Sanchez Helmuth A, Slavi Nefeli, Srinivas Miduturu, Verselis Vytas K
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.
Department of Biological Sciences, SUNY College of Optometry, New York, NY 10036.
J Gen Physiol. 2016 Jul;148(1):25-42. doi: 10.1085/jgp.201611585.
Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.
连接蛋白26（Cx26）是一种跨膜蛋白，可形成六聚体半通道，这些半通道在未受阻碍时即可发挥功能，或对接形成细胞间缝隙连接通道。功能异常的未受阻碍的半通道是与Cx26突变相关的综合征性耳聋的一个常见特征。在本研究中，我们检测了Cx26 N端结构域同一位置的两种不同突变，即N14K和N14Y，据报道这两种突变在患者中产生了不同的表型。我们发现，单独表达或与野生型（WT）Cx26共同表达时，N14K和N14Y都会导致具有广泛不同功能特性的功能性半通道。N14K电流很强，而N14Y电流很小。这两种突变体在电压依赖性环门控方面也表现出相反的变化，使得N14K和N14Y的激活分别向超极化和去极化方向偏移。失活动力学表明，N14K使开放状态稳定，而N14Y使开放状态不稳定。在低细胞外Ca(2+)条件下对单个N14K半通道的记录显示，没有证据表明与环门控相关的稳定关闭转变，并且N14K半通道对pH不敏感。这些特性共同导致N14K半通道特别难以关闭。尽管我们发现N14K的单通道电导与WT Cx26没有区别，但诱变和取代半胱氨酸可及性研究表明，N14残基暴露于孔中，并且N14K和N14Y半通道的不同特性可能是由于N端与相邻亚基中TM2的细胞质延伸之间的静电相互作用改变所致。我们观察到的对环门控和pH调节的综合影响可能解释了携带N14K突变患者不寻常颊部皮肤表现的原因。我们的工作还为控制质膜中半通道开放的环门控潜在分子机制提供了新的思考。
