Structure-function analysis of the ion channel selectivity filter in human annexin V

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Annexin V is a calcium-binding protein involved in membrane trafficking and regulation.
• Ion channels play critical roles in cellular physiology and disease.
• Understanding the structure-function relationship of ion channels is essential for therapeutic development.

Purpose of the Study:

• To investigate the role of glutamic acid-95 in the pore region of annexin V.
• To determine the effect of a glutamic acid-95 to serine mutation on ion conductance and selectivity.
• To analyze structural changes in the mutant annexin V.

Main Methods:

• Site-directed mutagenesis to create the E95S variant of annexin V.
• Electrophysiology (single-channel recordings) to measure ion conductance.
• X-ray crystallography to determine the structure of the mutant and wild-type proteins.
• Electron microscopy to analyze the membrane-bound form of annexin V.

Main Results:

• The E95S mutation significantly altered ion selectivity, reducing calcium conductance and increasing sodium and potassium conductance.
• Structural analysis revealed minor changes around the mutation site but significant differences elsewhere, including a novel calcium binding site in domain III.
• Electron microscopy showed no differences in the membrane-bound conformation between wild-type and mutant annexin V.

Conclusions:

• Glutamic acid-95 is a key component of the annexin V ion selectivity filter.
• The mutation induces conformational changes beyond the immediate mutation site, affecting protein structure and function.
• Annexin V's structure and ion-binding properties can be modulated by specific mutations.