Structure-dynamic and functional relationships in a Li⁺-transporting sodium‑calcium exchanger mutant

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Cell membrane (NCX) and mitochondrial (NCLX) Na⁺/Ca²⁺ exchangers are crucial for cellular calcium (Ca²⁺) homeostasis.
• NCXs exhibit high selectivity for Na⁺ and Ca²⁺ due to conserved ion-coordinating residues.
• NCLXs differ in nine out of twelve ion-coordinating residues, allowing exchange with Na⁺ or Li⁺, but the molecular basis is unclear.

Purpose of the Study:

• To investigate the molecular mechanisms underlying the distinct ion selectivity of NCLX compared to NCX.
• To analyze how specific residue substitutions in NCLX affect its ion binding, accessibility, and function.
• To elucidate the physiological relevance of these structure-function differences for Ca²⁺ homeostasis.

Main Methods:

• Created a mutant NCLX (NCLX_Mj) by substituting nine residues from NCX_Mj to mimic NCLX ion selectivity.
• Employed site-directed fluorescent labeling and ion flux assays to assess ion accessibility and binding kinetics.
• Utilized hydrogen-deuterium exchange mass-spectrometry (HDX-MS) to analyze structural changes and protein dynamics.

Main Results:

• NCLX_Mj showed nearly symmetric ion accessibility, unlike NCX_Mj's predominantly extracellular vestibule access.
• HDX-MS revealed symmetrically rigidified core helices in NCLX_Mj, contrasting with asymmetric rigidification in NCX_Mj.
• NCLX_Mj's Ca²⁺ site binds Na⁺, Li⁺, or Ca²⁺, with additional Na⁺/Li⁺ sites incompatible with NCX binding sites.

Conclusions:

• Substitution of ion-coordinating residues significantly alters NCLX's ion selectivity, binding capacity/affinity, and ion accessibility.
• These structural changes affect NCLX's response to membrane potential and its functional properties compared to NCX.
• The identified structure-controlled functional differences likely contribute to the distinct roles of NCX and NCLX in cellular Ca²⁺ regulation.