BioCurrents Research Center : Transporters

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Transporters: Slow or Electroneutral

The study of electroneutral and slow rate transporters has lagged behind the study of electrogenic transporters due to the difficulty in monitoring transport rates. Electrogenic transporters, that create a flow of net charge, are studied with voltage clamp methods that are used to measure current transfer across the membrane that is proportional to transport rates. This real-time assay enables monitoring transport kinetics in systems with a high density of electrogenic transporters that have high transport rates. Electroneutral transporters, however, do not generate current at all while slow-rate transporters generate current that is not detectable unless transporter density reaches 1-10 thousand times greater than that used with high rate electrogenic transporters. We have found that a sensitive, real-time assay for monitoring transport exists by using extracellular, electrochemical sensors to monitor gradients established during transport. By monitoring two points in the concentration gradient we are able to calculate flux of the analyte that is proportional to transport rate.

The table below lists specific transporters and exchangers (both electrogenic and electroneutral) that have been characterized using self-referencing with extracellular ion-selective microelectrodes. This list does not incorporate the larger number of extracellular ion fluxes measured with this method due to transport from as yet, unidentified transporters, exchangers or channels. Using this approach, we are able to measure small, relatively steady fluxes of H+, Ca2+, K+, Na+ and Cl- from ion transporters and exchangers in native cells and tissues. We are currently using self-referencing of ISMs for the near real-time characterization of slow rate and electroneutral transporters expressed in heterologous expression systems.

Ion Transporter	Source
PM Ca²⁺-ATPase	hair cells (hearing)^a
	horizontal cells (vision)^b
	molluscan cardiac muscle ^c
H⁺/K⁺ ATPase	Microglia ^d
V-ATPase	rat vas deferens (reproduction) ^{e, i}
V-ATPase	mosquito midgut ^f
Ion Exchanger	Source
Na⁺/Ca²⁺	mouse ova ^g
Na⁺/H⁺	horizontal cells (vision)^b
Cl^-/HCO3-	mosquito midgut ^h
Table 1. A list of ion transporters, exchangers and channels that have been characterized with self-referencing of ion-selective microelectrodes in native cells and tissues. In most cases the transport protein was known to exist using other methods or was identified with specific pharmacological inhibitors. a) (Yamoah et al., 1998), b) (Molina et al., 2004) c) (Devlin, 2001), d) (Shirihai et al., 1998), e) (Breton and Brown, 2007) f) (Boudko et al., 2001a), g) (Pepperell et al., 1999), h) (Boudko et al., 2001b), (Shum et al., 2008)

Related BRC publications

Boudko, D.Y., Moroz, L.L., Harvey, W.R. and Linser, P.J. 2001. Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut. Proceedings of the National Academy of Sciences, 98(26): 15354-15359.

Boudko, D., Moroz, L., Linser, P., Trimarchi, J., Smith, P. and Harvey. W. 2001. In situ analysis of pH gradients in mosquito larvae using non-invasive, self-referencing, pH-sensitive microelectrodes. Journal of Experimental Biology, 204(4): 691-9.

Breton, S. and Brown, D. 2007. New insights into the regulation of V-ATP ase- dependent proton secretion. Am J Physiol Renal Physiol, Jan;292(1):F1-10

Devlin, C. L. 2001. 5-Hydroxytryptamine stimulates Ca²⁺ flux in the ventricular muscle of mollusk (Busycon canaliculatum) during cardioexcitation. Biological Bulletin, 200(3): 344-350.

Molina, A.J.A., Verzi, M.P., Birnbaum, A.D., Yamoah, E.N., Hammar, K., Smith, P.J.S., Malchow, R.P. 2004. Neurotransmitter modulation of extracellular H⁺ fluxes from isolated retinal horizontal cells of the skate. Journal of Physiology-London 560:639-657.

Pepperell, J.R., Kommineni, K., Buradagunta, S., Smith, P.J.S. and Keefe, D.L. 1999. Transmembrane regulation of intracellular calcium by a plasma membrane Sodium/Calcium exchanger in mouse oocytes. Biology of Reproduction, 60: 1137-1143.

Shirihai, O., Smith, P.J.S., Hammar, K. and Dagan, D. 1998. H⁺ and K⁺ gradient generated by microglia H/K ATPase. Glia 23: 339-348.

Shum, W.W, Da Silva, N., McKee, M, Smith, P.J, Brown, D., Breton, S. 2008. Transepithelial projections from basal cells are luminal sensors in pseudostratified epithelia. Cell. Dec 12;135(6):1108-17.

Yamoah, E., Lumpkin, E.A., Dumont, R.A., Smith, P.J.S., Hudspeth, A.J. and Gillespie, P.G. 1998. Plasma-membrane Ca²⁺-ATPase ensures low Ca²⁺ concentration in hair-cell stereocilia. Journal of Neuroscience. 18(2): 610-624.