Abstract:binding to CBD2 alleviates Na + -dependent inactivation. A combination of mutually exclusive (A, B) and cassette (C-F) exons in CBD2 raises functionally diverse splice variants through unknown mechanisms. Here, the effect of exons on CBDs backbone dynamics were investigated in the 2-domain tandem (CBD12) of the brain, kidney, and cardiac splice variants by using hydrogen-deuterium exchange mass spectrometry and stopped-flow techniques. Mutually exclusive exons stabilize interdomain interactions in the apoprote… Show more
“…This result reveals that the alternative splicing segment (Fig. 1, located on CBD2) affects the folding stability of CBD12 in the apo state and thus, may predefine distinct conformational responses of CBDs to Ca 2+ -binding in a given splice variant 6, 29, 31 .Figure 4Effect of alternative splicing on the conformational dynamics of NCX3-CBD12. Deuterium uptake differences between NCX3-AC and NCX3-B in the apo form ( A ) or in the Ca 2+ -bound form ( B ) are color-coded onto the model of NCX3-AC.…”
Section: Resultsmentioning
confidence: 91%
“…Therefore, it remains unclear how Ca 2+ -dependent NCX regulation is diversified between the different isoforms and splice variants. To better understand the differential conformational dynamics of NCX-CBD12 isoforms and splice variants, the isolated preparations of NCX2-CBD12 and NCX3-CBD12 proteins were analyzed in the apo and Ca 2+ -bound states by HDX-MS 29, 31 .…”
Section: Resultsmentioning
confidence: 99%
“…This population shift of conformational states is essential for regulation, thereby representing a common mechanism for NCX1 splice variants regardless of their regulatory specificity 28 . Finally, hydrogen-deuterium exchange mass-spectrometry (HDX-MS) analyses of NCX1-CBD12 splice variants have shown that Ca 2+ binding to all tested variants mainly rigidifies the backbone dynamics of CBD2 (and not that of CBD1), where the alternative splicing of CBD2 secondarily modifies the strength and expansion of rigidification throughout CBD2 29, 31 . Importantly, the Ca 2+ -induced effects on CBD2 backbone dynamics correlate well with the regulatory specificity found in the matching splice variants of full-size NCX1s 29, 31 .…”
Section: Introductionmentioning
confidence: 99%
“…Thus far, NCX1 splice variants share a common mechanism for the initial decoding of the regulatory signal upon Ca 2+ occlusion and tethering of CBDs (i.e., through the population shift mechanism), whereas alternative splicing of CBD2 controls the propagation and strength of Ca 2+ -dependent rigidification and thereby, diversifies the regulatory response 28, 29, 31 . Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant.…”
Section: Introductionmentioning
confidence: 99%
“…Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant. To overcome this gap, here we analyzed the apo and Ca 2+ -bound forms of isolated NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B proteins with SAXS 19, 28, 29 and HDX-MS 29, 31 in order to identify their conformational dynamics and the effect of alternative splicing. The present analysis reveals a general mechanism for regulatory coupling associated with Ca 2+ interactions with regulatory CBD domains of NCX1, NCX2, and NCX3 isoforms and their splice variants, where the dynamic effects of alternative splicing on conformational dynamics of the two-domain regulatory tandem (CBD12) control the contributions of a given isoform/splice variant to dynamic handling of cytosolic Ca 2+ oscillations in distinct cell types.…”
Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.
“…This result reveals that the alternative splicing segment (Fig. 1, located on CBD2) affects the folding stability of CBD12 in the apo state and thus, may predefine distinct conformational responses of CBDs to Ca 2+ -binding in a given splice variant 6, 29, 31 .Figure 4Effect of alternative splicing on the conformational dynamics of NCX3-CBD12. Deuterium uptake differences between NCX3-AC and NCX3-B in the apo form ( A ) or in the Ca 2+ -bound form ( B ) are color-coded onto the model of NCX3-AC.…”
Section: Resultsmentioning
confidence: 91%
“…Therefore, it remains unclear how Ca 2+ -dependent NCX regulation is diversified between the different isoforms and splice variants. To better understand the differential conformational dynamics of NCX-CBD12 isoforms and splice variants, the isolated preparations of NCX2-CBD12 and NCX3-CBD12 proteins were analyzed in the apo and Ca 2+ -bound states by HDX-MS 29, 31 .…”
Section: Resultsmentioning
confidence: 99%
“…This population shift of conformational states is essential for regulation, thereby representing a common mechanism for NCX1 splice variants regardless of their regulatory specificity 28 . Finally, hydrogen-deuterium exchange mass-spectrometry (HDX-MS) analyses of NCX1-CBD12 splice variants have shown that Ca 2+ binding to all tested variants mainly rigidifies the backbone dynamics of CBD2 (and not that of CBD1), where the alternative splicing of CBD2 secondarily modifies the strength and expansion of rigidification throughout CBD2 29, 31 . Importantly, the Ca 2+ -induced effects on CBD2 backbone dynamics correlate well with the regulatory specificity found in the matching splice variants of full-size NCX1s 29, 31 .…”
Section: Introductionmentioning
confidence: 99%
“…Thus far, NCX1 splice variants share a common mechanism for the initial decoding of the regulatory signal upon Ca 2+ occlusion and tethering of CBDs (i.e., through the population shift mechanism), whereas alternative splicing of CBD2 controls the propagation and strength of Ca 2+ -dependent rigidification and thereby, diversifies the regulatory response 28, 29, 31 . Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant.…”
Section: Introductionmentioning
confidence: 99%
“…Currently it is unclear to what extent (if at all) the above-described mechanisms are also valid for NCX2 and NCX3 splice variants and how the exons control the CBD dynamics as related to shaping the regulatory specificity in a given isoform/splice variant. To overcome this gap, here we analyzed the apo and Ca 2+ -bound forms of isolated NCX2-CBD12, NCX3-CBD12-AC, and NCX3-CBD12-B proteins with SAXS 19, 28, 29 and HDX-MS 29, 31 in order to identify their conformational dynamics and the effect of alternative splicing. The present analysis reveals a general mechanism for regulatory coupling associated with Ca 2+ interactions with regulatory CBD domains of NCX1, NCX2, and NCX3 isoforms and their splice variants, where the dynamic effects of alternative splicing on conformational dynamics of the two-domain regulatory tandem (CBD12) control the contributions of a given isoform/splice variant to dynamic handling of cytosolic Ca 2+ oscillations in distinct cell types.…”
Mammalian Na+/Ca2+ exchangers, NCX1 and NCX3, generate splice variants, whereas NCX2 does not. The CBD1 and CBD2 domains form a regulatory tandem (CBD12), where Ca2+ binding to CBD1 activates and Ca2+ binding to CBD2 (bearing the splicing segment) alleviates the Na+-induced inactivation. Here, the NCX2-CBD12, NCX3-CBD12-B, and NCX3-CBD12-AC proteins were analyzed by small-angle X-ray scattering (SAXS) and hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to resolve regulatory variances in the NCX2 and NCX3 variants. SAXS revealed the unified model, according to which the Ca2+ binding to CBD12 shifts a dynamic equilibrium without generating new conformational states, and where more rigid conformational states become more populated without any global conformational changes. HDX-MS revealed the differential effects of the B and AC exons on the folding stability of apo CBD1 in NCX3-CBD12, where the dynamic differences become less noticeable in the Ca2+-bound state. Therefore, the apo forms predefine incremental changes in backbone dynamics upon Ca2+ binding. These observations may account for slower inactivation (caused by slower dissociation of occluded Ca2+ from CBD12) in the skeletal vs the brain-expressed NCX2 and NCX3 variants. This may have physiological relevance, since NCX must extrude much higher amounts of Ca2+ from the skeletal cell than from the neuron.
Ca 2+ homeostasis is essential for cell function and survival. As such, the cytosolic Ca 2+ concentration is tightly controlled by a wide number of specialized Ca 2+ handling proteins. One among them is the Na + -Ca 2+ exchanger (NCX), a ubiquitous plasma membrane transporter that exploits the electrochemical gradient of Na + to drive Ca 2+ out of the cell, against its concentration gradient. In this critical role, this secondary transporter guides vital physiological processes such as Ca 2+ homeostasis, muscle contraction, bone formation, and memory to name a few.Herein, we review the progress made in recent years about the structure of the mammalian NCX and how it relates to function. Particular emphasis will be given to the mammalian cardiac isoform, NCX1.1, due to the extensive studies conducted on this protein. Given the degree of conservation among the eukaryotic exchangers, the information highlighted herein will provide a foundation for our understanding of this transporter family. We will discuss gene structure, alternative splicing, topology, regulatory mechanisms, and NCX's functional role on cardiac physiology. Throughout this article, we will attempt to highlight important milestones in the field and controversial topics where future studies are required.
Sodium–calcium exchanger (NCX) proteins are ubiquitously expressed and play a pivotal role in cellular calcium homeostasis by mediating uphill calcium efflux across the cell membrane. Intracellular calcium allosterically regulates the exchange activity by binding to two cytoplasmic calcium‐binding domains, CBD1 and CBD2. However, the calcium‐binding affinities of these domains are seemingly inadequate to sense physiological calcium oscillations. Previously, magnesium binding to either domain was shown to tune their affinity for calcium, bringing it into the physiological range. However, while the magnesium‐binding site of CBD2 was identified, the identity of the CBD1 magnesium site remains elusive. Here, using molecular dynamics in combination with differential scanning fluorimetry and mutational analysis, we pinpoint the magnesium‐binding site in CBD1. Specifically, among four calcium‐binding sites (Ca1–Ca4) in this domain, only Ca1 can accommodate magnesium with an affinity similar to its free intracellular concentration. Moreover, our results provide mechanistic insights into the modulation of the regulatory calcium affinity by magnesium, which allows an adequate NCX activity level throughout varying physiological needs.
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