Katalin Kalman

Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1

Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail ofhSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1.3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed withhIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

Molecular basis of pH and Ca2+ regulation of aquaporin water permeability.

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca2+ increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can “tune” the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca2+ sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca2+ and pH modulation are separable and arise from processes on opposite sides of the membrane.

Lack of linkage or association between schizophrenia and the polymorphic trinucleotide repeat within the KCNN3 gene on chromosome 1q21

To determine the importance of a candidate gene KCNN3 (formerly named hSKCa3) in the susceptibility to schizophrenia, we have studied the genotypes of a (CAG)n polymorphism within this gene in the DNAs of the members of 54 multiplex families with this disease. Parametric and nonparametric linkage analysis did not provide evidence for linkage between KCNN3 (that we mapped to chromosome 1q21) and schizophrenia. Furthermore, we observed no difference in the distribution of the (CAG)n alleles between affected and normal individuals. These results do not support the hypothesis that larger KCNN3 alleles are preferentially associated with schizophrenia [Chandy et al. 1998 Mol Psychiatr 3:32-37] in individuals from multiply affected families.

Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer.

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777–6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573–580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.

Two distinct aquaporin 0s required for development and transparency of the zebrafish lens.

PURPOSE AQP0, formerly known as MIP26, likely has multiple separate functions in the mammalian lens, including water transport, formation of thin junctions, and interactions with other lens components. Although mammalian genomes contain only one Aqp0 gene, the zebrafish genome contains two, Aqp0a and Aqp0b, and the putative multiple functions of the single mammalian protein may be divided between these two genes. The purpose of this study was to exploit this gene duplication and divergence to illuminate the multiple functions of AQP0 in the lens. METHODS Wholemount in situ hybridization and Western blot analyses were used to determine the expression pattern of Aqp0a and Aqp0b. The role of both proteins was studied in vivo by microinjection of antisense morpholino oligonucleotides in zebrafish. The water permeability of both proteins was tested using the Xenopus oocyte swelling assay and a yeast shrinkage assay. RESULTS Both genes, like their mammalian counterpart, are expressed in the lens. Morpholino knock-down of either gene alone led to cataract formation, indicating that both genes are necessary for normal lens development and transparency. Full-length Aqp0a is a functional water channel when expressed in Xenopus oocytes and in yeast, whereas Aqp0b was not. However, the addition of an HA-tag at its N terminus converted Aqp0b to a water channel in Xenopus oocytes. CONCLUSIONS These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens.

Phosphorylation Determines the Calmodulin-mediated Ca2+ Response and Water Permeability of AQP0

In Xenopus oocytes, the water permeability of AQP0 (Pf) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca2+ regulation of Pf, we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 Pf manifests four distinct phenotypes: Group 1, with high Pf upon removal of external Ca2+ (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high Pf in elevated (5 mm) external Ca2+ (S235D and R241A); Group 3, with high Pf and no Ca2+ regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low Pf and no Ca2+ regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high Pf and no Ca2+ regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 Pf. CaM binding is associated with a low Pf state, and a lack of CaM binding is associated with a high Pf state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce Pf changes contributing to the development of a cataract.