Locke Darren, Koreen Irina V, Harris Andrew L
Department of Pharmacology and Physiology, New Jersey Medical School, 185 South Orange Ave., University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
FASEB J. 2006 Jun;20(8):1221-3. doi: 10.1096/fj.05-5309fje. Epub 2006 Apr 27.
The isoelectric points of the gap junction proteins connexin26 (Cx26) and connexin32 (Cx32) were determined by isoelectric focusing in free fluids. The isoelectric points were significantly more acidic than predicted from amino acid sequences and different from each other, allowing homomeric channels to be resolved separately. The isoelectric points of the homomeric channels bracketed the isoelectric points of heteromeric Cx26/Cx32 channels. For heteromeric channels, Cx26 and Cx32 were found in overlapping, pH-focused fractions, indicating quaternary structure was retained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify post-translational modifications of Cx26 and Cx32 cytoplasmic domains, including the first reported post-translational modifications of Cx26. Suspected modifications were hydroxylation and/or phosphorylation near the amino terminus of both connexins, gamma-carboxyglutamate residues in the cytoplasmic loop of both connexins, phosphorylation in the carboxyl-terminal domain of Cx32, and palmitoylation at the carboxyl-terminus of Cx32. These modifications contribute to the measured acidic isoelectric points of Cx26 and Cx32, whereas their low molecular masses would not appreciably change connexin SDS-PAGE mobility. Most of these modifications have not previously been identified for connexins and may be instrumental in guiding and understanding novel aspects of channel trafficking and molecular mechanisms of channel regulation.
通过在自由流体中进行等电聚焦来测定间隙连接蛋白连接蛋白26（Cx26）和连接蛋白32（Cx32）的等电点。等电点比根据氨基酸序列预测的明显更偏酸性，且彼此不同，这使得同聚体通道能够被分别解析。同聚体通道的等电点界定了异聚体Cx26/Cx32通道的等电点范围。对于异聚体通道，在重叠的、pH聚焦的组分中发现了Cx26和Cx32，表明四级结构得以保留。采用基质辅助激光解吸/电离飞行时间质谱法来鉴定Cx26和Cx32胞质结构域的翻译后修饰，包括首次报道的Cx26的翻译后修饰。疑似修饰包括两种连接蛋白氨基末端附近的羟基化和/或磷酸化、两种连接蛋白胞质环中的γ-羧基谷氨酸残基、Cx32羧基末端结构域的磷酸化以及Cx32羧基末端的棕榈酰化。这些修饰导致了所测得的Cx26和Cx32的酸性等电点，而它们的低分子量不会明显改变连接蛋白的SDS-PAGE迁移率。这些修饰中的大多数此前尚未在连接蛋白中被鉴定出来，可能有助于指导和理解通道运输的新方面以及通道调节的分子机制。
