Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Area of Science:

• Cellular Biology
• Biophysics
• Ion Channel Physiology

Background:

• Rat connexin 46 (rCx46) hemichannels are voltage-dependent and activated by low external Ca2+.
• These hemichannels exhibit time-dependent activation and spontaneous inactivation upon depolarization.
• Protein kinase C (PKC) is a key signaling enzyme involved in cellular regulation.

Purpose of the Study:

• To investigate the role of PKC in regulating rCx46 hemichannel activity.
• To elucidate the mechanisms underlying OAG-induced inactivation of rCx46 hemichannels.
• To determine the effects of PKC modulation on rCx46 hemichannel conductance and gating.

Main Methods:

• Expression of rCx46 in Xenopus laevis oocytes.
• Two-electrode voltage-clamp recordings to measure hemichannel currents.
• Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate PKC.
• Use of calphostin C and phloretin as specific PKC inhibitors.

Main Results:

• OAG treatment reversibly reduced rCx46-mediated current amplitude.
• OAG induced a time-dependent inactivation of voltage-dependent currents.
• PKC inhibition by calphostin C or phloretin removed both OAG-induced and spontaneous inactivation.
• Wash-out of OAG increased membrane conductance and abolished inactivation.

Conclusions:

• PKC activation negatively regulates rCx46 hemichannel conductance.
• PKC plays a crucial role in the voltage-dependent inactivation of rCx46 hemichannels.
• Modulation of PKC activity offers a potential mechanism to control rCx46 hemichannel function.