A "mix-and-match" approach to designing Ca(2+) microdomains at membrane-contact sites.

Ca(2+) microdomains are critical for regulating cellular activity and often form at membrane contact sites. Such sites between lysosomes and the ER potentially provide a platform for signaling by the Ca(2+) mobilizing messenger NAADP. However, at present we know little of how Ca(2+) release events are coordinated at these experimentally intractable junctions. We therefore developed a computational model of lysosome-ER microdomains, which suggested that small leaks of Ca(2+) from the lysosome couple to Ca(2+)-sensitive Ins(1,4,5)P 3 receptors on the ER to generate global, microdomain-dependent Ca(2+) signals. Here we discuss how the "mix-and-match" arrangement of different Ca(2+) signaling proteins on the "source" and "target" membranes might generate functionally heterogeneous Ca(2+) microdomains.

Membrane contact sites (MCS) are specialized structures, whereby cellular membranes are closely apposed (10–50 nm). Such an arrangement facilitates information flow between different cellular compartments. Ca2+ is a universal signaling currency that often makes use of MCS to direct cellular activity. The restricted space within MCS allows formation of local Ca2+ microdomains where the Ca2+ concentration ([Ca2+]) is thought to reach high, micromolar levels (~10 μM) levels. These microdomains facilitate activation of downstream Ca2+-dependent processes. A high [Ca2+] within the dyadic junction of cardiac cells, for example, couples excitation of the sarcolemma to cellular contraction, driven by Ca2+ release from the sarcoplasmic reticulum (SR). Similarly, high [Ca2+] microdomains at ER-mitochondria MCS drive excitation-metabolism coupling. Recently, MCS between lysosomes and the endoplasmic reticulum (ER) have been identified. These sites provide an anatomical basis for functional coupling of lysosomal Ca2+ stores with the ER through the Ca2+ mobilizing messenger, NAADP. NAADP is thought to act via 2-pore channels to evoke Ca2+ signals from acidic organelles that are then amplified by the ER to drive complex Ca2+ behavior., However, the function of lysosome-ER MCS and putative Ca2+ microdomains contained therein is currently unknown.

In our recent paper, we developed a model of Ca2+ dynamics within these putative lysosome-ER microdomains and the continuous bulk cytosol to simulate cellular Ca2+ responses. We modeled leaks of Ca2+ from the “source” lysosome membrane, such as those produced in response to NAADP. These leaks activated Ca2+-sensitive inositol trisphosphate receptors (Ins(1,4,5)P3Rs) on the “target” ER membrane and generated global Ca2+ responses that were microdomain-dependent. Interestingly, we also found that varying either the distribution or density of the lysosomal Ca2+ leak altered the mode of coupling. For example, selectively increasing the magnitude of the lysosomal Ca2+ leak into the microdomain, akin to channel clustering, broadened the concentration range over which NAADP produced responses. A uniform increase in Ca2+ leak density into both the microdomain and non-microdomain compartments, mimicking channel overexpression, also had a broadening effect. However, in these simulations, microdomains acted to modulate the frequency of global Ca2+ oscillations rather than initiate them. Lysosome-ER Ca2+ microdomains can either drive or shape complex Ca2+ signals, depending on their composition.

Unlike other microdomains,, the lysosome-ER microdomains simulated within our model did not achieve high [Ca2+]. Although kinetically distinct, Ca2+ signals in the microdomain were only marginally greater in amplitude than within the bulk cytosol. However, this result is not as counterintuitive as it may first seem. As Ca2+-release from Ins(1,4,5)P3Rs on the target membrane can be activated by relatively low [Ca2+], small leaks of Ca2+ from lysosomes can raise microdomain [Ca2+] sufficiently to initiate Ca2+-induced Ca2+-release via the Ins(1,4,5)P3R. This arrangement differs from other Ca2+ microdomains. For example, ryanodine receptors (RyRs) on the target membrane in the cardiac dyadic cleft have a lower affinity for Ca2+-induced activation than Ins(1,4,5)P3Rs (~0.8–1 μM, compared with ~0.1–0.3 μM). They therefore require higher [Ca2+] for activation to generate a high [Ca2+] microdomain. Moreover, the mitochondrial Ca2+ uptake machinery on the target membrane in ER-mitochondria microdomains has an even lower affinity for Ca2+ (estimated between 10–100 μM)., Thus, microdomain [Ca2+] may “match” Ca2+ affinity of target proteins.

The unexpectedly low microdomain [Ca2+] in our model was due to the presence of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pumps within the microdomains, which efficiently take up the released Ca2+. SERCA pumps were required to balance a basal Ca2+ leak through the Ins(1,4,5)P3R at a resting level of Ins(1,4,5)P3. This arrangement differs from other studies utilizing RyR models because, unlike Ins(1,4,5)P3Rs, RyRs can be gated directly by Ca2+. As such, these models contain no basal RyR leak and therefore do not require balancing Ca2+ uptake mechanisms within the microdomain. This allows for the accumulation of high [Ca2+] upon activation of the RyRs. Our hypothesis, that peak [Ca2+] within microdomains can be determined by the level of microdomain SERCA, may also extend to ER-mitochondria microdomains. Here, the ER acts as the source membrane whereby Ins(1,4,5)P3Rs create high microdomain [Ca2+] to facilitate low affinity mitochondrial Ca2+ uptake. It is therefore of note that Ins(1,4,5)P3Rs are enriched within ER-mitochondria MCS, while SERCA is not. The potential absence of SERCA within these microdomains might permit spontaneous activity within these junctions, potentially accounting for the proposed maintenance of mitochondrial bioenergetics by basally active Ins(1,4,5)P3Rs. Ca2+ signaling proteins may be “mixed” together to generate functionally heterogeneous microdomains.

Computational models of Ca2+ dynamics often have a modular design, whereby individual models for each Ca2+ transport process are assembled together to generate an appropriate, relevant system. This rule is consistent at different levels of computational complexity.,, We suggest that viewing “real” microdomains in this modular fashion can aid our understanding of their architecture in live cells. As discussed, the variable expression of individual Ca2+ channels and pumps within MCS, on either the source or target membrane, can profoundly alter the properties of microdomains. This “mix-and-match” approach may account for the functionally diverse behaviors that microdomains coordinate. (Fig. 1)

Figure 1. Heterogeneity of Ca2+ microdomains at membrane contact sites. MCS between the source (top) and target (bottom) membranes allow functional Ca2+ microdomains to form between them. Ca2+ influx through voltage gated Ca2+ channels (Cav) in the PM-SR MCS of the dyadic cleft (left) forms a high [Ca2+] microdomain (dark circle) to initiate Ca2+ release from low-affinity ryanodine receptors (RyR). Ca2+ release through inositol trisphosphate receptors (Ins(1,4,5)P3R) in ER-mitochondria MCS (center) also forms a high [Ca2+] microdomain to facilitate mitochondrial Ca2+ uptake by the low-affinity mitochondrial uniporter (MCU). Ca2+ release through 2-pore Channels (TPC) in lysosome-ER MCS (right) forms a low [Ca2+] microdomain (light circle) due to the presence of SERCA (S) but which is nevertheless able to activate high-affinity Ins(1,4,5)P3Rs.