# Signal Transduction and Smooth Muscle

**Edited by Mohamed Trebak Scott Earley**

ISBN: 978–1–4987–7422–2 (hbk) ISBN: 978–1–315–15458–9 (ebk)

First published 2019

## **Chapter 9**

#### **Mitochondria Structure and Position in the Local Control of Calcium Signals in Smooth Muscle Cells**

*John G. McCarron, Christopher Saunter, Calum Wilson, John M. Girkin, and Susan Chalmers*

(CC BY 4.0)

## 9 Mitochondria Structure and Position in the Local Control of Calcium Signals in Smooth Muscle Cells

*John G. McCarron, Christopher Saunter, Calum Wilson, John M. Girkin, and Susan Chalmers*

## **CONTENTS**


## **9.1 INTRODUCTION**

Features of Ca2+ signals including the amplitude, duration, frequency and location are encoded by various physiological stimuli. These features of the signals are decoded by cells to selectively activate smooth muscle functions that include contraction and proliferation [1–3]. Central, therefore, to an appreciation of how smooth muscle is controlled is an understanding of the regulation of Ca2+. In smooth muscle, Ca2+ signals arise from two major sources. The first is the extracellular space from which Ca2+ enters the cell via channels such as voltage-dependent Ca2+ channels, store-operated Ca2+ (SOC) channels and various members of the transient receptor potential channel family. The second major Ca2+ source is the internal Ca2+ store (sarcoplasmic reticulum; SR) [4–6]. The SR accumulates Ca2<sup>+</sup> using sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) and Ca2+ is released from the SR via the ligand-gated channel/receptor complexes, the IP3 receptor (IP3R) and ryanodine receptor (RyR). Release of Ca2+ via IP3R is activated by

IP3 generated in response to many G-protein or tyrosine kinase-linked receptor activators including drugs [7,8]. RyR may be activated pharmacologically (e.g., caffeine), by Ca2+ influx from outside the cell in the process of Ca2+-induced Ca2<sup>+</sup> release (CICR), or when the stores Ca2+ content exceeds normal physiological values, that is in store overload [2,9–12].

Activation of either Ca2+ influx or Ca2+ release results in an increase of the cytoplasmic Ca2+ concentration ([Ca2+]c) from the resting value of ~100 nM to ~1 μM for many seconds throughout the cell, and transiently (e.g., 100 ms) to much higher values (e.g., 50 µM) in small parts of the cytoplasm close to sites of influx or Ca2<sup>+</sup> release. These local Ca2+ signals begin with the opening of one or a few channels, allowing a large flux of the ion into the cytoplasm. Influx to the cytoplasm via voltage-dependent Ca2+ channels occurs at rates of ~0.6 million Ca2+ ions per second per channel (0.2 pA current). The influx generates a significant local concentration gradient near the channel in which [Ca2+] declines from ~10 μM to ~100 nM over a few hundred nanometers from the plasma membrane [2,13–17]. Voltage-dependent Ca2+ channel open time is brief (~1 ms) and the gradient dissipates rapidly with rates of change in the subplasma membrane space on the order of ~5000 μM s−1 [2] as compared to a much slower rate of ~0.5 μM s−1 in the bulk cytoplasm [2] after a global [Ca2+] rise [18,19]. The large difference in rate of decline in the subplasma membrane space and bulk cytoplasm arise because local changes are driven mostly by buffering and diffusion while the slower rate of decline in bulk cytoplasm is determined by pumps. High local [Ca2+] and the rapid rates of change near channels may target processes with rapid Ca2+ binding kinetics to selectively activate particular functions [20–23]. The high local [Ca2+] signals arising from influx also, in turn, may activate IP3R or RyR to amplify the local signals or propagate through the cell as global signals with slower but more widespread effects [24–30]. The transition of signals from those involving single to multiple channels and from local to global Ca2+ increases creates a multitude of signals with various locations, magnitudes and time courses [31–34] so that various cellular biological responses may be selectively activated.

It is acknowledged that a major way that Ca2+ signaling specifically targets particular biological processes is by increases in concentration of the ion being selectively localized to certain regions of the cell (Figure 9.1) [36,37]. In native smooth muscle cells, mitochondria contribute to the localization of Ca2+ signals and to the modulation of the amplitude of Ca2+ signals [38–42]. Mitochondria regulate these local signals by the organelles' ability to take up and release the ion. Ca2+ uptake occurs through the mitochondrial Ca2+ uniporter while efflux is mediated by the mitochondrial Na+/Ca2+ exchanger. Mitochondrial Ca2+ uptake and efflux may regulate cytoplasmic Ca2+ concentrations both directly and indirectly. Direct regulation occurs by alteration of bulk Ca2+ levels (Figures 9.2 and 9.3) [18,40,44–47]. Indirect regulation occurs as a result of mitochondrial influence on the activity of SR or plasma membrane Ca2+ channels. This chapter describes how the structure and positioning of mitochondria contribute to the control of Ca2+ signaling, including a previously unrecognized ability of the position of the organelles to increase local Ca2+ entry via voltage-dependent Ca2+ channels.

**FIGURE 9.1** Simultaneous wide-field epi-fluorescence and total internal reflection fluorescence (TIRF) [Ca2+] measurements in a voltage-clamped smooth muscle. Depolarization (−70 to +10 mV; e), activated a voltage-dependent Ca2+ current (ICa; d) to evoke a rise in [Ca2+] in both the subplasma membrane space (c) and bulk cytoplasm (b). The rise in [Ca2+] that occurred in the subplasma membrane space (measured by TIRF) (c) was more rapid in onset than that seen in the bulk cytoplasm (measured by wide-field epi-fluorescence) (b). Changes in the fluorescence ratio with time (b and c) are each derived from precisely the same 2 × 2-pixel boxes (regions 1–6 in (a), middle and right (expanded) panel; drawn at a 3 × 3-pixel size to facilitate visualization) and from a larger region encompassing the entire TIRF region (region 7). Significantly, while the cytosolic [Ca2+] increase that occurred in the bulk cytoplasm (b) was approximately uniform and simultaneous throughout the cell, those in subplasma membrane space (c) had a wide range of amplitudes and various time courses. Insets in (b) and (c) show the rising phase of the transients on an expanded time base. Black dotted lines are the average responses. (Reproduced from McCarron, J.G. et al., *J. Gen. Physiol*., 133, 439–457, 2009. With permission.)

**FIGURE 9.2** Preventing mitochondrial Ca2+ uptake does not alter the initial upstroke or peak cytosolic [Ca2+] after depolarization-evoked Ca2+ entry. Depolarization (−70 to 0 mV; b) of a voltage-clamped single smooth muscle cell activated a voltage-dependent Ca2+ current (not shown) and an increased cytosolic [Ca2+] (a). After carbonyl cyanide *m*-chlorophenyl hydrazone (CCCP; 5 µM; transients in red lines) neither the initial rate of cytosolic [Ca2+] rise (see inset) or peak cytosolic [Ca2+] achieved were significantly altered. The insets show the transients on an expanded time base. Cytosolic [Ca2+] was measured at a frequency of 50 Hz using the membrane-impermeable fura-2 (potassium salt, 50 μM) introduced into the cell from the patch pipette. (Reproduced from McCarron, J.G. et al., *Pflugers Arch*., 464, 51–62, 2012. With permission.)

#### **9.2 ROLE OF MITOCHONDRIA IN Ca2**+ **SIGNALING**

Mitochondria are major hubs for cellular signaling. In smooth muscle, mitochondria control contractility, proliferation, and growth through regulation of cytoplasmic Ca2+ concentrations. A critical feature of mitochondria's ability to control Ca2<sup>+</sup> signaling is the position and structure of the organelles within cells. Understanding of the precise relationship between Ca2+ signaling and mitochondrial structure and position in living, fully-differentiated, (native) cells is preliminary, which may be due to the uncertainty in mitochondrial structure in these cells. Most studies describing mitochondrial structure are derived from cells maintained in culture conditions. Yet, the structure and arrangement of mitochondria in cultured cells differ substantially from the organelles in fully differentiated smooth muscle cells [48–51]. In cultured cells the position of mitochondria contributes significantly to regulation of cytoplasmic Ca2+ signals and to the control of mitochondrial Ca2+ concentration ([Ca2+]m). For example, SOC increased the [Ca2+]m in the cultured endothelial cell culture line ECV304, suggesting the organelles accumulate this Ca2+ influx. In ECV304 cells, 14% of mitochondria are positioned close to the plasma membrane, a position that will facilitate Ca2+ uptake. Less than 4% of mitochondria are close to the internal

**FIGURE 9.3** Mitochondria contribute to cytosolic [Ca2+] decline following voltage-dependent Ca2+ entry in smooth muscle. (a) Depolarization (−70 to 0 mV) activated a voltage-dependent Ca2+ current (not shown) and increased cytosolic [Ca2+]. Carbonyl cyanide *m*-chlorophenyl hydrazone (CCCP; 5 μM) slowed the rate of decline of cytosolic [Ca2+] on repolarization compared with control. (b) The rate of decline of the two transients (a) is shown in (b). The derivative (b) was obtained from high-order polynomial fits to the declining phase of the transient and shows a significant slowing when mitochondria were prevented from accumulating Ca2+. (c) A summary of the rates of decline for ten cells in the presence and absence of CCCP. The inferred mitochondrial contribution to the decline of cytosolic [Ca2+] (red line) was obtained by subtracting control rates from those seen in CCCP and shows that mitochondrial Ca2+ uptake occurred above 200 nM [Ca2+]c. (Reproduced from McCarron, J.G. et al., *J. Physiol.*, 516: 149–161, 1999. With permission.)

store and store release of Ca2+ did not increase [Ca2+]m. Conversely in HeLa cells, where 65% of mitochondria are close to the internal store and <6% are in close proximity to the plasma membrane, store release of Ca2+ caused a much greater increase in [Ca2+]m than did SOC [52]. These observations suggest mitochondrial position is critical in determining the organelles' uptake of Ca2+. In turn, the position of the mitochondria regulates SOC [53,54]. Forced relocation of mitochondria away from

the cell periphery and towards the nucleus (by overexpressing dynamitin, a protein involved in mitochondrial movement) decreased SOC following store depletion in HeLa cells [55]. These results point to the position of mitochondria as being critical in regulation of Ca2+ signaling.

In native fully-differentiated cell types the distribution of mitochondria may also regulate Ca2+ signaling. For example, in fully-differentiated cardiac myocytes or neurons, mitochondria appear to be located particularly close to sites of initiation of Ca2+ signals from voltage-dependent Ca2+ channels on the plasma membrane [56]. At these sites mitochondria contribute to the clearance of Ca2+ from the mouth of the channel, limiting Ca2+-dependent inactivation and thus prolonging channel open time [57,58] and facilitating Ca2+ entry. In smooth muscle, however, mitochondrial Ca2+ uptake does not appear to alter the kinetics of voltage-dependent Ca2+ entry (Figure 9.2). This observation suggests the gating of voltage-dependent Ca2+ channels is not altered by mitochondria. Notwithstanding the absence of a direct effect on voltage-dependent Ca2+ channels, mitochondria may modulate Ca2+ rises arising from voltage-dependent Ca2+ entry in smooth muscle by modulating Ca2+-dependent feedback processes operative on other ion channels. Mitochondria appear to increase clearance of bulk cytoplasmic Ca2+ concentrations, and as a result decrease the activity of Ca2+-activated chloride channels (ClCa) [59] and increase the activity of Ca2+-activated K+ channels (KCa) [60]. Pharmacological inhibition of the ability of mitochondria to remove Ca2+ from the cytoplasm prolonged the activity of ClCa in portal vein smooth muscle cells [59] and inhibited KCa currents in cerebral artery smooth muscle [60]. Modulation of ClCa or KCa will alter the membrane potential to then regulate Ca2+ influx via voltage-dependent Ca2+ channels as a result.

By taking up Ca2+, mitochondria modulate the time course and amplitude of Ca2<sup>+</sup> signals to shape the resulting message [1]. For example, we have shown that the Ca2<sup>+</sup> transient arising from activation of voltage-dependent Ca2+ channels has an accelerated rate of decline as a consequence of mitochondrial Ca2+ uptake; when uptake is inhibited, the rate of Ca2+ decline is substantially slowed (Figures 9.2 and 9.3). In the example shown in Figure 9.3, mitochondria modulate Ca2+ signals over the cytosolic [Ca2+] range 600 nM–200 nM, which demonstrates that mitochondria have a high affinity for Ca2+ (in the sub-micromolar range; Figure 9.3). Mitochondrial Ca2+ uptake does not alter the rate, or extent, of Ca2<sup>+</sup> *influx* via voltage-dependent Ca2+ channels [1] (the likely reasons for this are discussed later on). Some studies have shown that increased mitochondrial reactive oxygen species (ROS) production can alter voltage-dependent Ca2+ influx [61], however, this is likely to be due to ROS-dependent alterations in channel open time or conductance, rather than altered mitochondrial Ca2+ uptake during voltage-dependent Ca2+ influx.

Mitochondria also have the capacity to modulate Ca2+ signals that are ~2 orders of magnitude larger than the global Ca2+ transient arising from voltage-dependent Ca2<sup>+</sup> channels and in the tens of micromolar range [62]. One notable example in smooth muscle is mitochondrial regulation of IP3-mediated Ca2+ signals that arise from the activity of a single IP3R cluster (*Ca2*+ *puffs*; Figure 9.4). When mitochondria are prevented from taking up Ca2+ (using uncouplers, complex I inhibitors or uniporter inhibitors), the upstroke of Ca2+ puffs is inhibited [42] (Figure 9.4). The duration of the upstroke of a puff is determined by the open time of IP3R in the cluster.

**FIGURE 9.4** Preventing mitochondrial Ca2+ uptake by depolarizing ΔΨ*m* with CCCP/ oligomycin inhibits Ca2+ release from IP*3*R clusters (Ca2+ puffs). At −70 mV, locally photolyzed caged InsP3 (25 µM) (↑, C) in a ~20 µm diameter region (a; bright spot in left-hand panel, see also whole cell electrode, left side) evoked Ca2+ puffs in an EGTA (300 µM) buffered smooth muscle cell (b, c). Note: There are two individual Ca2+ puff sites in response to photorelease of InsP3; one site releases Ca2+ just before the other site. Flash photolysis of InsP3 every ~60 s generated approximately comparable cytosolic [Ca2+] increases. (c) Superfusion of carbonyl cyanide *m*-chlorophenyl hydrazone (CCCP) and oligomycin (1 µM and 6 µM, respectively) while continuing to photolyze InsP3 at ~60 intervals, decreased the amplitude of InsP3 mediated Ca2+ puffs (b, c). The cytosolic [Ca2+] images (b) are derived from the time points indicated by the corresponding numbers in (c). Cytosolic [Ca2+]c changes in (b) are expressed by color; dark blue low and light blue high cytosolic [Ca2+]. Measurements were made from a single 3 × 3-pixel box (a; right hand panel, white square). The large increase in fluorescence at time 0 is the flash artefact triggering photolysis of caged IP3. (Reproduced from Olson, M.L. et al., *J. Biol. Chem.*, 285, 2040–2050, 2010. With permission.)

The observation [42] that mitochondria modulates the puff upstroke suggests that the organelle's uptake is fast enough to modulate Ca2+ concentration near IP3R clusters during the channels open time, requiring mitochondria to have a very low affinity for Ca2+ (Ca2+ puffs >10 µM) and to be positioned close to the channel.

Electron microscopy studies of porcine tracheal smooth muscle cells suggest that 99% of mitochondria were within 30 nm of SR and 48% of the mitochondrial outer membrane was within 30 nm with the SR [63] with some mitochondria fully ensheathed by SR [63,64]. Given the proximity between sites of Ca2+ release and mitochondria, the question arises as to whether or not there is a direct intermolecular link between the two structures. While there are no high-resolution studies of this link in smooth muscle, electron tomographs [65] and transmission electron micrographs [66] in other cells show tethers that connect the endoplasmic reticulum (ER) to the mitochondrial outer membrane that may be significant in Ca2+ signaling. Indeed, immunocytochemical studies show that those regions of the ER in close

proximity to mitochondria are also enriched with IP3R to create *hotspots* for the transfer of Ca2+ from the ER to mitochondria [62]. Several molecular candidates for the tethers have been identified in cell types other than smooth muscle cells. The IP3R itself has been shown to be tethered to the mitochondrial voltage-dependent anion channel (VDAC) via the chaperone glucose-regulated protein 75 (GRP75), an interaction that promotes mitochondrial accumulation of Ca2+ released by IP3R [67]. Interestingly, both *in vitro* treatment of epithelial cells with high glucose and longterm hyperglycemia in a mouse model of diabetes reduced GRP75 levels, reducing ER-mitochondrial contact sites [68]. The protein mitofusin-2 is located on both mitochondrial and ER membranes and is proposed to form homodimeric tethers between the organelles [69] that contribute to Ca2+ signaling. Disrupting linkages between the SR and mitochondria by gene silencing of mitofusin-2 decreased mitochondrial Ca2+ uptake during IP3-mediated [Ca2+]c rises [65,69]. The multifunctional sorting protein PACS-2, which is localized to the ER and may play a role in apoptotic pathway initiation, has also been proposed to be important for maintaining close apposition of ER and mitochondria in the A7 astrocyte cell line [70]. siRNA silencing of PACS-2 caused mitochondrial fragmentation and separation from the ER, altering Ca2+ signaling by increasing IP3-mediated Ca2+ release [70]. The sigma-1 receptor, a protein involved in cell stress responses and present on the ER at regions in close contact with mitochondria, may also couple the membranes of the two organelles [71]. The sigma-1 receptor may form a complex with another chaperone protein (BiP) to stabilize IP3R at regions of ER-mitochondrial proximity. Depletion of ER Ca2+ dissociates the sigma-1 receptor from BiP, leading to prolonged Ca2+ signaling into mitochondria via IP3Rs [71].

The question arises: how can mitochondria—by removing Ca2+ from the cytoplasm (i.e., lowering [Ca2+])—generate a larger cytoplasmic [Ca2+] rise? IP3R are regulated by Ca2+-dependent positive and negative feedback mechanisms [72,73]. By removing Ca2+, mitochondria may limit a negative feedback inhibition of Ca2+ on IP3R [42]. There are at least two types of Ca2+-dependent negative feedback mechanisms that may deactivate smooth muscle IP3R. In the first, a Ca2+-dependent deactivation of IP3R occurs at cytosolic [Ca2+] exceeding ~300 nM [73]. The onset is rapid and the deactivation is persistent and lasts for ~5 s after the cytosolic [Ca2+] increase has ended [74]. Another form of Ca2+-dependent deactivation of IP3R, once initiated by an increased cytosolic [Ca2+], persisted for tens of seconds after cytosolic [Ca2+] had regained resting values, that is, IP3R became, at least partially, refractory [30,75]. Each of these processes would persistently restrict Ca2+ release via IP3R. Mitochondrial Ca2+ uptake, by buffering the Ca2+ rise at IP3R, presumably prevented a persistent deactivation of IP3R that would otherwise limit the overall release of Ca2+ [30,75].

The control that mitochondria exert on Ca2+ puffs enables the organelle to exert particularly dramatic effects on the global Ca2+ events such as Ca2+ waves and oscillations. When mitochondria are prevented from taking up Ca2+, waves and oscillations halt [42,45], that is, modulation of the time course of a Ca2+ rise by the organelle determines whether or not some Ca2+ signals occur at all. The position and structure of the organelles, together with nano-architectural features of SR-mitochondria junctions, is critical in determining precisely how mitochondria regulate local and global Ca2+ signals.

## **9.3 MITOCHONDRIA STRUCTURE IN NATIVE CELLS**

The precise structure and position of mitochondria have been studied most extensively in cultured smooth muscle cells because of the relative ease that the organelles can be visualized in these cells. The thin axial depth of cultured cells and ability to transfect the cells to express unique, mitochondrially-targeted fluorophores result in exceptional signal to noise for imaging mitochondrial structure. In cultured cells, mitochondria exist in a wide range of sizes and shapes (Figure 9.5) and the organelle may change rapidly from solitary ovoid shapes to extensive branched networks and

**FIGURE 9.5** Mitochondrial structures in cultured and fully-differentiated smooth muscle cells. (a) A cultured single vascular smooth muscle cell showing the arrangement of mitochondria. The organelle is scattered through the cytoplasm and is arranged in various orientations. Mitochondria were labeled with the mitotracker green. (b) Example mitochondria showing the diverse phenotypes which include small spheres, swollen spheres, straight rods, twisted rods, branched rods, and loops. (a and b reproduced from McCarron, J. et al., *J. Vasc. Res.*, 5, 357–371, 2013. With permission.) (c) A native fully-differentiated smooth muscle cell from a cerebral resistance artery showing the arrangement of mitochondria as revealed by the fluorophore tetramethylrhodamine, ethyl ester (TMRE). The organelle is distributed throughout the cytoplasm and appears to be mainly organized parallel to the long axis of the cell. However, the significant axial depth of the cells (when compared to cultured smooth muscle) and multiple overlapping fluorescence point sources result in an optically-confused image with structures that are hard to interpret (d) FaLM permits the size, shape, and position of mitochondria to determined and shows the organelles comprise mainly spheres and short rods. The inset between c and d shows the same example region of the cell before and after application of FaLM. (c and d modified from Chalmers, S. et al., *Nat. Sci. Rep.*, 30, 2000–2013, 2015. With permission.) Scale bars = 10 µm.

even single continuous mitochondrial structure throughout the cell [62,76,77]. This almost continuous re-shaping creates a diversity of structures (Figure 9.5), presumably each with different physiological roles although the precise functions are not yet fully understood [51,78]. On the other hand, mitochondria within fully-differentiated live smooth muscle cells are difficult to visualize because of the significant axial cell depth. This depth results in multiple confused overlapping fluorescence point sources at different distances through the cell derived from various mitochondria. The overlapping fluorescence is difficult to interpret and may be single large organelles or multiple small mitochondria. Exclusive fluorophore targeting is also more difficult in native cells than in cultured cells. Whether or not the arrangements of mitochondria seen in cultured cells occur in fully-differentiated cells is uncertain.

Live cell imaging is required to appreciate the precise relationship between position and structure of mitochondria and the control of Ca2+ signaling in fully-differentiated cells. Confocal or other optical sectioning imaging methods do not provide sufficient resolution to determine detailed structure of the organelle in fully-differentiated cells. Detailed insights into the structure of individual mitochondria at fixed points in time have been provided by electron microscopy [EM; 79]. However, EM cannot easily resolve the entire cellular mitochondrial complement or provide information on dynamic changes or functional connectivity and is not compatible with Ca2+ imaging. EM [80] and super-resolution techniques [81–89] do not unambiguously reveal the extent of the electrically-continuous inner mitochondrial membrane and are less suited to simultaneously studying Ca2+ signaling in live cells, nor do they currently have the imaging speed frequently required.

We recently developed a technique to determine the structure of individual, electrically-discrete mitochondria in live, fully-differentiated cells using conventional fluorescence imaging. Our approach is rapid and quantifies the shape, size and location of mitochondria even when the organelles are densely clustered. The approach measures transient changes in the membrane potential that are unique to each mitochondrion to determine their individual shapes. Changes in membrane potential (ΔΨm) arise from transient openings of the mitochondrial permeability transition pore [mPTP; 90,91] and can be visualized using a rapidly-repartitioning cationic fluorophore such as TMRE, as *flickers* of fluorescence [92,93]. These flickers demarcate individual, electrically-discrete mitochondria [92,94]. Drawing inspiration from PALM/BaLM super-resolution techniques [81,82], we used these flicker events to extract structural information of each organelle, facilitating a more detailed analysis of the live-cell fluorescence images. These mitochondrial flickers differ from the correlation of photo-activation, bleaching, or blinking in PALM/BaLM because the flickering objects are not single molecule emitters with a well-defined point-spread function but extended organelles of unknown shape. Nonetheless, the size, shape, and position of the mitochondria can be rapidly determined to the conventional resolution limit from a pixel-by-pixel spatio-temporal covariance of the derived flickering fluorescence signals in an approach that we call Flicker-assisted Localization Microscopy (FaLM) [49,50].

Using FaLM, we found that mitochondria in fully-differentiated resistance artery smooth muscle cells were distributed through the cytoplasm as solitary, spherical-, or short rod-shaped entities (Figure 9.5) [50]. We found mitochondria had a median

area of 0.46 μm2, a median length of 0.9 μm, and a median length:width ratio of 1.5. We also found the median density of mitochondria in resistance artery smooth muscle cells was 0.125 mitochondria per μm2 and that mitochondria occupied 7.0% of the cell volume. Each mitochondrion had 2–5 immediate neighbors with ~2 µm between the organelle centers. The mean center distance of a mitochondria from the plasma membrane was ~2 µm [49]. Given a width of mitochondria of 0.6 µm, then, the edge distance of the organelle from the plasma membrane is 1.4 µm. The opening of a single voltage-dependent Ca2+ channel (0.2 pA current, mean open-time 1 ms) generates a high local [Ca2+] that decreases steeply with distance from the plasma membrane as the ion diffuses into an increasing hemispheric volume. At a distance of 1.4 µm from the plasma membrane the [Ca2+] arising from the opening of the channel is only ~5 nM above resting values (assuming a buffer power of 100). It is unlikely that this microdomain of Ca2+ will be significantly altered by mitochondria. Clearly, the local rise in [Ca2+], and potential contribution of mitochondria, will increase if more than one channel opens or if mitochondria position is different as occurs in some other cell types.

## **9.4 MITOCHONDRIAL POSITION AND SIGNALING VIA VOLTAGE-DEPENDENT Ca2+ CHANNELS**

Mitochondria situated immediately at sites of Ca2+ influx or release into the cytoplasm may form part of subcellular Ca2+-signaling microdomains and have a privileged access to Ca2+ from these sites [38,42,44,62,95]. While, in smooth muscle, Ca2<sup>+</sup> influx via voltage-dependent Ca2+ channels appears not to be tightly controlled at a local level by mitochondria (Figure 9.2) [44,45], nonetheless, the sites of mitochondria near the plasma membrane are associated with *larger* Ca2+ signals than elsewhere in the plasma membrane (Figure 9.6). Thus, a single voltage-clamped cerebral resistance artery smooth muscle cell, activated with a depolarizing pulse (−70 to 0 mV) responded with voltage-dependent Ca2+ entry and a rise in cytoplasmic Ca2<sup>+</sup> concentration (Figure 9.6). In this same cell, mitochondrial position was determined (Figure 9.6) using the ΔΨm-sensitive dye TMRE (see Section 9.3). Interestingly, the amplitude of the local rise in cytoplasmic Ca2+ concentration was largest at these sites close to mitochondria (Figure 9.6).

After the depolarization ended, at precisely the same sites, there was a substantial undershoot in the cytoplasmic Ca2+ concentration when compared to regions away from mitochondria (Figure 9.6). These results suggest that mitochondria may regulate (increase) voltage-dependent Ca2+ entry and aid the removal of Ca2+ from the cytoplasm positioned close to the plasma membrane.

The upstroke of the voltage-dependent Ca2+ transient is unaltered when mitochondria are prevented from taking up Ca2+ (Figure 9.2), which suggests that mitochondria are not regulating channel gating. The question arises as to how do mitochondria regulate voltage-dependent Ca2+ entry? One possibility is that mitochondria are not involved in the short-term regulation of Ca2+ entry but rather in the longer-term level of either expression or distribution of the voltage-dependent Ca2+ channel. IP3 mediated Ca2+ release has been shown to induce mitochondrial-ROS generation,

**FIGURE 9.6** Mitochondrial position and the magnitude of [Ca2+] changes arising from voltage-dependent Ca2+ entry. Depolarization (−70 to 0 mV; b), of a single voltage-clamped smooth muscle cell (a—upper left panel) activated a voltage-dependent Ca2+ current (not shown) and rise in [Ca2+] (a—upper right panel, b, and c). The rise in [Ca2+] declined after the depolarization ended. Mitochondrial position was determined at the same time by labeling the organelles with TMRE (a—lower left panel). The magnitude of the rise in [Ca2+] throughout the cell (b and c) was compared on a pixel-by-pixel basis to the center of mass of each nearest mitochondrion (a—lower left and right panels). The rise in [Ca2+] was largest close to a mitochondrion (c and d). In (c) the Ca2+ signal in each pixel is plotted as a function of time (x-axis; same scale as b) and distance to the nearest mitochondrion (left y-axis). The magnitude of the [Ca2+] is colored—red high, blue low. After the depolarization had ended, (b) there was an undershoot in [Ca2+] at those regions closest to a mitochondrion (c; shown as darker blue regions and indicated by the dotted lines). (d) Plots the pixel [Ca2+] as a function of distance from the nearest mitochondrion (0 on the x axis is the mitochondrial position) at 4 time points indicated by the arrows from (c). The shaded regions highlight the differences in Ca2+ signals close (darker gray) and further (lighter gray) from a mitochondrion. Those regions that are closest to mitochondria experience the highest [Ca2+] concentration after a depolarization and the greatest undershoot (indicated by the dotted lines) as [Ca2+] returns to resting values.

activate the transcription factor NF-kappaB, and stimulate voltage-dependent Ca2<sup>+</sup> channel transcription [96]. It is tempting to speculate that mitochondria may therefore control voltage-dependent Ca2+ channel expression following persistent entry via voltage-dependent Ca2+ channels. It is also tempting to speculate that the regional changes in Ca2+ (Figure 9.1) arising from voltage-dependent Ca2+ channel entry arise from mitochondrial control of the distribution of the channel. The localized decreases in Ca2+ near mitochondria also offer support to the proposed control mechanisms operating to regulate and maintain IP3-mediated Ca2+ release (Figure 9.4).

## **ACKNOWLEDGMENTS**

This work was funded by the Wellcome Trust (092292/Z/10/Z; 202924/Z/16/Z) and the British Heart Foundation (PG/16/54/32230; PG16/82/32439), whose support is gratefully acknowledged. CW is supported by a Sir Henry Wellcome Postdoctoral Research Fellowship (204682/Z/16/Z).

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