Structure and function of two-pore-domain K+ channels: contributions from genetic model organisms.

K+ channels that possess two pore domains in each channel subunit are common in many animal tissues. Such channels are generated from large families of subunits and are implicated in several functions, including temperature sensation, responses to ischaemia, K+ homeostasis and setting the resting potential of the cell. Their activity can be modulated by polyunsaturated fatty acids, pH and oxygen, and some are candidate targets of volatile anaesthetics. However, despite their potential as targets for novel drugs for human health, comparatively little is known about the molecular basis of their diverse physiological and pharmacological properties. Genetic model organisms have considerable potential for improving our understanding of these channels. In this article, we review the contributions of some of these genetic model organisms to recent advances in our knowledge of two-pore-domain K+ channels.

The effects of amyloid peptides on A-type K(+) currents of Drosophila larval cholinergic neurons: modeled actions on firing properties.

In a previous paper we described the actions of beta-amyloid on an A-type K(+) current from Drosophila 3(rd) Instar larval neurons. The results were a depolarizing shift in the steady-state voltage dependence of inactivation and an increase in the rate of recovery from inactivation of the current. In this work we have used the simulation program NEURON to construct a model cell. We then use the model to predict the effects of changing the A-type K(+) current as was observed in the amyloid treated neurons on the firing properties of the cell. We show that changing the steady-state voltage dependence of inactivation of the current to a more depolarized level as observed in experiments in beta-amyloid treated neurons causes an increase in the threshold for the initiation of repetitive firing. However, increasing the rate of recovery from inactivation had no effect. Changing both properties simultaneously had no additional effect over changing the voltage dependence of inactivation alone. Thus, a change in the steady-state properties of the A-type K(+ )current as seen in the amyloid-treated Drosophila cholinergic neurons is sufficient to alter the firing properties of the modeled cell.

Dimeric cystic fibrosis transmembrane conductance regulator exists in the plasma membrane.

CFTR (cystic fibrosis transmembrane conductance regulator) mediates chloride conduction across the apical membrane of epithelia, and mutations in CFTR lead to defective epithelial fluid transport. Recently, there has been considerable interest in determining the quaternary structure of CFTR at the cell surface, as such information is a key to understand the molecular basis for pathogenesis in patients harbouring disease-causing mutations. In our previous work [Ramjeesingh, Li, Kogan, Wang, Huan and Bear (2001) Biochemistry 40, 10700-10706], we showed that monomeric CFTR is the minimal functional form of the protein, yet when expressed in Sf 9 cells using the baculovirus system, it also exists as dimers. The purpose of the present study was to determine if dimeric CFTR exists at the surface of mammalian cells, and particularly in epithelial cells. CFTR solubilized from membranes prepared from Chinese-hamster ovary cells stably expressing CFTR and from T84 epithelial cells migrates as predicted for monomeric, dimeric and larger complexes when subjected to sizing by gel filtration and analysis by non-dissociative electrophoresis. Purification of plasma membranes led to the enrichment of CFTR dimers and this structure exists as the complex glycosylated form of the protein, supporting the concept that dimeric CFTR is physiologically relevant. Consistent with its localization in plasma membranes, dimeric CFTR was labelled by surface biotinylation. Furthermore, dimeric CFTR was captured at the apical surface of intact epithelial cells by application of a membrane-impermeable chemical cross-linker. Therefore it follows from the present study that CFTR dimers exist at the surface of epithelial cells. Further studies are necessary to understand the impact of dimerization on the cell biology of wild-type and mutant CFTR proteins.

Effects of amyloid peptides on A-type K+ currents of Drosophila larval cholinergic neurons.

Accumulation of amyloid (Abeta) peptides has been suggested to be the primary event in Alzheimer's disease. In neurons, K+ channels regulate a number of processes, including setting the resting potential, keeping action potentials short, timing interspike intervals, synaptic plasticity, and cell death. In particular, A-type K+ channels have been implicated in the onset of LTP in mammalian neurons, which is thought to underlie learning and memory. A number of studies have shown that Abeta peptides alter the properties of K+ currents in mammalian neurons. We set out to determine the effects of Abeta peptides on the neuronal A-type K+ channels of Drosophila. Treatment of cells for 18 h with 1 microM Abeta1-42 altered the kinetics of the A-type K+ current, shifting steady-state inactivation to more depolarized potentials and increasing the rate of recovery from inactivation. It also caused a decrease in neuronal viability. Thus it seems that alteration in the properties of the A-type K+ current is a prelude to the amyloid-induced death of neurons. This alteration in the properties of the A-type K+ current may provide a basis for the early memory impairment that was observed prior to neurodegeneration in a recent study of a transgenic Drosophila melanogaster line over-expressing the human Abeta1-42 peptide.

A heteromeric complex of the two nucleotide binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) mediates ATPase activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a member of the ABC superfamily of transporter proteins. Recently, crystal structures of intact, prokaryotic members of this family have been described. These structures suggested that ATP binding and hydrolysis occurs at two sites formed at the interface between their nucleotide binding domains (NBDs). In contrast to the prokaryotic family members, the NBDs of CFTR are asymmetric (both structurally and functionally), and previous to the present studies, it was not clear whether both NBDs are required for ATP hydrolysis. In order to assess the relative roles of the two NBDs of human CFTR, we purified and reconstituted NBD1 and NBD2, separately and together. We found that NBD1 and NBD2 by themselves exhibited relatively low ATPase activity. Co-assembly of NBD1 and NBD2 exhibited a 2-3-fold enhancement in catalytic activity relative to the isolated domains and this increase reflected enhanced ATP turnover (V(max)). These data provide the first direct evidence that heterodimerization of the NBD1 and NBD2 domains of CFTR is required to generate optimal catalytic activity.

CFTR directly mediates nucleotide-regulated glutathione flux.

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.

Paclitaxel affects cytosolic calcium signals by opening the mitochondrial permeability transition pore.

We have characterized the effects of the antimitotic drug paclitaxel (Taxol(TM)) on the Ca(2+) signaling cascade of terminally differentiated mouse pancreatic acinar cells. Using single cell fluorescence techniques and whole-cell patch clamping to record cytosolic Ca(2+) and plasma membrane Ca(2+)-dependent Cl(-) currents, we find that paclitaxel abolishes cytosolic Ca(2+) oscillations and in more than half of the cells it also induces a rapid, transient cytosolic Ca(2+) response. This response is not affected by removal of extracellular Ca(2+) indicating that paclitaxel releases Ca(2+) from an intracellular Ca(2+) store. Using saponin-permeabilized cells, we show that paclitaxel does not affect Ca(2+) release from an inositol trisphosphate-sensitive store. Furthermore, up to 15 min after paclitaxel application, there is no significant effect on either microtubule organization or on endoplasmic reticulum organization. The data suggest a non-endoplasmic reticulum source for the intracellular Ca(2+) response. Using the mitochondrial fluorescent dyes, JC-1 and Rhod-2, we show that paclitaxel evoked a rapid decline in the mitochondrial membrane potential and a loss of mitochondrial Ca(2+). Cyclosporin A, a blocker of the mitochondrial permeability transition pore, blocked both the paclitaxel-induced loss of mitochondrial Ca(2+) and the effect on Ca(2+) spikes. We conclude that paclitaxel exerts rapid effects on the cytosolic Ca(2+) signal via the opening of the mitochondrial permeability transition pore. This work indicates that some of the more rapidly developing side effects of chemotherapy might be due to an action of antimitotic drugs on mitochondrial function and an interference with the Ca(2+) signal cascade.