Lens Fiber Cell Gap Junction Gating

Abstract

Abstract: : Purpose: In normal lenses from all species studied (frog, rat, mouse), the coupling conductance in the outer shell of differentiating fibers (G_DF) is sensitive to pH and will go to near zero at values of pH ≤ 6, whereas coupling in the core of mature fibers (G_MF) is not pH sensitive. To better understand the regulatory mechanism, we studied pH gating of gap junctions in lenses from mice with decreases in amount and/or diversity of Cx46 and Cx50, and in lenses in which channels formed from Cx50 were pharmacologically blocked. Methods: The coupling conductance between fiber cells in intact mouse lenses was determined using intracellular microelectrodes and impedance analysis. pH gating was triggered by bubbling the bath solution with 100% CO₂. Lenses from connexin knockout (KO) or knockin (KI) mice were used to change amount/diversity of Cx46 and Cx50. Mefloquine (MFQ) was used to selectively block channels formed from Cx50. Results: In all conditions reported here, G_MF remained pH–insensitive. Following acidification, G_DF went to zero in wild type (WT) or Cx46 KO lenses, but was insensitive to pH in the Cx50 KO mouse lens. Treatment of WT lenses with MFQ caused G_DF to drop to about half its normal value, but the remaining open channels were pH–insensitive. These results suggested the presence of functioning Cx50 channels was required for pH–sensitivity of G_DF. In the Cx46 KI lenses, the coding region for Cx50 was replaced with that of Cx46, so these lenses expressed two copies of Cx46 on the normal gene locus and two copies of Cx46 on the Cx50 gene locus. In these lenses acidification caused G_DF to once again go to near zero, similar to that seen in WT lenses, even though they lacked Cx50. Conclusions: The MFQ and Cx50 KO results suggest that when Cx46 is synthesized on its normal gene locus, it is not intrinsically pH sensitive, probably due to post translational modifications. When either Cx46 or Cx50 is synthesized on the Cx50 gene locus, pH–sensitive channels are formed, and these channels appear to co–operatively interact with neighboring (intrinsically pH–insensitive) Cx46 channels to induce total closure of all channels. These results suggest that post translational modifications of connexin protein depend on the locus of expression, and that interactions between gap junction channels are more complex than previously thought.

Keywords: gap junctions/coupling • cell–cell communication • transgenics/knock–outs