Dan A. Klaerke

Aquaporin homologues in plants and mammals transport ammonia

Using functional complementation and a yeast mutant deficient in ammonium (NH4 +) transport (Δmep1–3), three wheat (Triticum aestivum) TIP2 aquaporin homologues were isolated that restored the ability of the mutant to grow when 2 mM NH4 + was supplied as the sole nitrogen source. When expressed in Xenopus oocytes, TaTIP2;1 increased the uptake of NH4 + analogues methylammonium and formamide. Furthermore, expression of TaTIP2;1 increased acidification of the oocyte‐bathing medium containing NH4 + in accordance with NH3 diffusion through the aquaporin. Homology modeling of TaTIP2;1 in combination with site directed mutagenesis suggested a new subgroup of NH3‐transporting aquaporins here called aquaammoniaporins. Mammalian AQP8 sharing the aquaammoniaporin signature also complemented NH4 + transport deficiency in yeast.

The human Na+–glucose cotransporter is a molecular water pump

1 The human Na+‐glucose cotransporter (hSGLT1) was expressed in Xenopus laevis oocytes. The transport activity, given by the Na+ current, was monitored as a clamp current and the concomitant flux of water followed optically as the change in oocyte volume. 2 When glucose was added to the bathing solution there was an abrupt increase in clamp current and an immediate swelling of the oocyte. The transmembrane transport of two Na+ ions and one sugar molecule was coupled, within the protein itself, to the influx of 210 water molecules. 3 This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. 4 The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein from the downhill fluxes of Na+ and sugar to the uphill transport of water, indicative of secondary active transport of water. 5 Unstirred layer effects were ruled out on the basis of experiments on oocytes treated with gramicidin or other ionophores. Na+ currents maintained by ionophores did not lead to any initial water movements. 6 The finding of a molecular water pump allows for new models of cellular water transport which include coupling between ion and water fluxes at the protein level; the hSGLT1 could account for almost half the daily reuptake of water from the small intestine.

Pharmacological modulation of SK3 channels.

Small-conductance, calcium-activated K+ channels (SK channels) are voltage-insensitive channels that have been identified molecularly within the last few years. As SK channels play a fundamental role in most excitable cells and participate in afterhyperpolarization (AHP) and spike-frequency adaptation, pharmacological modulation of SK channels may be of significant clinical importance. Here we report the functional expression of SK3 in HEK293 and demonstrate a broad pharmacological profile for these channels. Brain slice studies commonly employ 4-aminopyridine (4-AP) to block voltage-dependent K+ channels or a methyl derivative of bicuculline, a blocker of gamma-aminobutyric acid (GABA)-gated Cl- channels, in order to investigate the role of various synapses in specialized neural networks. However, in this study both 4-AP and bicuculline are shown to inhibit SK3 channels (IC50 values of 512 microM and 6 microM, respectively) at concentrations lower than those used for brain slice recordings. Riluzole, a potent neuroprotective drug with anti-ischemic, anticonvulsant and sedative effects currently used in the treatment of amyotrophic lateral sclerosis, activates SK3 channels at concentrations of 3 microM and above. Amitriptyline, a tricyclic antidepressive widely used clinically, inhibits SK3 channels with an IC50 of 39.1 +/- 10 microM (n=6).

WATER TRANSPORT BY THE NA+/GLUCOSE COTRANSPORTER UNDER ISOTONIC CONDITIONS

Abstract Solute cotransport in the Na + glucose cotransporter is directly coupled to significant water fluxes. The water fluxes are energized by the downhill fluxes of the other substrates by a mechanism within the protein itself. In the present paper we investigate the Na + glucose cotransporter expressed in Xenopus oocytes. We present a method which allows short-term exposures to sugar under voltage clamp conditions. We demonstrate that water is cotransported with the solutes despite no osmotic differences between the external and intracellular solutions. There is a fixed ratio of 195:1 between the number of water molecules and the number of Na+ ions transported, equivalent to 390 water molecules per glucose molecule. Unstirred layer effects are ruled out on the basis of experiments on native oocytes incubated with the ionophores gramicidin D or nystatin.

KCNE4 Is an Inhibitory Subunit to Kv1.1 and Kv1.3 Potassium Channels

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.

Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE β-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38-40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel.

The voltage-gated potassium channel subunit, Kv1.3, is expressed in epithelia

The Shaker-type voltage-gated potassium channel, Kv1.3, is believed to be restricted in distribution to lymphocytes and neurons. In lymphocytes, this channel has gained intense attention since it has been proven that inhibition of Kv1.3 channels compromise T lymphocyte activation. To investigate possible expression of Kv1.3 channels in other types of tissue, such as epithelia, binding experiments, immunoprecipitation studies and immunohistochemical studies were performed. The double-mutated, radiolabeled peptidyl ligand, (125)I-HgTX(1)-A19Y/Y37F, which selectively binds Kv1.1, Kv1.2, Kv1.3 and Kv1.6 channels, was used to perform binding studies in epithelia isolated from rabbit kidney and colon. The equilibrium dissociation constant for this ligand was found to be in the sub-picomolar range and the maximal receptor concentration (in fM/mg protein) 1.68 for colon and 0.61-0.75 for kidney epithelium. To determine the subtype of Kv1 channels, immunoprecipitation studies with (125)I-HgTX(1)-A19Y/Y37F labeled epithelial membranes were performed with specific antibodies against Kv1.1, Kv1.2, Kv1.3, Kv1.4 or Kv1.6 subunits. These studies demonstrated that Kv1.3 subunits constituted more than 50% of the entire Kv1 subunit population. The precise localization of Kv1.3 subunits in epithelia was determined by immunohistochemical studies.

Differential expression of BK channel isoforms and β-subunits in rat neuro-vascular tissues

We investigated the expression of splice variants and beta-subunits of the BK channel (big conductance Ca2+-activated K+ channel, Slo1, MaxiK, KCa1.1) in rat cerebral blood vessels, meninges, trigeminal ganglion among other tissues. An alpha-subunit splice variant X1(+24) was found expressed (RT-PCR) in nervous tissue only where also the SS4(+81) variant was dominating with little expression of the short form SS4(0). SS4(+81) was present in some cerebral vessels too. The SS2(+174) variant (STREX) was found in both blood vessels and in nervous tissue. In situ hybridization data supported the finding of SS4(+81) and SS2(+174) in vascular smooth muscle and trigeminal ganglion. beta-subunits beta2 and beta4 showed high expression in brain and trigeminal ganglion and some in cerebral vessels while beta1 showed highest expression in blood vessels. beta3 was found only in testis and possibly brain. A novel splice variant X2(+92) was found, which generates a stop codon in the intracellular C-terminal part of the protein. This variant appears non-functional as a homomer but may modulate the function of other splice-variants when expressed in Xenopus oocytes. In conclusion a great number of splice variant and beta-subunit combinations likely exist, being differentially expressed among nervous and vascular tissues.

Cell volume and membrane stretch independently control K+ channel activity

A number of potassium channels including members of the KCNQ family and the Ca2+ activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell‐attached patch clamp studies (∼50 μm2 macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume‐insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of −5.0 ± 0.1 mmHg the increase amounted to 381 ± 146% (mean ±s.e.m., n= 6, P < 0.025). In contrast, in oocytes expressing the strongly volume‐sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.

Localization of Ca2+-activated big-conductance K+ channels in rabbit distal colon

Big-conductance Ca2+-activated K+ channels (BK channels) may play an important role in the regulation of epithelial salt and water transport, but little is known about the expression level and the precise localization of BK channels in epithelia. The aim of the present study was to quantify and localize the BK channels in the distal colon epithelium by iberiotoxin (IbTX) binding using the radiolabeled iberiotoxin analogue 125I-IbTX-D19Y/Y36F, by autoradiography and by immunohistochemical studies. The results showed that the surface cells, responsible for Na+ absorption, contained a high number of BK channels, whereas the abundance of the channels in the Cl−-secreting crypt cells was very low or absent. Surprisingly, the 125I-IbTX-D19Y/Y36F binding and immunohistochemical studies showed expression of BK channels in the apical as well as in the basolateral membranes of the surface cells. In conclusion, the significant and distinct expression of BK channels in epithelia, combined with their strict regulation, indicate that these channels may play an important role in the overall regulation of salt and water transport.