement in the Ca_V/CaM interaction upon Ca²⁺ entry into the cell affecting channel gating. Unfortunately, the lack of structural data for many of the binding states presents a challenging mechanistic problem. However, additional insight into these structural aspects may be gained by looking outside the Ca_V channel family. Knowledge of Ca_V Ca²⁺ regulation has already informed on the function of Na_V channel Ca²⁺ regulation as described previously.⁸⁶ Given the homology between the Ca_V and Na_V channel carboxyl termini, it is reasonable to apply the structural insights gained in the Na_V channel field to the Ca_V channel.^110-112 Such comparisons of function and structure between the two channel families have created a synergistic relationship in which both fields have prospered.

Ca²⁺ in Systems

A fine mechanistic understanding of the role of Ca²⁺ signaling in cellular processes hints at its broader impact in biological systems and disease states. Indeed, the highly coordinated and complex nature of Ca²⁺ signaling and its involvement in a wide array of biological functions – spanning the gamut from neural excitability and synaptic transmission to excitation-contraction coupling – argues for the necessity of its study. A firmer grasp of the biological impact of Ca²⁺ signaling requires linking single-molecule mechanisms described previously in this review with their impact on higher order functions of whole systems.

In neurons, Ca²⁺ orchestrates a number of functions, such as vesicle fusion¹¹³ and synaptic plasticity.¹¹⁴ Moreover, transient increases in intracellular Ca²⁺ are directly related to neural activity.¹¹⁵ Thus the development of robust genetically encoded calcium indicators (GECIs) is enabling for the study of neural activity. While much work has focused on the study of single neuron activity, the study of cortical activity on larger scales is useful for addressing the question of cortical organization such as the tonotopic arrangement of auditory cortex. To address this question, the Yue lab has pioneered a multiscale approach that takes advantage of transgenic mice expressing GCaMP3, a GECI, in neurons.¹¹⁶ These mice allowed for large-scale transcranial mapping of auditory cortex followed by fine-scale 2-photon imaging of Ca²⁺ activity in individual neurons within single trials (Fig. 6A–C). Frequency tuning curves of individual neurons in the primary auditory cortex, as seen by 2-photon imaging, adhered tightly to tuning predicted by their location in the large-scale maps (Fig. 6D–F), confirming the presence of a tonotopic axis in mouse cortex.¹¹⁷ These experiments brought the CSL full circle from David Yue's early experiments as a graduate student injecting aequorin into cardiac cells.¹⁸

Figure 6.

Multiscale Ca²⁺ imaging of mouse auditory cortex. (A) Baseline fluorescence image obtained via widefield transcranial imaging of GCaMP3 transgenic mice. (B) Ca²⁺ transients in response to tones as obtained from widefield imaging. Responses taken from region delineated by red square in A. (C) Tonotopic map obtained from transcranial imaging. ‘L’ and ‘H’ highlight low- and high-frequency landmarks, respectively. The red square indicates region scrutinized in D-E under 2-photon microscopy. AI: primary auditory cortex; AII: secondary auditory cortex; AAF: anterior auditory field; UF: ultrasonic field. (D) Two-photon imaging field showing individual neurons. (E) Ca²⁺ transients from a single neuron (indicated by black square in D). Average response to tones (dark trace) and individual responses (gray traces) are shown, indicating low-frequency tuning in this particular neuron. (F) Population data for tonotopic axis in AI. Each square indicates the average best frequency (BF) for a field of view and each dot indicates BF for an individual neuron. The red square is the same as in (C–E). (0 = ‘L’ pole and 1 = ‘H’ pole). Reproduced from Issa et al.¹¹⁷ © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder.

Beyond an understanding of healthy cardiac and neural systems, Ca²⁺ signaling also affords a means of studying disease states, which not only helps in understanding the disease itself but can also aid in recognizing the general underlying biological principles and in deciphering which biophysical features are most relevant in the disease states. For example, owing to their diverse physiological roles, it is perhaps not surprising that disruption of Ca_V function has been implicated in numerous human pathologies. Diseases associated with Ca_Vs are referred to as Ca²⁺ channelopathies and to date there have been 10 human channelopathies associated with 5 of the 10 Ca_V α-subunit genes. The consequences of disease causing mutations on Ca_V channel function varies widely and includes many loss-of-function and gain-of-function effects on voltage dependency and kinetics of channel gating.¹¹⁸ However much of the pioneering work of the Yue lab on calmodulin regulation of Ca_V channels has inspired subsequent work exploring the effects of human disease mutations on calcium regulation of Ca_V channels.

Several mutations in the CACNA1A gene encoding the P/Q-type channel α-subunit (Ca_V2.1) enriched within central nervous system cause channelopathies with severe neurological pathology. Such disorders include Familial Hemiplegic Migraine (FHM1), Episodic Ataxia type 2 (EA2)¹¹⁹ and Spinocerebellar Ataxia type 6 (SCA6).¹²⁰ In fact, mutations associated with FHM1and EA2 have been shown to modify CDF and CDI of Ca_V2.1 channels and alter short-term synaptic plasticity.¹²¹ Furthermore, the L-type channel α₁ subunit (Ca_V1.4) is localized within retinal cells and mutations in the CACNA1F gene encoding this channel are implicated in the vision related disorders incomplete X-linked congenital stationary night blindness (IXLCSNB) and X-linked cone-rod dystrophy (CORDX).^{122, 123} One IXLCSNB mutation within the carboxyl tail of the Ca_V1.4 enables CaM binding, resulting in robust CDI in these channels¹²⁴ and a boost in channel open probability¹⁰⁹ Furthermore, mutations in the CACNA1C gene encoding the α₁ subunit Ca_V1.2 enriched in cardiac tissue have been associated with a severe arrhythmic disorder called Timothy syndrome, ¹²⁵ and implicated in autism spectrum disorder (ASD).¹²⁶ These mutations have been shown to have significant effect on Ca_V1.2 CDI (Dick et al, BPS abstract).

The recent work by the Yue lab demonstrating the conservation of CaM regulation between calcium and sodium channels has already shown that channelopathic disease mutations in sodium channels^{127, 128} alter sodium channel CDI, ⁸⁶ and likely channel open probability.¹⁰⁹ The consequences could be extensive, as the Na_V1 superfamily governs excitability in brain, heart, and skeletal muscle, ¹²⁹ and related diseases encompass epilepsy, autism, pathological pain, cardiac arrhythmias, and skeletal muscle myotonias.^{127, 128} Specifically, a number of mutations in Na_V1.4, an isoform commonly found in skeletal muscle, are associated with myotonia, difficulty voluntarily relaxing skeletal muscle. Genetic screening revealed a variety of mutations in Na_V1.4 corresponding to a wide spectrum of the severity of myotonia.^130–132 Since a subset of these mutations lie in the carboxyl-tail of Na_V1.4, ^{133, 134} one might postulate that the disruption of CDI could be one of the mechanisms for myotonia in this subgroup as CaM orchestrates CDI of Na_V1.4 through interacting with the channel's carboxyl tail.⁸⁶

Beyond skeletal Na_V1.4 channels, mutations in cardiac Na_V1.5 channels have also been implicated in several prominent cardiac channelopathies such as Brugada syndrome and congenital long QT syndrome (LQTS).¹³⁵ While the mechanism behind Brugada syndrome has yet to be firmly established, decreased Na current density is generally accepted as the functional consequence.^135-137 In contrast, in LQTS associated with mutations in SCN5A, a gene encoding the Na_V1.5 channel, an increase in persistent inward sodium current is often implicated as the mechanism of action potential lengthening in LQTS.^{135, 138-140} Interestingly, many Brugada syndrome and LQTS mutations occur within the carboxyl-terminus of Na_V1.5 channels, ^141-145 thus calmodulation and Ca²⁺ regulation dysfunction of Na_V1.5 present as promising potential mechanisms behind the 2 prominent cardiac channelopathies with disparate functional effects on Na channel function. For example, a A1924T mutation associated with Brugada syndrome leads to decreased CaM binding affinity to the Na_V1.5 carboxyl-terminus and may alter Na_V1.5 channel gating kinetics.^{80, 85, 146} Taken together, recent insights and elucidation in calmodulation and Ca²⁺ regulation of Na_V1.5 channels leads to exciting frontiers in understanding cardiac channelopathy mechanisms.

Not only do disruptions in Ca_V channels themselves lead to disease, but alterations to CaM itself can underlie pathophysiology. For instance, the open probability of the long variant of Ca_V1.3 is strongly dependent on the concentration of CaM within cells (Fig. 7A, middle). When CaM levels were increased within dopaminergic neurons (where the long variant of Ca_V1.3 is found), the action potential duration of these pacemaking cells were dramatically increased (Fig. 7A, right). With increased Ca²⁺ exposure linked to neuronal toxicity, ¹⁴⁷ the finding that the concentration of CaM can significantly alter Ca²⁺ influx raises the possibility of CaM's involvement in the pathogenesis of Parkinson's disease.

Figure 7.

Biophysical and physiological impact of altered CaM. (A) CaM boosts a channel open probability (red). In neurons, overexpression of CaM increases the magnitude of Ca²⁺ current leading to depolarized resting membrane potential and the lengthening of action potential duration. Reproduced from Adams et al., 2014.¹⁰⁹ (B) CaM orchestrates CDI of the L-type Ca²⁺ channel. LQTS-associated mutant CaM has diminished Ca²⁺ binding affinity slows current inactivation (red), thus prolonging cardiac APD, a cellular correlate of LQTS. Reproduced from Limpitikul et al., 2014. © Elsevier. Reproduced by permission of Elsevier. Permission to reuse must be obtained from the rightsholder.

Additionally, given the importance of CaM in triggering the CDI of L-type Ca²⁺ channels in the heart, mutations within CaM might disrupt CDI, prolonging the cardiac action potential via an increase in the inward current during the plateau phase. Indeed, myocytes overexpressing CaM₁₂₃₄ exhibit markedly prolonged action potentials.^{148, 149} These studies highlighted the importance of CaM in the heart, adding credence to the widespread belief that naturally-occurring mutant CaMs would likely be so deleterious to be incompatible with life. However, it was not until a decade later, that a rare group of diseases resulting from heterozygous missense mutations within one of the 3 genes (CALM1-3) encoding identical CaM proteins was discovered. These calmodulinopathies result in catecholaminergic polymorphic ventricular tachycardia (CPVT) and severe long-QT syndrome (LQTS) with recurrent cardiac arrest.¹⁵⁰

Expressing these CaM mutants in myocytes (Fig. 7B, left), the Yue lab found that the CDI of cardiac L-type Ca²⁺ channels was attenuated (Fig. 7B, middle). Although a diminished CDI could explain the QT interval prolongation, the severity of the disease in human patients was puzzling, given that the number of wild-type CaM molecules is thought to greatly outnumber the number of mutant CaMs. Using FRET-based binding assays, the Yue lab demonstrated that the mutant CaMs had preserved binding with the L-type channel, suggesting that a fraction of L-type channels were occupied by a mutant CaM. Through modeling and further electrophysiology, they showed that the large effect that mutant CaM has on a small fraction of channels was sufficient to produce a significant reduction of CDI, ²² leading to a large prolongation of action potential duration (Fig. 7B, right).

Remembering a Great Scientist

From his early childhood as a ham radio operator, David T. Yue was fascinated by electrical signals and delighted by transistors. It is no wonder then, that as he began his scientific career he gravitated to the biological equivalent of a transistor, the voltage gated Ca²⁺ channel. He brought with him a desire to apply detailed quantitative methods to biological function, a dream he made a reality. He sought to understand Ca²⁺ regulation at its most fundamental level, and yet he never lost sight of the biological implications of these mechanistic discoveries. As a result, David's scientific contributions spanned the spectrum all the way from atomic structure and single molecule recordings to live brain imaging. He was able to gain mechanistic insight within a single channel which could then be applied not only across channel subtypes, but across families of channels, ultimately linking 2 channel fields.

The story of Ca²⁺ regulation presented here is but a glimpse into the world of David T. Yue. He was a seeker of truth who pursued science with a sense of wonder which was inspirational to all who knew him. He was passionate about his research, relating experimental discovery with “a syllable that God spoke.” All who passed through the Calcium Signals Lab have fond memories of late nights of exciting research, punctuated with humorous and loving stories of David's three exceptional sons, Michael, Daniel and Jonathan. David's wife Nancy was such a strong and supportive presence in the CSL that she earned the nickname ‘Saint Nancy’ from the lab members. So now, as David's Calcium Signals Lab family looks to the future, we remember the lessons he taught us and strive to carry his legacy forward.

“As for you, you mustn't let what could have been destroy the dream and wonder of what is and what will be. Keep your bow pointed – Good luck.” – David T. Yue, 1980

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank David T. Yue for his years of dedicated and enthusiastic support, guidance and friendship. He was a brilliant scientist, caring mentor and inspiring teacher who will be greatly missed.

Funding

This work was supported by grants from the NHLBI (R37HL076795), NIMH (R01MH065531), NINDS (R01NS073874) and NINDS (R01NS08074)to David T. Yue; NHLBI(HL50411) to GT, AHA(13PRE16500029) to WL, NSF(DGE-1232825) to JN and MSTP fellowships to JBI and SRL.