How to study transporter using voltage clamp

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connie_2006
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How to study transporter using voltage clamp
Fraser Moss
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You most definitely can study

You most definitely can study Transporters using voltage clamp.

To explain this Ill use the Na+/Cl--dependent SLC6 family of transporters as an example. These include the GABA (GAT), 5HT (SERT), Dopamine (DAT) and norepinephrine (NET) transporters. These transporters couple the movement of substrate and other ions, typified by Na, across the plasma membrane. The proteins are Na+ co-transporters, with the energy for transport of transmitter against its concentration gradient provided by the electrochemical gradient for Na+. Na+ is an absolute prerequisite. Cl- is also cotransported, although this requirement varies across members. Precisely 2 Na+, and one Cl- are co transported with the substrate. Therefore there is a net movement of one positive charge into the cell per functional transporter unit per molecule of substrate transported into the cell. Despite the fact that this charge transfer per transporter molecule (1-10000/s, depending on the transporter) is much less than the elementary current for a channel (typically 10000000/s), macroscopic currents can be measured with modern voltage-clamp techniques for nearly all transporters. Steady-state substrate-induced currents have been observed in many members of the Na/Cl-dependent SLCL6 gene family (Mager et al., 1994; Sonders et al., 1997; Mager et al., 1993; Sonders and Amara, 1996). The current amplitude depends on the membrane potential and the GABA concentration.

From Mager et al 1993 Neuron (10) pp177

Steady-state measurements of transporter currents can yield the following types of data. (a) In the usual application of electrophysiological analysis, steady-state currents are measured as a function of the nature and concentration of all substrates and the resulting information resembles that from radiotracer flux. (b) In conjunction with tracer fluxes, current measurements can provide stoichiometric information. (c) Electrophysiological measurements in conjunction with ligand binding to quantitate transporter number
can yield turnover numbers. (d) Because transporter current would vanish at equilibrium, appropriately designed electrophysiological experiments could reveal how the equilibrium depends on substrate concentrations, membrane potential, and other factors. This thermodynamic measurement requires careful isolation of the transporter currents and is not often performed.

I could write you a lecture on this topic but I suggest you read the references below and if you cannot get hold of them from your library please private message me through Scientist Solutions, and I'll see what I can do you help get you copies.

References

Mager, S., C. Min, D. J. Henry, C. Chavkin, B. J. Hoffman, N. Davidson, and H. A. Lester. 1994. Conducting states of a mammalian serotonin transporter. Neuron. 12:845859.

Sonders, M. S., S. J. Zhu, N. R. Zahniser, M. P. Kavanaugh, and S. G. Amara. 1997. Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants. J. Neurosci. 17:960974.

Mager, S., J. Naeve, M. Quick, C. Labarca, N. Davidson, and H. A. Lester. 1993. Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes. Neuron. 10:177188.

Sonders, M. S., and S. G. Amara. 1996. Channels in transporters. Curr. Opin. Neurobiol. 6:294302.

Blakely R.D., De Felice L.J., Hartzell H.C. 1994. Molecular physiology of norepinephrine and serotonin transporters. J. exp. Biol. 196, 263281 (1994)

Galli A, Blakely RD, DeFelice LJ. 1996. Norepinephrine transporters have channel modes of conduction. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8671-6.

Nicholls D, Attwell D. 1990. The release and uptake of excitatory amino acids. Trends Pharmacol Sci. Nov;11(11):462-8.

Grewer C, Rauen T. 2005. Electrogenic glutamate transporters in the CNS: molecular mechanism, pre-steady-state kinetics, and their impact on synaptic signaling. J Membr Biol. Jan;203(1):1-20.

Richerson GB, Wu Y. 2003. Dynamic equilibrium of neurotransmitter transporters: not just for reuptake anymore. J Neurophysiol. Sep;90(3):1363-74.

Fairman WA, Amara SG. 1999 Functional diversity of excitatory amino acid transporters: ion channel and transport modes. Am J Physiol. Oct;277(4 Pt 2):F481-6.

Sonders MS, Quick M, Javitch JA. 2005. How did the neurotransmitter cross the bilayer? A closer view. Curr Opin Neurobiol. Jun;15(3):296-304.