Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.
نویسندگان
چکیده
The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites.
منابع مشابه
Functional characterization of a human purine-selective, Na+-dependent nucleoside transporter (hSPNT1) in a mammalian expression system.
Nucleosides and nucleoside analogs are actively transported in the human kidney. With the recent cloning of a purine-selective, Na+-dependent, nucleoside transporter (hSPNT1, also termed hCNT2) from human kidney, it is now possible to study the interaction of nucleosides and nucleoside analogs with this transport protein and gain a more detailed knowledge of the underlying mechanisms of nucleos...
متن کاملEffect of Purine Nucleoside Analogue-Acyclovir on The Sperm Parameters and Testosterone Production in Rats
متن کامل
Electrophysiological characterization of the human Na(+)/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function.
We previously reported that the human Na(+)/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5'-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine...
متن کاملDrug-Resistant HIV-1 RT Gene Mutations in Patients under Treatment with Antiretroviral Drugs (HAART) in Iran
Abstract Background and Objective: Highly Active Antiretroviral Therapy (HAART) can effectively prevent the progression of HIV-1 replication and increase life expectancy. There are numerous causes of treatment failure and the leading one is drug resistance. Thus, we aimed to determine the HIV RT gene drug resistance mutations in patients treated with antiretroviral medications. Material...
متن کاملAn ancient prevertebrate Na+-nucleoside cotransporter (hfCNT) from the Pacific hagfish (Eptatretus stouti).
The human concentrative (Na+-linked) plasma membrane transport proteins hCNT1, hCNT2, and hCNT3 are pyrimidine nucleoside-selective (system cit), purine nucleoside-selective (system cif), or broadly selective for both pyrimidine and purine nucleosides (system cib), respectively. All have orthologs in other mammalian species and belong to a gene family (CNT) that has members in insects, nematode...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- American journal of physiology. Cell physiology
دوره 291 6 شماره
صفحات -
تاریخ انتشار 2006