Engineered Cx40 Variants Showed Heterotypic Colocalization and Increased GAP Junctional Coupling with Cx43

Abstract

to the extracellular medium. Hemichannels are gated by changes in extracellular Ca þ2 concentration and membrane voltage. Several human connexin mutations that produce diseases increase hemichannel opening (e.g., gain of function due to decreased sensitivity to extracellular Ca þ2 ). Using Xenopusoocytes and two-electrode voltage clamp techniques, we studied the voltage and calcium sensitivity of a connexin 26 (Cx26) mutation, N14K, located in the N-terminal domain (NT). The NT is thought to fold into the pore and form part of the pore lining; position N14 is approximately at the ''hinge'' region at which the NT bends toward the pore. We observe a significant leftward shift in the voltage-dependence N14K (midpoint at -6.7 mV 5 0.7) mutants with respect to wild type channels (midpoint at þ 9.6 mV 5 0.9). Rearrangements of the NT are associated with voltage dependent sub-conductances in wild type Cx26 hemichannels, however, N14K mutant channels did not display voltage dependent subconductances. The apparent calcium affinity was also significantly reduced in N14K (K D = 0.74 mM) compared to wild type channels (K D = 0.34 mM). Deactivation kinetics in wild type channels show single mono-exponential relaxations that are accelerated as a function of calcium concentration, but not voltage. The Cx26 mutant N14K displays two slower components. One of these components is both Ca þ2 and voltage dependent but the other is not. Overall, our data indicate that under physiological conditions the N14K mutation stabilizes the open state relative to wild type channels by reducing both calcium and voltage sensitivity. R01GM099490/ R01GM101950.

Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N‐terminal domains

“…The steady state G j was normalized to the peak G j to obtain a normalized steady-state junctional conductance (G j,ss ) for each tested V j . The G j,ss values were then plotted with V j values to obtain a G j,ss -V j plot, which could sometimes fit well with a two-state Boltzmann equation for each V j polarity to obtain gating parameters, V 0 , G min , G max and A (Jassim et al 2016). V 0 is the voltage when the G j,ss is reduced by half [(G max − G min )/2], G min is the normalized minimum residual conductance, while G max represents the maximum normalized conductance, and A is the slope of the curve which reflects V j gating sensitivity (Spray et al 1981):…”