Sesti F, Nizzari M, Torre V
Dipartimento di Fisica, Università di Genova, Italy.
Biophys J. 1996 Jun;70(6):2616-39. doi: 10.1016/S0006-3495(96)79832-6.
Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects.
在虎纹钝口螈幼体的视杆外段研究了天然的环磷酸鸟苷（cGMP）门控通道。牛视杆cGMP门控通道的α亚基，此处称为野生型（w.t.），以及突变通道在非洲爪蟾卵母细胞中进行了异源表达。这些通道在内面向外模式的切除膜片中进行研究，并通过添加100或500微摩尔/升的cGMP来激活。研究了温度对离子通透的影响。在+100毫伏时，通过天然通道的宏观电流在分别存在锂离子、钠离子、钾离子、铷离子、铯离子和铵离子的情况下，活化能分别为35.8、30、31.8、34.5、41.3和22.4千焦/摩尔。在+100毫伏时，通过野生型通道的宏观电流在分别存在锂离子、钠离子、钾离子、铷离子、铯离子和铵离子的情况下，活化能分别为45.2、38.2、37.5、47.3、49.4和38.9千焦/摩尔。当通透离子的离子浓度在2.5至110毫摩尔之间变化时，通过天然通道和野生型通道的宏观电流的活化能没有显著变化。在+100毫伏时，野生型通道单通道电流对于钠离子和铵离子的活化能分别为40.4和33千焦/摩尔。双离子溶液的反转电位随温度显著变化。这些结果可用于估计离子在通过开放通道渗透过程中对吉布斯自由能势垒的焓和熵贡献。这些估计表明，cGMP门控通道的离子通透和选择性受焓和熵因素共同控制，并且天然通道对锂离子比对钠离子的选择性主要是由熵效应引起的。
