Andrea J. Yool

Forskolin stimulation of water and cation permeability in aquaporin 1 water channels

Aquaporin1, a six-transmembrane domain protein, is a water channel present in many fluid-secreting and -absorbing cells. In Xenopus oocytes injected with aquaporin1 complementary RNA, the application of forskolin or cyclic 8-bromo- adenosine 3′,5′-monophosphate increased membrane permeability to water and triggered a cationic conductance. The cationic conductance was also induced by direct injection of protein kinase A (PKA) catalytic subunit, reduced by the kinase inhibitor H7, and blocked by HgCl2, an inhibitor of aquaporin1. The cationic permeability of the aquaporin1 channel is activated by a cyclic adenosine monophosphate-dependent mechanism that may involve direct or indirect phosphorylation by PKA.

EXCESS POTASSIUM INDUCES LARVAL METAMORPHOSIS IN FOUR MARINE INVERTEBRATE SPECIES

An increase in the concentration of K+ in defined seawater medium induces settlement and metamorphosis in larvae of the marine molluscs Phestilla sibogae, Haliotis rufescens, and Astraea undosa, and in larvae of the marine annelid Phragmatopoma californica. The effect is dose-dependent, optimal at approximately double the normal concentration of K+ in seawater, and specific for the K+ ion. The ability of K+ to directly influence cell membrane potential is proposed as an explanation for its broad effectiveness as a metamorphic inducer for larvae that recruit to different habitats. Depolarization of externally accessible, excitable cells thus is suggested to be a mechanism common to the induction of settlement and metamorphosis of a number of species. For Phestilla and Haliotis, the inductive effect of excess K+ is additive with that of the substratum-derived inducers or analogs. The sensitivity of induction by K+ to external tetraethylammonium (TEA, a K+-channel blocker) reported previously for Haliotis (B...

Inhibition of Aquaporin-1 and Aquaporin-4 water permeability by a derivative of the loop diuretic bumetanide acting at an internal pore-occluding binding site

Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na+-K+-2Cl- cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (≥100 μM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC50 ∼20 μM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC50 ∼8 μM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 μM had no effect, and 20 μM caused 20% block of human Na+-K+-2Cl- cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl- currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Ion Channel Function of Aquaporin-1 Natively Expressed in Choroid Plexus

Aquaporins are known as water channels; however, an additional ion channel function has been observed for several including aquaporin-1 (AQP1). Using primary cultures of rat choroid plexus, a brain tissue that secretes CSF and abundantly expresses AQP1, we confirmed the ion channel function of AQP1 and assessed its functional relevance. The cGMP-gated cationic conductance associated with AQP1 is activated by an endogenous receptor guanylate cyclase for atrial natriuretic peptide (ANP). Fluid transport assays with confluent polarized choroid plexus cultures showed that AQP1 current activation by 4.5 μm ANP decreases the normal basal-to-apical fluid transport in the choroid plexus; conversely, AQP1 block with 500 μm Cd2+ restores fluid transport. The cGMP-gated conductance in the choroid plexus is lost with targeted knockdown of AQP1 by small interfering RNA (siRNA), as confirmed by immunocytochemistry and whole-cell patch electrophysiology of transiently transfected cells identified by enhanced green fluorescent protein. The properties of the current (permeability to Na+, K+, TEA+, and Cs+; voltage insensitivity; and dependence on cGMP) matched properties characterized previously in AQP1-expressing oocytes. Background K+ and Cl− currents in the choroid plexus were dissected from AQP1 currents using Cs-methanesulfonate recording salines; the background currents recorded in physiological salines were not affected by AQP1–siRNA treatment. These results confirm that AQP1 can function as both a water channel and a gated ion channel. The conclusion that the AQP1-associated cation current contributes to modulating CSF production resolves a lingering concern as to whether an aquaporin ionic conductance can have a physiologically relevant function.

Tetraethylammonium block of water flux in Aquaporin-1 channels expressed in kidney thin limbs of Henle's loop and a kidney-derived cell line.

BackgroundAquaporin-1 (AQP1) channels are constitutively active water channels that allow rapid transmembrane osmotic water flux, and also serve as cyclic-GMP-gated ion channels. Tetraethylammonium chloride (TEA; 0.05 to 10 mM) was shown previously to inhibit the osmotic water permeability of human AQP1 channels expressed in Xenopus oocytes. The purpose of the present study was to determine if TEA blocks osmotic water flux of native AQP1 channels in kidney, and recombinant AQP1 channels expressed in a kidney derived MDCK cell line. We also demonstrate that TEA does not inhibit the cGMP-dependent ionic conductance of AQP1 expressed in oocytes, supporting the idea that water and ion fluxes involve pharmacologically distinct pathways in the AQP1 tetrameric complex.ResultsTEA blocked water permeability of AQP1 channels in kidney and kidney-derived cells, demonstrating this effect is not limited to the oocyte expression system. Equivalent inhibition is seen in MDCK cells with viral-mediated AQP1 expression, and in rat renal descending thin limbs of Henles loops which abundantly express native AQP1, but not in ascending thin limbs which do not express AQP1. External TEA (10 mM) does not block the cGMP-dependent AQP1 ionic conductance, measured by two-electrode voltage clamp after pre-incubation of oocytes in 8Br-cGMP (10–50 mM) or during application of the nitric oxide donor, sodium nitroprusside (2–4 mM).ConclusionsTEA selectively inhibits osmotic water permeability through native and heterologously expressed AQP1 channels. The pathways for water and ions in AQP1 differ in pharmacological sensitivity to TEA, and are consistent with the idea of independent solute pathways within the channel structure. The results confirm the usefulness of TEA as a pharmacological tool for the analysis of AQP1 function.

Aquaporins: Multiple Roles in the Central Nervous System

Aquaporins (AQPs) represent a diverse family of membrane proteins found in prokaryotes and eukaryotes. The primary aquaporins expressed in the mammalian brain are AQP1, which is densely packed in choroid plexus cells lining the ventricles, and AQP4, which is abundant in astrocytes and concentrated especially in the end-feet structures that surround capillaries throughout the brain and are present in glia limitans structures, notably in osmosensory areas such the supraoptic nucleus. Water movement in brain tissues is carefully regulated from the micro- to macroscopic levels, with aquaporins serving key roles as multifunctional elements of complex signaling assemblies. Intriguing possibilities suggest links for AQP1 in Alzheimers disease, AQP4 as a target for therapy in brain edema, and a possible contribution of AQP9 in Parkinsons disease. For all the aquaporins, new contributions to physiological functions are likely to continue to be discovered with ongoing work in this rapidly expanding field of research. NEUROSCIENTIST 13(5):470—485, 2007.

Roles for novel pharmacological blockers of aquaporins in the treatment of brain oedema and cancer

1. Aquaporins (AQPs) are targets for drug discovery for basic research and medicine. Human diseases involving fluid imbalances and oedema are of major concern and involve tissues in which AQPs are expressed. The range of functional properties of AQPs is continuing to expand steadily with ongoing research in the field.

A fascinating tail: cGMP activation of aquaporin-1 ion channels

Aquaporin-1 (AQP1) is a member of the diverse major intrinsic protein family of water and solute channels. AQP1 is known as an osmotic water channel in kidney, brain, vascular system and other tissues, and recently has been demonstrated to function as a cation channel gated by cGMP. Electrophysiology and binding assays implicate direct cGMP binding in the AQP1 C-terminus and sequence similarities with cyclic-nucleotide-gated channels support the idea that the AQP1 C-terminus mediates ion channel activation. In this article, new data show that the AQP1 C-terminus also exhibits homology, at key residues, with the substrate-selectivity subdomain of cyclic nucleotide phosphodiesterases. Distinct pathways for fluxes of water and ions in the tetrameric AQP1 channel indicate an intriguing multifunctional capacity. The physiological role of AQP1 in transmembrane signaling remains to be elucidated for these channels expressed in native tissues.

Functional domains of aquaporin-1 : Keys to physiology, and targets for drug discovery

Aquaporins (AQPs) are expressed in physiologically essential tissues and organs in which edema and fluid imbalances are of major concern. Potential roles in brain water homeostasis and edema, angiogenesis, cell migration, development, neuropathological diseases, and cancer suggest that this family of membrane proteins is an attractive set of novel drug targets. A problem in pursuing therapeutic and basic research strategies for dissecting contributions of AQPs to cell and tissue functions is that little is known regarding the pharmacology of AQP channels; currently defined agents such as tetraethylammonium and phloretin as blockers for aquaporins suffer from a lack of specificity and potency. Subtypes of AQPs modulated by signaling pathways could enable discrete localized control of fluid homeostasis, volume and morphology in cells and intracellular organelles, and might be found to participate in many different aspects of physiology, such as the control of paracellular permeability, process extension, growth, migration, and other responses involving changes in cell shape or surface to volume ratios. Recognizing that AQP1 is a water channel and, under permissive conditions, also a cGMP-gated cation channel, evidence in various tissues for a coupling of the cGMP signaling cascade to a physiological outcome that might involve AQP1 dual ion-and-water channel functions is of interest. Groundbreaking advances in defining aquaporin gating mechanisms suggest conformational changes are important elements in regulation and gating across classes of aquaporins. With a rapidly expanding knowledge of aquaporin structure and functional regulation, new avenues for manipulation of aquaporin channels are likely to be discovered. In parallel, a discovery for novel compounds with specificity and potency for aquaporins is a compelling goal. The need for pharmacological agents to dissect the roles of aquaporins in physiological and pathological processes is a clear call for further research in the field.

Structure, function and translational relevance of aquaporin dual water and ion channels.

Aquaporins have been assumed to be selective for water alone, and aquaglyceroporins are accepted as carrying water and small uncharged solutes including glycerol. This review presents an expanded view of aquaporins as channels with more complex mechanisms of regulation and diverse repertoires of substrate permeabilities than were originally appreciated in the early establishment of the field. The role of aquaporins as dual water and gated ion channels is likely to have physiological and potentially translational relevance, and can be evaluated with newly developed molecular and pharmacological tools. Ion channel activity has been shown for Aquaporins -0, -1, and -6, Drosphila Big Brain, and plant Nodulin-26. Although the concept of ion channel function in aquaporins remains controversial, research advances are beginning to define not only the ion channel function but also the detailed molecular mechanisms that govern and mediate the multifunctional capabilities. With regard to physiological relevance, the adaptive benefit of expression of ion channel activity in aquaporins, implied by amino acid sequence conservation of the ion channel gating domains, suggests they provide more than water or glycerol and solute transport. Dual ion and water channels are of interest for understanding the modulation of transmembrane fluid gradients, volume regulation, and possible signal transduction in tissues expressing classes of aquaporins that have the dual function capability. Other aquaporin classes might be found in future work to have ion channel activities, pending identification of the possible signaling pathways that could govern activation.