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ABSTRACT: KAAT1 and CAATCH1 are amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta. They are members of the SLC6/NSS family, which groups membrane proteins that use Na(+), K(+), and Cl⁻ gradients for the coupled transport of amines and amino acids. The report of the atomic-resolution x-ray crystal structure of the eubacterium Aquifex aeolicus leucine transporter (AaLeuT) has contributed significantly to understanding of the structure-function relationship in NSS proteins. Transport by AaLeuT is Cl⁻ independent, whereas many neurotransmitter:sodium symporters like serotonin transporter (SERT), GABA transporter (GAT1), dopamine transporter, and norephinephrine transporter, among others, are strongly Cl⁻ dependent.
Channels (Austin, Tex.) 10/2008; 2(5):358-62. · 1.91 Impact Factor
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ABSTRACT: Atomic force microscopy (AFM) was used to investigate the native plasma membrane of Xenopus laevis (X. laevis) oocyte purified by means of ultracentrifugation on sucrose gradient and subsequently adsorbed on mica leaves through a physisorption process. Reproducible AFM topography images were collected, analyzed, and compared. AFM images showed the presence of large single or double bilayer membrane sheets covered with protein complexes. The lateral dimension and height of protein complexes imaged in air showed a normal distribution centred on 15.4 +/- 0.4 nm (mean +/- SE; n = 59) and 3.9 +/- 0.2 nm (mean +/- SE; n = 57), respectively. A density of about 270 protein complexes per square micron was calculated. Less frequently, ordered nanometer domains with densely packed protein complexes arranged in hexagonal patterns were also visualized in AFM images, confirming previously published data. Their lateral dimension and height showed a normal distribution centred on 23.0 +/- 0.4 nm (mean +/- SE; n = 42) and 1.5 +/- 0.6 nm (mean +/- SE; n = 90), respectively. A density of about 870 protein complexes per square micrometer was calculated. Advantages and drawbacks of this new sample preparation for AFM imaging are discussed.
Microscopy Research and Technique 07/2008; 71(6):397-402. · 1.79 Impact Factor
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ABSTRACT: The substrate specificity of KAAT1, a Na+- and K+-dependent neutral amino acid cotransporter cloned from the larva of the invertebrate Manduca sexta and belonging to the SLC6A gene family has been investigated using electrophysiological and radiotracer methods. The specificity of KAAT1 was compared to that of CAATCH1, a strictly related transporter with different amino acid selectivity. Competition experiments between different substrates indicate that both transporters bind leucine more strongly than threonine and proline, the difference between KAAT1 and CAATCH1 residing in the incapacity of the latter to complete the transport cycle in presence of leucine. The behaviour of CAATCH1 is mimicked by the S308T mutant form of KAAT1, constructed on the basis of the atomic structure of a leucine-transporting bacterial member of the family, which indicates the participation of this residue in the leucine-binding site. The reverse mutation T308S in CAATCH1 conferred to this transporter the ability to transport leucine in presence of K+. These results may be interpreted by a kinetic scheme in which, in presence of Na+, the leucine-bound state of the transporter is relatively stable, while in presence of K+ and at negative potentials the progression of the leucine-bound form along the cycle is favoured. In this context serine 308 appears to be important in allowing the change to the inward-facing conformation of the transporter following substrate binding, rather than in determining the binding specificity.
The Journal of Physiology 07/2007; 581(Pt 3):899-913. · 4.72 Impact Factor
