Human Connexin 26 (Cx26) N14K Mutant Alters Hemichannel Calcium and Voltage Sensitivity

Abstract

Unpaired hemichannels, those not forming gap junction channels, play an autocrine/paracrine role by releasing transmitters, such as glutamate and ATP, to the extracellular medium. Hemichannels are gated by changes in extracellular Ca+2 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+2). 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 ± 0.7) mutants with respect to wild type channels (midpoint at + 9.6 mV ± 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 sub-conductances. The apparent calcium affinity was also significantly reduced in N14K (KD = 0.74 mM) compared to wild type channels (KD = 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+2 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.