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Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations
C Castro1, J M Gómez-Hernandez, K Silander
1Unidad de Neurología Experimental-Consejo Superior de Investigaciones Científicas, Departamento de Investigación, Hospital "Ramón y Cajal," 28034 Madrid, Spain.
Abstract:
Hexamers of connexins (Cxs) form hemichannels that dock tightly in series via their extracellular domains to give rise to gap junction channels. Here we examined the ability of a variety of C-terminal Cx32 mutations, most of which have been identified in X-linked Charcot-Marie-Tooth disease, to form hemichannels and to complete gap junction channels using the Xenopus oocyte system. First, we show that undocked wild-type Cx32 hemichannels at the plasma membrane can be detected as opening channels activated by depolarization. We have been able to estimate the efficiency of assembly of complete channels by measuring the time-dependent incorporation of preformed hemichannels into gap junction channels after cell-to-cell contact. These data offer strong evidence that hemichannels are the direct precursors of gap junction channels. Of 11 Cx32 mutants tested, a group of 5 mutations prevented the formation of functional hemichannels at the cell surface, whereas 4 mutations were fully able to form precursors but reduced the ability of hemichannels to assemble into complete channels, and 2 mutants formed channels normally. The data revealed that a minimum length of human Cx32 including the residue Arg-215 is required for the expression of hemichannels at the cell surface and that the efficiency of hemichannel incorporation into complete channels decreased gradually with the progressive shortening of the cytoplasmic C-terminal domain.
Insights
Connexin (Cx) hemichannels are precursors to gap junction channels. Cx32 mutations linked to Charcot-Marie-Tooth disease impair hemichannel formation or assembly, impacting channel function.
Area of Science:
- Cell biology
- Molecular neuroscience
- Biophysics
Background:
- Connexins (Cxs) assemble into hexameric hemichannels.
- Hemichannels dock to form gap junction channels, crucial for intercellular communication.
- X-linked Charcot-Marie-Tooth disease is associated with mutations in Cx32.
Purpose of the Study:
- To investigate the functional consequences of C-terminal Cx32 mutations on hemichannel and gap junction channel formation.
- To determine if hemichannels are direct precursors to gap junction channels.
- To identify structural requirements for Cx32 hemichannel assembly and function.
Main Methods:
- Utilized the Xenopus oocyte expression system.
- Assessed hemichannel formation and function at the plasma membrane via depolarization.
- Quantified gap junction channel assembly by measuring hemichannel incorporation after cell-cell contact.
Main Results:
- Wild-type Cx32 hemichannels, when undocked, can function as open channels activated by depolarization.
- Demonstrated that hemichannels are direct precursors to gap junction channels.
- Five of eleven Cx32 mutants failed to form cell surface hemichannels.
- Four mutants formed hemichannel precursors but had reduced gap junction assembly efficiency.
- Two mutants exhibited normal channel formation.
- A minimum Cx32 length, including Arg-215, is necessary for cell surface hemichannel expression.
- Progressive shortening of the C-terminal domain reduced hemichannel incorporation into complete channels.
Conclusions:
- Hemichannels serve as direct precursors for functional gap junction channels.
- C-terminal Cx32 mutations associated with Charcot-Marie-Tooth disease disrupt hemichannel formation or subsequent gap junction assembly.
- The Cx32 C-terminal domain length is critical for both hemichannel surface expression and efficient gap junction formation.