Microarray-based screening system identifies temperature-controlled activity of Connexin 26 that is distorted by...

Area of Science:

• Molecular biology
• Biophysics
• Cellular physiology

Background:

• Human Connexin 26 (hCx26) forms hemichannels that facilitate intercellular communication.
• These hemichannels are implicated in various physiological and pathological processes.
• Understanding the regulation of hCx26 hemichannel activity is crucial for cellular function.

Purpose of the Study:

• To investigate the role of temperature in regulating hCx26 hemichannel activity.
• To analyze the impact of specific point mutations on hCx26 hemichannel function and temperature sensitivity.
• To elucidate the molecular mechanisms underlying temperature-dependent gating of hCx26 hemichannels.

Main Methods:

• Optical microarray-based Lucifer Yellow uptake assay.
• Two-electrode voltage clamp (TEVC) on frog oocytes.
• Liposome flux assay (LFA) for purified hemichannels.

Main Results:

• hCx26 hemichannel opening and small molecule transport are triggered by temperature changes and calcium (Ca2+) blockade compensation.
• Point mutations (L90P, F161S, R184P, K188N) significantly altered temperature-dependent activity.
• The K188N mutation destabilized a temperature-sensitive salt bridge, impairing hemichannel function at elevated temperatures (>30°C).

Conclusions:

• The K188 position in hCx26 forms a temperature-sensitive salt bridge with E47, crucial for gating.
• Temperature sensitivity of hCx26 hemichannels likely protects cells from uncontrolled molecular transport.
• Mutations affecting this temperature sensitivity have significant implications for hCx26 channel function.