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Molecular dissection of a basic COOH-terminal domain of Cx32 that inhibits gap junction gating sensitivity
1Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642-8711, USA.
Abstract:
Connexin32 (Cx32) mutants were studied by double voltage clamp in Xenopus oocytes to determine the role of basic COOH-terminal residues in gap junction channel gating by CO2 and transjunctional voltage. Replacement of five arginines with N (5R/N) or T residues in the initial COOH-terminal domain (CT1) of Cx32 enhanced CO2 sensitivity. The positive charge, rather than the R residue per se, is responsible for the inhibitory role of CT1, because mutants replacing the five R residues with K (5R/K) or H (5R/H) displayed CO2 sensitivity comparable to that of wild-type Cx32. Mutants replacing R with N residues four at a time (4R/N) showed that CO2 sensitivity is strongly inhibited by R215 and mildly by R219, whereas R220, R223, and R224 may slightly increase sensitivity. Neither the 5R/N nor the 4R/N mutants differed in voltage sensitivity from wild-type Cx32. The possibility that inhibition of gating sensitivity results from electrostatic interactions between CT1 and the cytoplasmic loop is discussed as part of a model that envisions the cytoplasmic loop of Cx32 as a key element of chemical gating.
Insights
Basic residues in Connexin32 (Cx32) COOH-terminal domains regulate gap junction channel gating by CO2. Positive charge, not specific arginines, inhibits CO2 sensitivity, suggesting electrostatic interactions are key.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Biology
Background:
- Connexin32 (Cx32) forms gap junctions, crucial for intercellular communication.
- The COOH-terminal domain (CT1) of Cx32 plays a role in channel gating.
- Understanding Cx32 gating mechanisms is vital for cellular signaling.
Purpose of the Study:
- To investigate the role of basic COOH-terminal residues in Cx32 gap junction channel gating.
- To determine the influence of CO2 and transjunctional voltage on Cx32 mutants.
- To elucidate the contribution of positive charges versus specific arginine residues in CT1.
Main Methods:
- Utilized double voltage clamp electrophysiology in Xenopus oocytes.
- Created and analyzed various Cx32 mutants, including substitutions of arginine residues (5R/N, 5R/K, 5R/H, 4R/N).
- Assessed CO2 sensitivity and transjunctional voltage sensitivity of wild-type and mutant Cx32 channels.
Main Results:
- Replacing five arginines with asparagine (5R/N) significantly enhanced CO2 sensitivity.
- The positive charge of arginine, not the residue itself, mediates inhibition of CO2 sensitivity.
- Mutants with lysine or histidine substitutions (5R/K, 5R/H) showed wild-type CO2 sensitivity.
- Specific arginines (R215, R219) were identified as major inhibitors of CO2 sensitivity.
- No significant differences in voltage sensitivity were observed between mutants and wild-type Cx32.
Conclusions:
- Positive charges within the Cx32 CT1 domain are critical for CO2-mediated inhibition of gap junction gating.
- Electrostatic interactions between CT1 and the cytoplasmic loop likely mediate chemical gating.
- The cytoplasmic loop of Cx32 is proposed as a key component in chemical gating mechanisms.