Diferentes enfoques sobre las competencias del profesorado 24

Capítulo 2: Marco Teórico 10

2.3 Diferentes enfoques sobre las competencias del profesorado 24

Crystal structure studies, SCAM experiments, and functional studies indicate NT and E1

as pore-lining domains which may determine γ

and V

-gating properties of GJs, however exact

factors governing these channel properties remain largely unknown (Kronengold et al. 2012;

Maeda et al. 2009; Oh et al. 2008). Mutations on the residues in the NT domain alter γ

and V

-

gating properties (Peracchia et al. 2005; Xin et al. 2012a). Increasing negative charge of

proposed pore-lining residue Cx50G46, i.e. G46D or G46E, resulted in increased γ

to as high as

~293 pS in G46E GJ, which is almost equivalent to the largest Cx37 GJ γ

, indicating Cx50 GJs

are not fully optimized for the rate of ion permeation at individual channels and mutations of

putative pore-lining residue to negatively charged glutamate (E) substantially increase γ

(Tong

between Cx50 and Cx37, especially on NT and E1 domains. We found key differences in two

positions in Cx50, i.e. G8 and V53 residues, which in the corresponding positions of Cx37 they

are E8 and E53. It is possible that these putative pore-lining residues in Cx37 are partially

responsible for its larger γ

and that mutations of these two residues in Cx50 could make γ

of

Cx50 channel even larger, especially in combination with Cx50G46E GJs which already showed

a γ

similar to that of Cx37. Exchanging these residues in Cx50 in order to have individual and

combination mutations, G8E, G46E, and/or V53E, may further increase the mutant GJ γ

.

Additionally, each of these mutants involves an increase in negative charges along the pore,

which could significantly change the pore electrostatic properties, which had been shown to be

critical not only on γ

, but also for other channel properties, such as V

-gating and modulation by

magnesium (Palacios-Prado et al. 2013; Tong et al. 2015b; Tong et al. 2014; Xin et al. 2013).

1.7 Hypothesis

We hypothesize that mutations on putative pore-lining residues of Cx50 G8, G46, and

V53 into glutamic acid (E) residue individually or in combination affect γ

, V

-gating properties

and sensitivity to intracellular Mg

.

1.8 Objectives

To test our hypothesis experimentally we have the following objectives:

1)

Investigate effects of the Cx50 mutants G8E-G46E, G8E-V53E, G46E-V53E, and G8E-

G46E-V53E on γ

and V

-gating.

2)

Examine the intracellular Mg

sensitivity of the mutant Cx50 mutant GJ properties (γ

and

V

-gating)

3)

Study mechanisms responsible for the altered channel properties in these Cx50 mutants.

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