Gap junctions provide a direct intercellular pathway for cell-to-cell signaling and impulse conduction and play an important role in normal physiology. However, mutations in their connexin proteins have been implicated in many hereditary diseases, including nervous system disorders, deafness, cataracts and some skin diseases. Mutations in connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD) which manifests with bone deformities and accompanying neurological complications. In another case, Cx40 mutations have been correlated with atrial fibrillation and other arrhythmias. Most of the disease associated mutations are single amino acid substitutions and we hypothesize that they could potentially alter the specific perm-selectivity properties of the affected connexin channel. We wish to quantify second messenger permeability for wild-type Cx43 and Cx40 and compare them to human mutations of Cx43 and Cx40 associated with ODDD and arrhythmia. The cyclic nucleotides, cAMP and cGMP are of particular interest as both are important in normal bone development and play a role in cardiac myocyte contractility and pacing.
In Aim1 the perm-selectivity properties of gap junction channels formed by Cx40 and Cx43 to second messengers cAMP and cGMP will be determined. A reporter gene SpIH will be used to monitor cAMP/cGMP permeability while simultaneously monitoring junctional conductance by dual whole cell patch clamp.
Aim2 will determine the cyclic nucleotide perm-selectivity properties of gap junction channels formed by disease linked mutations in Cx40 (A96S, M163V and G38D) and Cx43 (L90V, I130T and K134E). We will quantify and compare the permeability of gap junction channels formed by mutated connexins to their wild-type counterparts.
Aim3 will characterize the intercellular trafficking, gating properties and perm-selectivity properties of gap junction channels formed by Cx40 and Cx43 mutants. We will test for changes in channel open probability, voltage and pH dependent gating, intercellular trafficking and permeability as these parameters represent alternative ways that mutations could diminish the magnitude of cell-to-cell communication. In this proposal, the cyclic nucleotide perm-selectivity of wild-type and disease associated mutant connexins will be compared qualitatively and quantitatively. The results of the proposed research will establish a baseline for understanding the role of perm-selectivity as a potential determinant of disease states such as ODDD and atrial fibrillation. Based on the results of Aims 1-3, we will determine which channel properties (perm-selectivity, open probability, or trafficking) are responsible for functional alterations in mutated connexins.

Public Health Relevance

Gap junctions play an important role in normal physiology and mutations in their connexin proteins have been implicated in many human hereditary diseases. This proposal seeks to evaluate the perm-selectivity of wild-type and disease associated mutant connexins qualitatively and quantitatively, including permeability to known signaling molecules like cyclic nucleotides. The results of the proposed research will establish a baseline for understanding the role of perm-selectivity as a potential determinant of disease states such as oculodentodigital dysplasia and atrial fibrillation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088181-03
Application #
8464148
Study Section
Intercellular Interactions (ICI)
Program Officer
Nie, Zhongzhen
Project Start
2011-05-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
3
Fiscal Year
2013
Total Cost
$287,860
Indirect Cost
$104,510
Name
State University New York Stony Brook
Department
Physiology
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
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Clausen, Chris; Valiunas, Virginijus; Brink, Peter R et al. (2013) MATLAB implementation of a dynamic clamp with bandwidth of >125 kHz capable of generating I Na at 37 °C. Pflugers Arch 465:497-507
Mese, Gulistan; Sellitto, Caterina; Li, Leping et al. (2011) The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome. Mol Biol Cell 22:4776-86
Jia, Zhiheng; Valiunas, Virginijus; Lu, Zongju et al. (2011) Stimulating cardiac muscle by light: cardiac optogenetics by cell delivery. Circ Arrhythm Electrophysiol 4:753-60