We hypothesize that there are specific charge groups that preferentially influence the permeation of molecules in the size and charge range of second messengers while having little influence on monovalent cation selectivity. We hypothesize that there are multiple sites able to affect the permselectivity of gap junction channels.
The specific aims are:
Aim la) Transfect mouse N2A cells or rat insulinoma cells (RIN) with mutant versions of Cx43, Cx40, and Cx37 cDNA and determine expression via Western and Northern blot analysis. We will also use the GFP tag strategy to determine cellular distribution. We have chosen Cx43 and Cx40 because of their ubiquity in the SA an AV nodes of the heart and hence their potential to affect cardiac arrhythmias. Cx37 is the major connexin of endothelium.
Aim i b) Monitor single channel conductance in homotypic mutants in KC 1, CsCI, NaCI and TEACI to assess changes in cation/anion selectivity using dual whole cell patch clamp.
Aim 2) Monitor single channel conductance in which one hemichannel is composed of a mutant connexin (muCx43 or muCx4O) and the other is composed of a wild type connexin of Cx43 or Cx40 in Kcl, CsCI, NaCl and TEAC1 to assess changes in cation/anion selectivity. We will use cysteine scanning mutation to determine if a mutated site lines the poor wall.
Aim 3) Simultaneously determine junctional conductance and junctional permeability to charged fluorescent probes for wild type and mutant connexins (homotypic and heterotypic forms). This will allow the determination of the permeation rates of charged probes in the 0.8-1.2 nm size range relative to K+/Cs+. Inert probes (Lucifer Yellow, DAPI, carboxyfluorescein) are probes of choice because they are not rapidly removed from the cytosol, as is the case for lP3 (Tau less than 60s).
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