Lum Kevin, Ingólfsson Helgi I, Koeppe Roger E, Andersen Olaf S
Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas.
Biophys J. 2017 Oct 17;113(8):1757-1767. doi: 10.1016/j.bpj.2017.08.049.
The canonical mechanism of gramicidin (gA) channel formation is transmembrane dimerization of nonconducting subunits that reside in opposite bilayer leaflets. The channels do not open and close; they appear and disappear due to subunit association and dissociation. Many different types of experiments support this monomer ↔ dimer mechanism. Recently, however, this mechanism was challenged, based on experiments with lipid vesicle-incorporated gA under conditions where vesicle fusion could be controlled. In these experiments, sustained channel activity was observed long after fusion had been terminated, which led to the proposal that gA single-channel current transitions result from closed-open transitions in long-lived bilayer-spanning dimers. This proposal is at odds with 40 years of experiments, but involves the key assumption that gA monomers do not exchange between bilayers. We tested the possibility of peptide exchange between bilayers using three different types of experiments. First, we demonstrated the exchange of gA between 1,2-dierucoyl-sn-glycero-3-phosphocholine (DCPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DCPC) lipid vesicles using a fluorescence assay for gA channel activity. Second, we added gA-free DCPC vesicles to both sides of planar DCPC bilayers preincubated with gA, which reduced channel activity up to 10-fold. Third, we added gA-containing DCPC vesicles to one or both sides of DCPC planar bilayers, which produced much higher channel activity when the gA-containing vesicles were added to both sides of the bilayer, as compared to one side only. All three types of experiments show that gA subunits can exchange between lipid bilayers. The exchange of subunits between bilayers thus is firmly established, which becomes a crucial consideration with respect to the mechanism of channel formation.
短杆菌肽（gA）通道形成的经典机制是位于相对双层小叶中的非导电亚基跨膜二聚化。通道不会打开和关闭；它们由于亚基的缔合和解离而出现和消失。许多不同类型的实验支持这种单体↔二聚体机制。然而，最近，基于在可以控制囊泡融合的条件下对脂质囊泡掺入的gA进行的实验，这一机制受到了挑战。在这些实验中，在融合终止很长时间后仍观察到持续的通道活性，这导致有人提出，gA单通道电流转变是由长寿命跨双层二聚体中的关闭-开放转变引起的。这一观点与40年的实验结果不一致，但涉及一个关键假设，即gA单体不会在双层之间交换。我们使用三种不同类型的实验测试了双层之间肽交换的可能性。首先，我们使用gA通道活性的荧光测定法证明了gA在1,2-二芥酰基-sn-甘油-3-磷酸胆碱（DCPC）或1,2-二油酰基-sn-甘油-3-磷酸胆碱（DOPC）脂质囊泡之间的交换。其次，我们将不含gA的DCPC囊泡添加到预先用gA孵育的平面DCPC双层的两侧，这使通道活性降低了多达10倍。第三，我们将含有gA的DCPC囊泡添加到DCPC平面双层的一侧或两侧，与仅添加到一侧相比，当将含有gA的囊泡添加到双层两侧时，产生的通道活性要高得多。所有三种类型的实验都表明，gA亚基可以在脂质双层之间交换。因此，双层之间亚基的交换得到了确凿的证实，这对于通道形成机制来说是一个至关重要的考虑因素。
