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(Circulation Research. 2004;94:1011.)
© 2004 American Heart Association, Inc.

Reviews

Imaging Microdomain Ca²⁺ in Muscle Cells

Shi-Qiang Wang, Chaoliang Wei, Guiling Zhao, Didier X.P. Brochet, Jianxin Shen, Long-Sheng Song, Wang Wang, Dongmei Yang, Heping Cheng

From the Laboratory of Cardiovascular Science (S.-Q.W., D.X.P.B., J.S., L.-S.S., W.W., D.Y., H.C.), National Institute on Aging, NIH, Baltimore, Md; National Laboratory of Biomembrane and Membrane Biotechnology (S.-Q.W., C.W., D.Y.) and The Institute of Molecular Medicine (C.W., H.C.), Peking University, Beijing, China; and the Department of Physiology (G.Z.), The First Military Medical University, Guangzhou, China.

Correspondence to Heping Cheng, PhD, Laboratory of Cardiovascular Science, NIA, NIH, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail chengp{at}grc.nia.nih.gov

This Review is part of a thematic series on Imaging of Cardiovascular Cells and Tissues, which includes the following articles:



Use of Chimeric Fluorescent Proteins and Fluorescence Resonance Energy Transfer to Monitor Cellular Responses
Imaging Microdomain Ca²⁺ in Muscle Cells
Optical Imaging of the Heart
Examining Intracellular Organelle Function Using Fluorescent Probes
Two-Photon Microscopy of Cells and Tissues

Brian O’Rourke Guest Editor

	Abstract

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Ca²⁺ ions passing through a single or a cluster of Ca²⁺-permeable channels create microscopic, short-lived Ca²⁺ gradients that constitute the building blocks of cellular Ca²⁺ signaling. Over the last decade, imaging microdomain Ca²⁺ in muscle cells has unveiled the exquisite spatial and temporal architecture of intracellular Ca²⁺ dynamics and has reshaped our understanding of Ca²⁺ signaling mechanisms. Major advances include the visualization of "Ca²⁺ sparks" as the elementary events of Ca²⁺ release from the sarcoplasmic reticulum (SR), "Ca²⁺ sparklets" produced by openings of single Ca²⁺-permeable channels, miniature Ca²⁺ transients in single mitochondria ("marks"), and SR luminal Ca²⁺ depletion transients ("scraps"). As a model system, a cardiac myocyte contains a 3-dimensional grid of 10⁴ spark ignition sites, stochastic activation of which summates into global Ca²⁺ transients. Tracking intermolecular coupling between single L-type Ca²⁺ channels and Ca²⁺ sparks has provided direct evidence validating the local control theory of Ca²⁺-induced Ca²⁺ release in the heart. In vascular smooth muscle myocytes, Ca²⁺ can paradoxically signal both vessel constriction (by global Ca²⁺ transients) and relaxation (by subsurface Ca²⁺ sparks). These findings shed new light on the origin of Ca²⁺ signaling efficiency, specificity, and versatility. In addition, microdomain Ca²⁺ imaging offers a novel modality that complements electrophysiological approaches in characterizing Ca²⁺ channels in intact cells.

Key Words: Ca²⁺ signaling • Ca²⁺ sparks • Ca²⁺ channels • excitation-contraction coupling • sarcoplasmic reticulum

	Introduction

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
As a ubiquitous intracellular messenger, Ca²⁺ plays critical roles in a myriad of physiological processes, including muscle contraction, synaptic transmission, hormone secretion, gene transcription, cell survival, and cell death.^1–5 Ca²⁺ as an inorganic divalent cation exists in only one chemically and biologically relevant form and is diffusible in the cytosolic milieu. A fundamental issue has arisen as to how the same ion can fulfill different and even opposing physiological functions in a given cell. Over the last decade, it has been increasingly appreciated that spatial and temporal patterning endows Ca²⁺ signaling with efficiency, specificity, and unparalleled versatility. Optical visualization of microdomain (0.1 to 10 µm) Ca²⁺ thus holds the promise to dissect out the space-time architecture of Ca²⁺ dynamics and, thereby, to unravel Ca²⁺ signaling mechanisms in different physiological contexts.

Since the first recording of Ca²⁺ sparks⁶ in cardiac myocytes in 1993, imaging microdomain Ca²⁺ in muscles has offered unique insights into the molecular mechanisms of excitation-contraction (EC) coupling and revolutionized our understanding of Ca²⁺-mediated signal transduction (see reviews^2,5,7–10). In this review, we intend to summarize advances in this rapidly evolving field, with an emphasis on cardiac microdomain Ca²⁺ imaging. In keeping with the aims of this review series, pertinent technical aspects will be discussed in an attempt to identify merits, potentials, and limitations of the various Ca²⁺ imaging techniques used. Selected terminologies used to describe cellular signaling Ca²⁺events are listed in the Table.

View this table: [in this window] [in a new window]   Table 1. Select Terminology of Ca2+ Signaling Events

	Ca2+ Gradients in Living Cells: Theoretical Considerations

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Sustained gradients of free Ca²⁺ concentration ([Ca²⁺]) can be found between membrane-delimited cellular and subcellular compartments. A 10⁴-fold [Ca²⁺] gradient exists across the plasma membrane as well as the membrane of the endoplasmic and sarcoplasmic reticulum (ER/SR). The cytosolic [Ca²⁺] is actively maintained at a very low level around 100 nmol/L, by a system of Ca²⁺ homeostatic regulatory mechanisms, including the ER/SR Ca²⁺ ATPase and the sarcolemmal Na⁺-Ca²⁺ exchanger and Ca²⁺ ATPase. The signal Ca²⁺ can then be rapidly mobilized, often via Ca²⁺-permeable channels, from the exterior or the intracellular Ca²⁺ reservoirs, resulting in [Ca²⁺] transients in the cytosol.

At one time, the cytosolic Ca²⁺ was conceptualized as a "common pool" in which [Ca²⁺] rises and falls uniformly. A corollary of this notion is that Ca²⁺ signaling in a given cell is fully determined by the magnitudes and the temporal dynamics of spatially averaged [Ca²⁺]. At the molecular level, however, transmembrane Ca²⁺ translocation is a discrete process mediated by Ca²⁺-permeable channels and Ca²⁺ transporters. When in action, these molecular entities function as either Ca²⁺ sources (admitting Ca²⁺ into a subcellular compartment) or Ca²⁺ sinks (removing Ca²⁺ from the compartment) and create dynamic [Ca²⁺] gradients in their immediate vicinity. Theoretically,^11–15 many cellular and molecular determinants shape the microscopic [Ca²⁺] gradients. These include (1) unidirectional Ca²⁺ fluxes, (2) intracellular Ca²⁺ buffers, many of which are also Ca²⁺ effectors, and (3) Ca²⁺ diffusion in the cytosolic milieu. In addition, [Ca²⁺] gradients will be accentuated and confined by spatial restriction, eg, disc-shaped dyadic clefts,^16,17 tortuous ER/SR network, or membrane-bound subcellular organelles such as mitochondria. The physiological relevance of such Ca²⁺ microdomains had not been fully appreciated until about a decade ago, when they were first visualized and investigated experimentally.

	Imaging the Elementary Intracellular Ca2+ Release Events

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Ca²⁺ signals in living cells were first "seen" in the late 1960s as [Ca²⁺] transients from single twitch barnacle muscle fibers¹⁸ and first imaged in the late 1970s as [Ca²⁺] waves in fertilizing medaka fish eggs.¹⁹ In both cases, [Ca²⁺] was measured by means of chemiluminescent aequorin. Shortly after, [Ca²⁺] transients during cardiac EC coupling were seen as aequorin signals in canine Purkinje fibers.²⁰ Measurement of [Ca²⁺] transients began to seriously expand with metallochromic dyes, first arsenazo III, used by Brown et al in squid axon²¹ and then in muscle,²² and later antipyrylazo III used by Kovacs et al.²³ In heart muscle cells, the use of fluorescent reporter, fura-2, was first introduced in conjunction with digital imaging to document spontaneous [Ca²⁺] waves by Wier et al.²⁴ With the advent of confocal microscopy and the new generation of fluorescent indicator, fluo-3, microdomain Ca²⁺ in muscles was first visualized as Ca²⁺ sparks in quiescent cardiac myocytes (Figure 1A) in 1993.⁶

View larger version (48K): [in this window] [in a new window]   Figure 1. Imaging the elementary intracellular Ca2+ release events. A, 3-D contour plot of a spontaneous Ca2+ spark in a rat cardiac myocyte (courtesy of M.B. Cannell at the University of Aukland, New Zealand). B, Time-resolved (16-ms) 2-dimensional visualization of an evoked Ca2+ spark beneath the loose-seal patch pipette. RP indicates resting potential (–80 mV). Tip of the patch pipette, seen as the dark rim (arrowhead), was placed in focus.

A Ca²⁺ spark appears abruptly amid a seemingly featureless background, reaches its peak within 10 ms, and dissipates in another 20 ms; it reflects the spontaneous activation of ryanodine receptors (RyRs)^25,26 in a single Ca²⁺ release unit (CRU) in the ER/SR. In spite of the Ca²⁺-induced Ca²⁺ release (CICR) mechanism^27–29 operating in these cells, a Ca²⁺ spark normally remains solitary and is confined to an area of 2.0 µm in diameter. Under Ca²⁺ overload conditions, however, a Ca²⁺ spark can sometimes activate release from neighboring CRUs to form a compound spark^6,30–32; ordered spatiotemporal activation of sparks and compound sparks can then evolve into propagating [Ca²⁺] waves.^6,31

Ca²⁺ sparks with identical properties can be evoked by Ca²⁺ influx through voltage-operated L-type Ca²⁺ channels (LCCs), known also as dihydropyridine receptors (DHPRs), via the CICR mechanism (Figure 1B).^33–37 To resolve individual events, Ca²⁺ sparks should be activated at a low density, by near-threshold or very brief depolarization,^35,37 with reduced extracellular [Ca²⁺],³⁸ or under conditions where LCC availability is reduced pharmacologically.^35–40 Both the trigger Ca²⁺ currents (I_Ca) and the probability of Ca²⁺ spark activation display a bell-shaped voltage dependence,^36,37,40 whereas the properties of individual Ca²⁺ sparks do not vary with the membrane voltage, or the duration and amplitude of I_Ca.^35–37,40 This indicates that, once activated, a Ca²⁺ spark evolves independently of its trigger. During full-fledged cardiac EC coupling, about 10⁴ sparks are activated within a few tens of milliseconds in a single myocyte,³³ summating into a global [Ca²⁺] transient of 1 µmol/L.

Similar Ca²⁺ sparks have also been recorded in intact cardiac trabeculae.⁴¹ In atrial myocytes, which lack transverse tubules (TT) and contain both peripheral junctional and central nonjunctional SR, spontaneous Ca²⁺ sparks are greater and longer than the ventricular counterparts (300 000 Ca²⁺ ions in 12 ms versus 100 000 Ca²⁺ ions in 7 ms), with a high prevalence at the periphery.^42,43 The action potential directly evokes subsurface Ca²⁺ sparks in a stochastic fashion, which subsequently activate inwardly propagating waves of CICR.⁴² Rhythmic Ca²⁺ sparks and compound Ca²⁺ sparks are also found to regulate diastolic depolarization and thereby the pacemaker activity in sinoatrial nodal cells.^44,45

High Ca²⁺ microdomains, resulting from intracellular Ca²⁺ release, are now found in many other cell types. In particular, Ca²⁺ sparks are present in skeletal^46,47 and smooth muscle cells⁴⁸ containing different isoforms of RyRs. In addition to CICR, depolarization-induced Ca²⁺ sparks in amphibian skeletal muscle fibers are generated by mechanical coupling between RyRs and DHPRs, the voltage sensors.^46,47 IP₃ receptor (IP₃R)–mediated "Ca²⁺ puffs"⁴⁹ or "Ca²⁺ blips"⁵⁰ have been shown to originate from discrete CRUs in Xenopous oocytes. Even in nonexcitable cells, local Ca²⁺ release events with typical spark characteristics have been visualized and attributed to the activation of RyRs and/or IP₃Rs.^51,52 However, there is less information about the existence and role of Ca²⁺ sparks in neurons. Although excessive "noise" or bright pixels have been detected by histogram analysis of neuronal Ca²⁺ images, no discernible spark events were actually resolved.⁵³ The elementary Ca²⁺ release events in nerve growth factor–differentiated PC12 cells or cultured hippocampal neurons exhibit distinctly greater width (4 µm) and duration (400 ms),⁵⁴ suggesting that they arise from spatially extended CRUs or represent compound sparks.

As a technical note, fluo-3 and its high-fluorescence derivative fluo-4 have thus far been the indicator of choice in spark experiments, This is because of their unique combination of superb signal-to-background contrast (200 times more brilliant on Ca²⁺ association), visible light excitation, appropriate kinetics, and dissociation constant (K_d=0.4 µmol/L in physiological saline⁵⁵; 1.1 µmol/L in skeletal muscle cells due to protein-binding of the indicator⁵⁶). However, neither indicator displays a significant shift in excitation or emission spectrum on Ca²⁺ association, which precludes the use of ratiometric measurement and poses a problem in determination of the absolute [Ca²⁺] involved. Wide ranges of image techniques have been applied to investigate Ca²⁺ sparks in different cell types. These include single-photon and two-photon excitation confocal microscopy,⁵⁷ and nonconfocal methods such as total internal reflection fluorescent microscopy (TIRFM)⁵⁸ and wide-field microscopy coupled with a low-noise CCD camera.⁵⁹ In fact, cardiac Ca²⁺ sparks are visible to the human eye with the aid of a conventional epifluorescence microscope (H.C., personal observation, 2003).

	Subspark and Sparkless Ca2+ Release Events

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Interestingly, ectopic expression of cardiac⁶⁰ or skeletal⁶¹ RyR in CHO cells is associated with a sparkless type of Ca²⁺ release. In guinea pig ventricular myocytes, Lipp and Niggli first reported sparkless release in response to photolysis of caged Ca²⁺,⁶² and later demonstrated subtler Ca²⁺ release events, named "Ca²⁺ quarks."⁶³ The sparkless appearance indicates that the elementary release events involve a quantity of Ca²⁺ that is well beyond the detection limit.

Mammalian adult skeletal muscle fibers manifest a sparkless form of release under physiological conditions.⁶⁴ The irony is that the same release machinery can also generate spark-featured release after chemical permeabilization of the surface membrane.⁶⁵ In immature skeletal muscle cells from mice, EC coupling at spatially segregated sites^66,67 displays a sparkless type of release, whereas discrete sparks occur at locations devoid of direct EC coupling.⁶⁶ These observations led to the proposal that DHPRs function also to suppress the mechanism that activates discrete release events.⁵⁴ Because repolarization can abruptly terminate on-going Ca²⁺ sparks in amphibian skeletal muscle cells,⁶⁸ a rapid "turn-on" and "turn-off" of RyRs by DHPRs could possibly explain the apparent lack of discrete Ca²⁺ sparks in the mammalian species.

	Morphometric Analysis of Microdomain Ca2+

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Generic problems for fluorescence-based imaging of microdomain Ca²⁺ include (1) out-of-focus blurring, particularly when the placement of the Ca²⁺ sources in relation to the focal volume is uncertain, (2) optical blurring, as defined by the point spread function (PSF) of the imaging system, and (3) kinetics and diffusion of the indicator. In this section, we will use morphometric analysis of Ca²⁺ sparks as a showcase to discuss these issues in quantitative measurements of microdomain Ca²⁺.

Confocal sampling theory states that out-of-focus blurring will produce a reduced spark amplitude, broadened spatial spreading, and blunted kinetics with the degree of distortion depending on the relative positioning.^12,13 Because there are more sparks at out-of-focus than in-focus places, it was predicted that the apparent spark amplitude always obeys a monotonically decaying distribution, regardless of the true spark amplitude.^69,70 Experiments aided with an automated spark detection algorithm corroborated this prediction.^69,71

Several approaches have been developed to curtail or correct for the effects of out-of-focus blurring. In an effort to restore the true population statistics of spark amplitude, Izu et al⁷⁰ and Rios et al⁷² have attempted to deconvolve the blurring from the apparent spark amplitude distribution. Some investigators have exploited spark repeats from hyperactive CRUs^73,74 or repeatedly evoked Ca²⁺ sparks at fixed TT-SR junctions³⁸ to analyze the variability of spark parameters. We have recently provided a solution to this problem by activating Ca²⁺ sparks from in-focus CRUs.^75–77 Specifically, a low-resistance (20 to 50 M) seal was formed by gently pressing a patch pipette against the surface membrane without disrupting the exquisite EC coupling machinery. A population of in-focus Ca²⁺ sparks (Figure 1B) can then be evoked by repeated patch depolarization. A more rigorous characterization of spark properties revealed that the true amplitude of Ca²⁺ sparks exhibits a broad, modal distribution,^75,76 in contrast to the notion that Ca²⁺ sparks are all-or-none or stereotypical.^6,35,36

Because of the finite PSF of the optical system, even the in-focus Ca²⁺ spark is not blur-free. A typical PSF used for confocal Ca²⁺ spark recording is 0.3 to 0.4 µm (measured as the full width at half-maximum, FWHM) in the radial direction and 0.7 to 1.5 µm in the axial direction, the latter being comparable to the FWHM of a spark. The sharpest optical focus is attainable with TIRFM, which is about 200 nm into the cell. In future studies, digital deblurring in conjunction with ultrafast 3-dimensional (x-y-z) confocal imaging may further improve the measurement morphometrics of in-focus sparks.

Apart from the cellular determinants of local [Ca²⁺] gradients discussed earlier, the fluorescence signal is further shaped up by the reaction kinetics and diffusion of the indicator.^12–14 Thus, even the blur-free fluorescence signal differs markedly from the underlying Ca²⁺ signal. A direct conversion of the fluorescence signal to [Ca²⁺] (assuming equilibrium between local Ca²⁺ and the indicator) would grossly underestimate [Ca²⁺] at the origin, but slightly overestimate [Ca²⁺] at the far site, while its time-to-peak tracks the duration of Ca²⁺ release.¹³ Finally, it should be cautioned that inclusion of the Ca²⁺ indicator disturbs the microscopic [Ca²⁺] gradients per se; photochemical and metabolic products of the indicator may introduce additional complications to the [Ca²⁺] measurement and cause significant damage to the cell.

	Cellular Organization of Ca2+ Microdomains

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
The cellular organization of spark ignition sites has been investigated in ventricular myocytes by linescan imaging along the transverse and longitudinal axes of the cell,^32,78 fast 2-dimensional (x-y) imaging in the presence of excessive Ca²⁺ buffers,⁷⁹ and 2-dimensional mapping of evoked Ca²⁺ sparks in high K⁺ depolarized myocytes (Figure 2). Simultaneous staining of the TT space^7,31 or memebrane^32,78 has consistently shown that Ca²⁺ sparks are predominantly localized to the Z-line/TT regions of sarcomeres, with a spacing of 1.8 µm (a sarcomere length) in the longitudinal direction. Laterally, they are, on average, 0.8 µm apart³² (Figure 2). The disposition of functional CRUs provides the basis for intrasarcomeric [Ca²⁺] gradients and spatial inhomogeneities of [Ca²⁺] transients seen during EC coupling.^33,79–81

View larger version (28K): [in this window] [in a new window]   Figure 2. Map of Ca2+ spark ignition sites in a ventricular myocyte. A, Resting staining of fluo-4. The terminal SR was stained brighter, presumably because of SR retention of the indicator.110 Green overlay shows the cell edge (lines), and pink overlay marks the z-lines. B, Distribution of "in-focus" sparks (F/F0>1.4). 550 sparks were detected in the presence of 15 mmol/L extracellular K+. C, Location of spark ignition sites on TTs, after grouping clustered events. Color bar codes the number of sparks at a given site.

The density of functional CRUs is estimated to be 1 CRU/µm³, or 10⁴ CRUs per myocyte. The estimated density of functional CRUs is thus in remarkable agreement with the data from thin-section electronic microscopic micrographs.⁸² Given that a cell contains about 10⁶ RyRs,⁵ this suggests an average grouping of 100 RyRs in a CRU. This estimate, however, differs considerably from the ultrastructure data (267 RyRs per CRU in rat ventricular myocytes, assuming a round CRU geometry).⁸² This discrepancy may stem from the lack of knowledge of the exact shape of CRUs. Similarly, a 3-dimensional grid of CRUs spaced at 2 µm apart has been deduced from functional data and immunostaining of RyRs in atrial myocytes.⁴²

Thus, in cardiac myocytes as a model system, it is no longer considered adequate to depict cellular Ca²⁺ signaling as a common-pool system governed by deterministic laws. Rather, it is a discrete, stochastic system that encompasses tens of thousands of Ca²⁺ microdomains operating relatively independently. In this new paradigm, cellular Ca²⁺ signaling is orchestrated by these localized, short-lived Ca²⁺ microdomains. Hence, regulation of Ca²⁺ signaling can be achieved by varying the number of microdomains recruited (from 1 to 10⁴) and by modulating the amount of Ca²⁺ released in individual microdomains through both global and local control mechanisms. Assuming each microdomain can exist in at least two distinct states ("on" and "off"), the number of patterned activation of Ca²⁺ microdomains in a cell would be astronomical (10³⁰⁰⁰). Furthermore, some Ca²⁺ transducing molecules such as Ca²⁺/calmodulin-dependent protein kinase II (CaMKII)⁸³ can retain a "memory" of the rate and duration of recent Ca²⁺ pulses, and render a frequency-encoding phosphorylation of downstream effectors (eg, phospholamban in heart cells),⁸⁴ thereby enriching the versatility of spatiotemporal Ca²⁺ signaling.

	Ca2+ Spikes: Visualization of Local Ca2+ Fluxes

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Injecting Ca²⁺ into a heavily buffered medium would produce a surge of free [Ca²⁺], dubbed a "Ca²⁺ spike," reflecting a kinetic imbalance between Ca²⁺ ions and the buffer.⁸⁵ In the limit of a high concentration of Ca²⁺ buffer, the Ca²⁺ spike follows the waveform of Ca²⁺ injection flux.⁸¹ By exploiting this principle, we devised a method to visualize localized Ca²⁺ release flux or Ca²⁺ spikes (Figure 3).⁸¹ This involves an admixture of a fast, low-affinity Ca²⁺ indicator (eg, Oregon Green 488 BAPTA 5N, 1 mmol/L, K_d=31 µmol/L) and an excess of high affinity, but slow Ca²⁺ chelator (eg, EGTA, 5 mmol/L, K_d=150 nmol/L at pH 7.2). As Ca²⁺ ions are emitted from an effective point source, the initial ratio of Ca²⁺ binding to the fluorescent indicator (F) and to EGTA is determined by k_on,Fx[F]/k_on,EGTAx[EGTA]. Because k_on,F is typically about 100-fold faster than k_on,EGTA, EGTA should exert little effect on the indicator signal in the close vicinity of the channel pore. As Ca²⁺ ions diffuse outward, however, nearly all of them will quickly be captured by the nonfluorescent EGTA at excess. As a result, this rather unusual experimental setup helps to track the time course and pinpoint the origin of local Ca²⁺ flux.

View larger version (24K): [in this window] [in a new window]   Figure 3. Ca2+ spikes and the gain function of EC coupling. Whole-cell patch clamp was established with the dialysis of 1 mmol/L Oregon Green 488 BAPTA 5N and 4 mmol/L EGTA. Ca2+ spikes were elicited at –30 mV. Data are shown as surface plot and vertical dashed lines mark the beginning and end of the voltage pulse. B. Bell-shaped voltage dependence for SR Ca2+ release flux (JSR, measured by spatially averaged Ca2+ spikes) (open symbols) and ICa (solid symbols). C, Voltage dependence of the gain function (JSR/ICa) (data from Cheng and Wang10 and Song et al81).

For the same reasoning that EGTA does not suppress the local indicator signal, inclusion of EGTA at mmol/L concentrations should not significantly affect CICR between LCCs and RyRs^81,86,87 nor the retrograde inactivation of LCCs by Ca²⁺ through RyRs,⁸⁸ both occurring on the nanometer scale. Nevertheless, excessive Ca²⁺ buffering would hamper Ca²⁺ communication on the micrometer scale, such as CICR between adjacent CRUs. Additionally, high [EGTA] would "clamp" the bulk Ca²⁺, suppress global [Ca²⁺] transient, and cause a slow refilling of the SR.^81,86,87

In heart muscle cells, Ca²⁺ spikes have been used to track Ca²⁺ release flux during spontaneous Ca²⁺ sparks⁷⁴ and TT-SR junction activation during full-fledged EC coupling (Figure 3).^81,86,87 The TT-SR Ca²⁺ spike reflects a single or a compound Ca²⁺ spark, depending on the number of CRUs recruited. The magnitude of spatially averaged Ca²⁺ spikes or the SR release function (J_SR) exhibits a bell-shaped voltage dependence (Figure 3B), but the gain function, defined as J_SR/I_Ca, is a monotonic decaying function of voltage (Figure 3C).⁸¹ This reaffirms the notion that CICR efficacy depends on the microscopic properties of the trigger I_Ca.⁸⁹ By examining consecutive TT-SR junction activation at 50-ms intervals, we have investigated possible mechanisms for termination of SR Ca²⁺ release in the heart.⁸⁶ At a TT-SR junction that just underwent maximal release, a greater trigger Ca²⁺ produced by a large-tail Ca²⁺ current failed to trigger additional release, indicating absolute local refractoriness. When the initial release is submaximal, however, tail current does activate succeeding Ca²⁺ spikes, perhaps from those RyRs that were unfired initially. These observations support the notion that a highly localized, use-dependent inactivation of RyRs underlies termination of cardiac Ca²⁺ sparks (see review⁹⁰).

	Optical Recordings of Ca2+ Flux Through Single Ca2+-Permeable Channels

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
Ca²⁺ ions passing through a single opening of a single Ca²⁺-permeable channel create a Ca²⁺ microdomain that is fundamental to intracellular Ca²⁺ signaling. Although Ca²⁺ sparks were initially thought to be a single-channel phenomenon,⁶ a consensus has evolved that most Ca²⁺ sparks are of multi-RyR origin.^{6,7,10,15,75–77,91} Niggli and colleagues have attributed Ca²⁺ quarks to single-RyR events^62,63 and Parker and colleagues have attributed Ca²⁺ blips to single-IP₃R events;⁵⁰ but experimental evidence thus far is equivocal as to the single-channel nature of the subtler release events they observed. Using confocal microscopy combined with the cell-attached patch-clamp technique, we provided an unequivocal demonstration of the feasibility of recording Ca²⁺ sparklets, microscopic [Ca²⁺] transients produced by single Ca²⁺-permeable channel openings in intact cells.⁷⁵ This was done for cardiac LCCs with the aid of the channel agonist FPL64176 to prolong the channel open duration (Figure 4). Simultaneous recording of unitary Ca²⁺ currents indicates that the total fluorescence of a Ca²⁺ sparklet in the linescan image correlates linearly with the amount of Ca²⁺ entry through the channel (Figure 4B). ⁷⁵ This indicates that Ca²⁺ sparklet serves as a faithful optical readout of single-channel Ca²⁺ flux.

View larger version (28K): [in this window] [in a new window]   Figure 4. Visualization of single-channel Ca2+ sparklets. A, Ca2+ sparklets (top) due to single LCC Ca2+ current, iCa (bottom). Cell-associated G-seal patch clamp was established with the pipette containing 10 µmol/L FPL64176 and 20 mmol/L Ca2+. SR Ca2+ release was disabled by caffeine (10 mmol/L) and thapsigargin (10 µmol/L). B, Correlation between sparklet signal mass (IIF/F0dxdt) (in arbitrary unit) and the integral of the corresponding qCa (number of Ca2+ ions) (data from Wang et al75). C, Ca2+ sparklet evoked at –50 mV from a TT inside a rat ventricular myocyte. Whole-cell voltage-clamp configuration was established in the presence of 10 µmol/L FPL64176 and 10 mmol/L extracellular Ca2+ and confocal plane was focused 5 µm into the cell. SR Ca2+ release was inhibited as in A. D, Parallel optical recording of LCC Ca2+ sparklets from deep inside a cell.

Using a wide-field imaging system equipped with a high-speed, low-noise CCD camera, Zou et al have independently visualized Ca²⁺ sparklets (single-channel Ca²⁺ fluorescence transients) arising from the openings of caffeine-sensitive cation channels⁹² or stretch-activated cation channels in smooth muscle myocytes.⁹³ Because a wide-field microscope collects light from both in-focus and out-of-focus planes, the 2-dimensional image it acquires is a spatial integration itself. While this may not be desirable for looking at localized fluorescence transient in its focal plane, it is valuable in determining the local Ca²⁺ flux by "signal mass" (total fluorescence signal).⁹³

Most recently, Demuro and Parker⁹⁴ succeeded in confocal recording of Ca²⁺ sparklets produced by individual N-type voltage-gated Ca²⁺ channels expressed in Xenopous oocytes. Peng et al⁹⁵ have developed an optical bilayer system that is capable of simultaneously visualizing single-channel Ca²⁺ flux and recording unitary ionic current in planar lipid bilayers. Using this experimental system, they detected bilayer Ca²⁺ sparklets from reconstituted RyRs carrying 0.25 to 14 pA of unitary Ca²⁺ current. This "bottom-up" approach provides an opportunity to dissect cellular and experimental variables that determine the space-time characteristics of microdomain Ca²⁺, to calibrate the fractional Ca²⁺ current through nonselective Ca²⁺ channels (eg, RyRs), and to investigate possible coordinated operation of RyRs in a 2-dimensional array.^96,97 By analysis of the quantal substructure found in the distribution of Ca²⁺ currents in a spark (I_spark), we have identified a subpopulation (12%) of Ca²⁺ sparks that consist of a single quantum of 1.2 pA with a mean release duration of 16.7 ms.⁷⁷ If a quantum reflects a single-RyR phenomenon, this represents the first measurement of unitary current and gating kinetics of RyR in intact cells.

	Optical Single-Channel Recording: Advantages, Limitations, and Potentials

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
The introduction of the patch-clamp technique in early 1980s has revolutionized our understanding of ion channel function by allowing measurement of single-channel currents at unprecedented resolution.⁹⁸ As illustrated earlier, modern optics, photonics, and the use of ion-selective indicators have extended the horizon by affording a new modality for monitoring single Ca²⁺ channel activity. Complementing the electrophysiological approaches, optical single-channel recording possesses unique advantages along with its own set of limitations.

First, optical single-channel recording is penetrative. It is widely applicable to channels inaccessible to electrophysiological means, such as those on intracellular organelles (Figure 1) or invaginations (TTs) of the plasma membrane (Figures 4C and 4D), or some distance into a tissue or organ preparation. Second, it is inherently nondestructive. No mechanical stress is exerted to disrupt the integrity of membrane features (eg, cavoeli), cytoskeleton, channel array assembly, and macromolecular channel complexes. However, the inclusion of exogenous Ca²⁺ indicator and light radiation may perturb Ca²⁺ signaling processes and even damage the cell. Third, the optical channel recording is capable of parallel readout, while retaining resolution for individual events (Figure 4D). For instance, a longitudinal linescan of a cardiac cell can survey 10⁴ RyRs at the same time.⁶ This is useful to catch rare, serendipitous, yet biologically relevant events (eg, cyclic activation of RyRs at fixed sites^45,73,74) and to map regional differences of channel behavior.⁹⁴ Last, but not least, the ion selectivity of the indicator offers the ability to discriminate among ion species through the same channels. This feature has been exploited to measure the fractional Ca²⁺ flux through nonselective Ca²⁺-permeable channels, at the whole-cell⁹⁹ or single-channel level.⁹³

Ca²⁺ sparklets produced by a known Ca²⁺ current can be used as a natural "yardstick" to calibrate any unknown local Ca²⁺ flux, such as I_spark. Sparklet-based measurements show an average I_spark of 2 to 3 pA.^75,76 This value falls to the lower bound of the I_spark (1.5 to 20 pA) derived from forward modeling,^12–15 model-based fitting,⁵⁷ or backward-calculation of the release flux from Ca²⁺ sparks.¹⁰⁰ Because Ca²⁺ sparklets and Ca²⁺ sparks share common microenvironments in terms of Ca²⁺ buffering, indicator binding, and Ca²⁺ and indicator diffusion, the optical calibration relies on no assumption other than a linear extrapolation.

A fundamental distinction between the optical and electrophysiological approaches exists in respect to the physical principles used. A single Ca²⁺ ion moving through a channel contributes two positive electron charges to the electrical current, whereas a Ca²⁺-bound fluorochrome can emit 10⁵ photons per millisecond with saturating excitation.⁵⁵ This represents a staggering power of amplification in terms of the number of information carriers involved. Although only a small fraction of these photons can be harvested and contribute to the output signal, future innovations might bring out the vast potential inherent in the optical single-channel recording technique.

At present, a major limiting factor for optical Ca²⁺ channel recording is insufficient temporal resolution, which is due to the reaction and diffusion kinetics of microdomain Ca²⁺ and the indicator as well as the photon collection efficiency. For confocal detection of Ca²⁺ sparklets, the smallest quantity of Ca²⁺ resolved in an in-focus single-channel event was about 8000 Ca²⁺ ions,⁷⁵ corresponding to a unitary current of 1 pA lasting 2.5 ms (Figures 4A and 4B). The temporal resolution achieved in another study was at best about 10 ms.⁹⁴ The limited temporal resolution has hampered efforts to resolve Ca²⁺ sparklets under physiological conditions where LCC current is 0.12 pA at 0 mV (2 mmol/L Ca²⁺ as the charge carrier)¹⁰¹ and lasts 0.3 ms,¹⁰² carrying a packet of 110 Ca²⁺ ions. Future technical innovations, particularly in the area of information-carrier amplification, are needed to improve the temporal resolution of optical single-channel recording in situ and in vitro.

Visualization of Intermolecular Ca²⁺ Signaling
In heart cells, LCCs and RyRs colocalize to dyadic junctions where the surface membrane or TT comes into direct apposition to the SR membrane at a 12-nm distance. Within the nanoscale junctional cleft, stochastic gating of single LCCs is expected to deliver the trigger Ca²⁺ pulses to activate Ca²⁺-gated RyRs. Two groups have independently suggested that single LCC excitation is sufficient to trigger a Ca²⁺ spark. Under conditions where most LCCs are inhibited (for resolution of solitary sparks), Lopez-Lopez et al³⁴ demonstrated kinetic similarities for I_Ca and spark activation. We have noticed that the voltage dependence of Ca²⁺ spark activation (P_s) is proportional to LCC activation (P_o,L) at near-threshold voltages (from –60 to –40 mV), displaying an e-fold increment per 7-mV depolarization.^35,37 Both lines of evidence suggest that spark activation does not require cooperative interaction among LCCs; otherwise, P_s would be expected to be a power function of P_o,L, or P_sP_o,L^x, with x>1.

Attempts have been made to demonstrate that a single LCC can trigger a spark.^75–77,103 By using simultaneous optical and electrophysiological single-channel recording techniques, we have directly visualized the triggering of a Ca²⁺ spark by at the opening of a single LCC (Figure 5).⁷⁵ This represents a real-time demonstration of nanoscale crosstalk between two sets of molecules functioning in cells at the molecular level of resolution.

View larger version (24K): [in this window] [in a new window]   Figure 5. Sparklet-spark coupling in heart cells. Data were obtained in the loose-seal patch-clamp conditions. A, Triggering Ca2+ spark by single LCC excitation. Experimental conditions were the same as in Figure 4A, except that the cell was bathed in normal physiological saline. RP indicates resting potential. B, Representative example showing that a Ca2+ sparklet directly triggers a Ca2+ spark. Note that succeeding Ca2+ sparklets in the wake of the spark fail to trigger SR Ca2+ release, suggestive of local refractoriness (from Wang et al75).

Simultaneous visualization of LCC Ca²⁺ sparklets and RyR Ca²⁺ sparks (Figure 5B) permitted us to determine the kinetics, fidelity, and stoichiometry of the intermolecular coupling. In contrast to the robust EC coupling at the cellular level, Ca²⁺ sparklets do not always trigger a spark. The latency from the onset of the sparklet to the ignition of the spark is well described by a single exponential function with a time constant of 6.7 ms.⁷⁵ This indicates that the LCC-to-RyR coupling is governed by first order kinetics.

Based on these results and macroscopic properties of EC coupling, we have estimated that, on average, one out of 50 LCC openings at 0 mV triggers a Ca²⁺ spark in the absence of the LCC channel agonist FPL64176.^10,104 At high voltages and increased LCC open probability, there could be more than one LCC opening simultaneously in a junctional cleft. Ca²⁺ spark activation under these conditions may involve two or more LCCs, as has been suggested recently.¹⁰⁵

Recent work has also begun to explore crosstalk of arrayed RyRs within a CRU in cardiac^76,77 and skeletal muscle cells.⁹¹ In a model recently advanced by us,⁷⁷ cardiac spark genesis involves variable cohorts of RyRs ranging from one to eight, as evidenced by the varying number of quanta in I_spark. A negative feedback or autoregulation mechanism results in an inverse relationship between Ca²⁺ release duration and the number of RyRs activated.⁷⁷ A third well-characterized case of intermolecular Ca²⁺ signaling involves RyRs in the SR and large-conductance Ca²⁺-sensitive K⁺ (BK_Ca) channels in the plasma membrane of smooth muscle myocytes.^48,59,106 This will be briefly addressed later.

	Ca2+ in Subcellular Compartments

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
[Ca²⁺] transients immediately beneath the cell membrane are critically involved in physiological processes such as EC coupling, hormone secretion, and neurotransmitter release, which are thought to be driven by Ca²⁺ concentration 10 to 100 times higher than those measured in the bulk cytosol. However, near-membrane Ca²⁺ signal is usually obscured by signals from the bulk cytosol when diffusive water-soluble indicators are used (except for under conditions of Ca²⁺ spike measurement). Llinas et al¹⁰⁷ used the low-affinity, highly nonlinear chemiluminescent Ca²⁺ indicator, n-aequorin-J, to demonstrate the existence of high subsurface [Ca²⁺] domains during neuronal transmitter release. Etter et al have made several attempts to resolve near-membrane [Ca²⁺] using membrane-associated fluorescent Ca²⁺ indicators, C₁₈-fura-2 (K_d=150 nmol/L)¹⁰⁸ and FFP18 (K_d 400 nmol/L, depending on indicator concentration used),¹⁰⁹ in smooth muscle cells. They found that [Ca²⁺] transients exhibit a greater rising rate and briefer duration in the submembrane layer than in the bulk cytosol as expected from the membrane association of Ca²⁺ sparks and sparklets.

As discussed earlier, microscopic [Ca²⁺] transients might also exist in membrane-bound subcellular organelles. Recently, Shannon et al¹¹⁰ developed a technique to image SR luminal [Ca²⁺] in rabbit cardiac myocytes (Figure 6) that involves AM-loading of the SR with a low-affinity indicator, fluo-5N, by an empirical method. During an action potential–triggered release, there was about 50% global depletion of the SR Ca²⁺. They named the negative SR Ca²⁺ depletion transients "Ca²⁺ scraps."¹¹⁰ Importantly, there is no detectable intraluminal [Ca²⁺] gradient between the junctional SR at the TT region (the release site) and the longitudinal SR (the uptake site).¹¹⁰ This suggests a rapid translocation of Ca²⁺ inside the SR network and challenges local Ca²⁺ depletion as the mechanism underlying the termination of Ca²⁺ sparks.^111,112 Direct visualization of local SR Ca²⁺ during a Ca²⁺ spark should be informative in elucidating Ca²⁺ spark termination mechanism as well as SR luminal Ca²⁺ regulation.

View larger version (80K): [in this window] [in a new window]   Figure 6. SR Ca2+ release transients in rabbit cardiac myocytes. A, Staining of the SR by fluo-5N, as described by Shannon et al.110 SR retention of fluo-5N gives rise to the striated appearance. B, SR Ca2+ release transient elicited by an action potential at time marked by an arrow.

Albeit inconsequential to beat-to-beat EC coupling, Ca²⁺ trafficking across the mitochondrial membrane is crucial to noncontractile cardiac function, such as energy metabolism and Ca²⁺-mediated cell death. Using rhod-2AM loading of mitochondria, Pacher et al¹¹³ demonstrated single mitochondria [Ca²⁺] transients, dubbed "Ca²⁺ marks," in cardiac myotubes differentiated from H9c2 cells. Ca²⁺ marks are thought to be triggered by RyR Ca²⁺ sparks in close proximity to the mitochondria, but they usually outlast the trigger sparks by hundreds of milliseconds. The relevance and significance of Ca²⁺ marks, and the exact relationship between Ca²⁺ sparks and Ca²⁺ marks, remain to be established in native cardiac myocytes.

	Physiological Significance of Microdomain Ca2+

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
As mentioned earlier, the demonstration of dynamic microdomain Ca²⁺ in living cells has reshaped our understanding of cellular Ca²⁺ signaling. In this section, we would like to present several prime examples of this to illustrate general Ca²⁺ signaling principles revealed by imaging microdomain Ca²⁺.

CICR Paradox
CICR as the control mechanism of SR Ca²⁺ release was once thought to be paradoxical. Because CICR is intrinsically a positive feedback mechanism, it is expected to operate in an all-or-none fashion, unless the amplification gain is sufficiently low.¹¹⁴ In contrast, cardiac EC coupling is characterized by both high-gain amplification (10 at 0 mV) (Figure 3C)^81,89 and a smoothly-graded response to the trigger I_Ca (Figure 3B).^{81,89,115–117} To solve this CICR paradox, early experimental¹¹⁸ and theoretical work¹¹⁴ challenged the wisdom of the "common pool" model and invoked the "local control theory" of CICR. This theory consists of four postulates, each of which has now been validated experimentally.

(1) RyRs in situ are relatively insensitive to physiological levels of global Ca²⁺. The rate of occurrence of spontaneous Ca²⁺ sparks is about 100 sparks per cell per second.⁶ Provided that 10⁶ RyRs are present in a single myocyte,⁵ this translates into an opening rate of only 10^–4 s^–1 for RyR at the resting [Ca²⁺] of 100 nmol/L. At the cellular level, a sudden uniform increase in cytosolic [Ca²⁺] produced by photolysis has also shown that global CICR is intrinsically a low-gain amplification system.¹¹⁸ (2) Yet, RyRs are effectively activated by local trigger Ca²⁺. Indeed, individual Ca²⁺ sparks can be evoked by LCC Ca²⁺ sparklets.^75–77 The peak rate of LCC-triggered spark activation was estimated to be on the order of 10⁶ sparks per cell per second.³³ That is, cardiac CICR manifests both high-gain (when triggered by local high [Ca²⁺]) and low-gain behavior (when triggered by global low [Ca²⁺]). Additionally, owing to channel "adaptation"¹¹⁹ or inactivation,⁸⁶ RyR is highly responsive to pulse rather than sustained Ca²⁺ signals.^119,120 The initial responsiveness of RyRs strongly depends on the [Ca²⁺] raised to the power of 2 or higher.^37,120 These provide critical mechanisms for a CRU to discriminate between local trigger Ca²⁺ pulses and the ambient Ca²⁺. (3) There is little or no communication between discrete CRUs under physiological conditions. The discreteness of Ca²⁺ sparks confirms that SR Ca²⁺ release does not always initiate spatially regenerative CICR. Cellular and molecular factors that contribute to the uncoupling between CRUs include spatial segregation, steep [Ca²⁺] gradients at a point source, and strong Ca²⁺ buffering of the cytoplasm. (4) There is a robust mechanism terminating CICR within a CRU. The brevity of Ca²⁺ sparks and the negative regulation of spark rise time by spark Ca²⁺ release flux^76,77 are suggestive of a strong negative feedback or autoregulation in a CRU. However, the exact nature of spark termination mechanism remains elusive⁹⁰ and merits future investigation.

Opposing Roles of Ca²⁺ in Regulation of Smooth Muscle Contraction
In arterial smooth muscle myocytes, an increase in global [Ca²⁺] is expected to initiate muscle contraction, as is the case in all types of muscles. Paradoxically, inhibition of Ca²⁺ sparks causes vessel constriction.⁴⁸ This is because subsurface Ca²⁺ sparks are associated with hyperpolarizing spontaneous transient outward currents (STOCs)¹⁰⁶ due to activation of clusters of BK_Ca channels (Figure 7).⁴⁸ The resultant membrane hyperpolarization shuts off tonic Ca²⁺ entry through the voltage-operated LCCs. Therefore, the net result of the spark-STOC coupling is a reduction in global [Ca²⁺] and vessel relaxation.

View larger version (23K): [in this window] [in a new window]   Figure 7. Ca2+ spark-STOC coupling in arterial smooth muscle cells. Perforated patch-clamp technique and confocal microscopy were applied for simultaneous recording of Ca2+ sparks and STOCs in smooth muscle cells. A, Ca2+ sparks in rat mesenteric arteriole smooth muscle myocytes. B, Ca2+ sparks evoke STOCs through large-conductance Ca2+-activated K+ channels (IBK). Inset, Membrane potential was held at –40 mV and the scan line was placed right beneath the patch membrane.

Local Activation of High-Threshold Ca²⁺ Signaling
It is now appreciated that cardiac RyRs and smooth muscle BK_Ca channels are intrinsically insensitive to the levels of [Ca²⁺] attainable in the bulk cytosol, as are the processes of hormone secretion and neurotransmitter release. They would remain quiescent if they were not localized to high [Ca²⁺] microdomains. To this end, the aforementioned 3-dimensional grid of CRUs and near-membrane high [Ca²⁺] compartments would serve as the primary system for high-threshold Ca²⁺ signal transduction.

	Perspectives

Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
In spite of the many advances in microdomain Ca²⁺ imaging, there are new emerging examples of physiologically relevant Ca²⁺ signaling mechanisms concealed in nanodomains (1 to 100 nanometers). In this regard, a fundamental limitation of the optical approach is its spatial resolution, which is 200 nm at best (as in TIFRM). Visualization of nanodomain Ca²⁺ would require supraoptical resolution, which demands new and innovative approaches. A possible strategy might involve tethering or docking Ca²⁺ indicators to molecularly defined locations through molecular and genetic manipulations.

It is predictable that next generation of Ca²⁺ indicators and imaging methods will be called on to perfect the science and technology of cellular Ca²⁺ imaging. Likewise, new areas of cellular Ca²⁺ signaling will be vigorously explored in the near future. In particular, the targeted expression of protein-based Ca²⁺ indicators to subcellular compartments,^121,122 in conjunction with time-lapsed Ca²⁺ imaging, holds promise for uncovering new dimensions of the space-time architecture of cellular Ca²⁺ dynamics. Moreover, these approaches should be useful in elucidating roles of Ca²⁺ in long-term regulation of noncontractile cellular functions (eg, myocyte differentiation, growth, hypertrophy, migration, and apoptosis). We eagerly await the grander horizons that lie ahead.

	Acknowledgments

 
This work was supported by NIH intramural research program (H.C.), NIA STAR Award (S.-Q.W.), National Natural Science Foundation of China (G.Z., D.Y., H.C.), and the Major State Basic Research Development Program of China (H.C.). We thank Drs Jeffery Froehlich, Edward Lakatta, and Rui-Ping Xiao for critical reading of this manuscript and Drs Eduardo Rios and Hui Zou for valuable suggestions.

	Footnotes

 
Original received November 25, 2003; resubmission received February 6, 2004; revised resubmission received February 25, 2004; accepted March 2, 2004.

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Top Abstract Introduction Ca2+ Gradients in Living... Imaging the Elementary... Subspark and Sparkless Ca2+... Morphometric Analysis of... Cellular Organization of Ca2+... Ca2+ Spikes: Visualization of... Optical Recordings of Ca2+... Optical Single-Channel... Ca2+ in Subcellular Compartments Physiological Significance of... Perspectives References

 
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