Further characterization of cation channels present in the chicken red blood cell membrane.

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd(3+) and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.

Female gender-specific inhibition of KCNQ1 channels and chloride secretion by 17beta-estradiol in rat distal colonic crypts.

The estrogen sex steroid 17beta-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl(-) secretion in the female rat distal colonic crypt by the inhibition of basolateral K(+) channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K(+) current was shown to be attenuated by pretreatment with rottlerin, a PKCdelta-specific inhibitor. In whole cell patch-clamp analysis, 17beta-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCdelta in comparison to male tissue. PKCdelta and PKA were activated at 5 min in response to 17beta-estradiol in female distal colonic crypts only. Both PKCdelta- and PKA-associated with the KCNQ1 channel in response to 17beta-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCdelta as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCdelta- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.

Modulation of whole-cell currents in Plasmodium falciparum-infected human red blood cells by holding potential and serum.

Recent electrophysiological studies have identified novel ion channel activity in the host plasma membrane of Plasmodium falciparum-infected human red blood cells (RBCs). However, conflicting data have been published with regard to the characteristics of induced channel activity measured in the whole-cell configuration of the patch-clamp technique. In an effort to establish the reasons for these discrepancies, we demonstrate here two factors that have been found to modulate whole-cell recordings in malaria-infected RBCs. Firstly, negative holding potentials reduced inward currents (i.e. at negative potentials), although this result was highly complex. Secondly, the addition of human serum increased outward currents (i.e. at positive potentials) by approximately 4-fold and inward currents by approximately 2-fold. These two effects may help to resolve the conflicting data in the literature, although further investigation is required to understand the underlying mechanisms and their physiological relevance in detail.

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane.

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.

A stretch-activated anion channel is up-regulated by the malaria parasite Plasmodium falciparum.

A recent study on malaria-infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host- or parasite-derived and their molecular nature have not been resolved. Here we report a comparison of a malaria-induced anion channel with an endogenous anion channel in Plasmodium falciparum-infected human RBCs. Ion channel activity was measured using the whole-cell, cell-attached and excised inside-out configurations of the patch-clamp method. Parasitised RBCs were cultured in vitro, using co-cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (~15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I(-) > Br(-) > Cl(-). In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (~80 pS, outward conductance) was spontaneously active. The host membrane of malaria-infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria-infected RBCs were shown to have a low open-state probability (P(o)) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole-cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up-regulated by the malaria parasite P. falciparum.

Age-dependent changes in cation transport in the chicken erythrocyte.

Previous work has shown that the transport phenotype of chicken erythrocytes changes with the age of the chicken. Here, we report changes in the transport of choline and K+ in erythrocytes from chickens at different developmental ages. The transport of choline in chicken erythrocytes was predominantly via saturable transport systems, was highest in erythrocytes from 1-day-old chickens and declined with chicken age when tested at 2 weeks of age and in mature chickens. Both Km and Vmax values for choline transport in chicken erythrocytes declined with chicken age. Similarly, the total unidirectional influx of K+ was highest in erythrocytes from 1-day-old chickens and declined with chicken age, as did ouabain-sensitive K+ influxes, which can be attributed to the Na+/K+ pump. In isotonic conditions, bumetanide-sensitive K+ influxes, which can be attributed to the Na+-K+-2Cl- cotransporter, were only measurable in erythrocytes from 1-day-old chickens. However, when stimulated by hypertonic conditions, bumetanide-sensitive K+ influxes were essentially identical in erythrocytes from 1-day- and 2-week-old chickens but decreased in erythrocytes from mature chickens. We conclude that both choline and K+ influxes decrease significantly in erythrocytes from chickens with increasing age. The changes are substantial but complex and may involve both regulation of existing transporters, and substitution or deletion of specific transporter isoforms.

Two functionally distinct organic osmolyte pathways in Plasmodium gallinaceum-infected chicken red blood cells.

Red cells infected with the human malaria parasite Plasmodium falciparum have an increased permeability to a range of small, structurally unrelated solutes via a malaria-induced pathway. We report here a similar pathway present in parasitised red cells from chickens infected with the avian malaria parasite, Plasmodium gallinaceum. Parasitised cells showed a marked increase in the rate of influx of sorbitol (76-fold) and, to a lesser degree, taurine (3-fold) when compared with red cells from uninfected chickens. Pharmacological data suggest that both sorbitol and taurine are transported via a single malaria-induced pathway, which is sensitive to inhibition by 5-nitro-2-(3-phenylpropylamino)benzoic acid (IC(50) approximately 7 microM). The malaria-induced pathway differed in its inhibition by a range of anion channel inhibitors when compared to the endogenous, volume-activated osmolyte pathway of chicken red cells. There were also differences in the selectivity of sorbitol and taurine by the two permeation routes. The data presented here are consistent with the presence of two distinct organic solute pathways in infected chicken red cells. The first is an endogenous volume-activated pathway, which is not activated by the parasite and the second is a malaria-induced pathway, similar to those that are induced by other types of malaria in other host species.