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First published online January 18, 2008
Journal of Experimental Biology 211, 409-422 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011213

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Gap junctions in Malpighian tubules of Aedes aegypti

Xing-He Weng¹, Peter M. Piermarini¹, Atsuko Yamahiro¹, Ming-Jiun Yu², Daniel J. Aneshansley³ and Klaus W. Beyenbach^1,*

¹ Department of Biomedical Sciences, Cornell University, Ithaca, NY 14853, USA
² National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
³ Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY 14853, USA

View larger version (17K): [in this window] [in a new window]   Fig. 1. Block diagram for investigating electrical coupling of principal cells through gap junctions in isolated Malpighian tubules. Cell 1 was voltage clamped at a desired command voltage and the voltage deflections in cells 1, 2 and 3 were recorded. The input resistance Rinput of cell 1 was calculated from the values of (1) current injected into cell 1 to hold it at the desired command voltage, and (2) the change in the basolateral membrane voltage of cell 1 (Vbl1).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Transepithelial secretion of NaCl and KCl by Malpighian tubules of the yellow fever mosquito. (A) Minimal molecular transport model. Electroneutral Na/H exchange and a cAMP-activated Na+ conductance allow the entry of Na+ from the hemolymph into principal cells. K+ enters via K+ channels. Na+ and K+ are moved across the apical membrane via a hypothetical cation/H exchanger that in turn is driven by the transmembrane H+ electrochemical potential generated by the vacuolar type H+-ATPase located in the apical membrane. The lumen-positive transepithelial voltage generated by transcellular Na+ and K+ secretion drives the transepithelial secretion of Cl– through the paracellular pathway. (B) Minimal electrical transport model that illustrates the active transport pathway through the cell and the passive transport pathway between the cells. Basolateral (bl) and apical (a) membranes are represented by an electromotive force (E) and a resistance (R). The paracellular resistance is represented by the shunt resistance Rsh.

View larger version (28K): [in this window] [in a new window]   Fig. 3. The Malpighian tubule modeled as a single electrical cable (A) and as a double cable (B). In the single cable model, the single core resistance (Rco) is the axial resistance along the length of the tubule. It includes the tubule lumen and epithelial cells. In the double cable model, there are two axial resistances: the gap-junction resistance (Rgj) and the lumen resistance (Rlu). E, electromotive force; R, resistance; a, apical membrane; bl, basolateral membrane; sh, paracellular shunt pathway.

View larger version (33K): [in this window] [in a new window]   Fig. 4. Measurement of the gap-junction resistance between principal cells of isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. (A) The measuring circuit in the tubule modeled as a double cable; (B) reduction of the circuit by eliminating Rsh via the short-circuiting effects of leucokinin-VIII; (C) further reduction of the circuit by combining the parallel resistances in circuit in B. The electromotive forces (E) are neglected in C as they should not be affected by voltage-clamping cell 1. V, R and I have their usual meaning; a, apical membrane; bl, basolateral membrane; lu, tubule lumen; gj, gap junction; nj, non-junction. The non-junctional resistance Rnj includes Ra, Rbl and Rlu.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Estimate of the input resistance (Rinput) from the non-junctional (nj) and gap-junctional (gj) resistances (R) of principal cells. Rinput can also be measured directly as the ratio Vbl1/Iinject. The comparison of estimated values and values measured directly tests the short-circuit assumption needed to obtain measurements of the gap-junction resistance. In previous studies we have used Rpc to refer to Rinput (Masia et al., 2000). The non-junctional resistance Rnj includes Ra, Rbl and Rlu.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Spontaneous oscillations of the basolateral membrane voltage (Vbl) in an isolated Malpighian tubule of Aedes aegypti. Three principal cells were impaled with conventional microelectrodes. The remaining epithelial cells (more than 120) are not shown in the tubule diagram. Note that cell 2 separates cell 1 (red trace) from cells 3 (blue trace) and 4 (green trace) from which voltages are recorded.

View larger version (5K): [in this window] [in a new window]   Fig. 7. Profiles of the basolateral membrane voltage deflections (Vbl) along the length of Malpighian tubules of Aedes aegypti. Cell 1 was voltage-clamped at a hyperpolarizing voltage of 40 mV, and the voltage deflections across the basolateral membranes of cells 2 and 3 recorded in the absence (control) and presence of leucokinin-VIII. Values are mean ± s.e.m. of 14 experiments.

View larger version (39K): [in this window] [in a new window]   Fig. 8. Inability of Lucifer Yellow to pass through gap junctions into neighboring epithelial cells. Green fluorescence identifies the principal cell injected with Lucifer Yellow at time 0 min. The image at 10 min is supplemented to outline the Malpighian tubule; bar, 100 µm.

View larger version (7K): [in this window] [in a new window]   Fig. 9. Neighbor-joining tree showing phylogenetic relationships between innexins in Drosophila and Aedes. Innexin1 from Caenorhabditis elegans (CeInx1) is the outgroup. The tree was constructed using MEGA 3 software (Kumar et al., 2004), based on the Poisson-corrected distance estimates. In this analysis, the cumulative branch length between the node of a branch (e.g. arrow) and two genes represents the proportion of amino acids that differ between them per residue (scale bar=0.1). For example, if two genes are separated by a cumulative branch length of `0.1', then one amino-acid residue differs between them for every ten amino acids. The number at each node indicates the bootstrap score (i.e. reliability) over 1000 replicates for that node. For example, a score of `94' indicates that the node occurred in 94% of the 1000 replicates. Accession numbers for Aedes innexins are listed in Table 5. Accession numbers (GenBank) for other innexins are as follows: DrInx1, NP_524824; DrPassover, NP_728361; DrInx3, NP_524730; DrInx4, NP_648049; DrInx5, NP_573353; DrInx6, NP_572374; DrInx7, NP_788872; CeInx1, NP_741826.

View larger version (51K): [in this window] [in a new window]   Fig. 10. RT-PCR analysis of Aedes Malpighian tubules for innexin transcripts. The image shows PCR products separated on a 1% agarose gel and stained with ethidium bromide. On the gel are results for PCRs designed to amplify each Aedes innexin using the primer pairs indicated in Table 1. For each innexin, the Malpighian tubule cDNA was used as a template in lane `a', and Malpighian tubule RNA was used as a template in lane `b'. The lane `mw' is a 1 Kb Plus DNA Ladder (Invitrogen), in which the first seven bands (starting from the bottom of gel) correspond to 100, 200, 300, 400, 500, 650 and 850 bp, respectively.

View larger version (41K): [in this window] [in a new window]   Fig. 11. Estimates of electrophysiological variables in the Malpighian tubule of the yellow fever mosquito. All values are normalized to cm tubule length. (A) The tubule modeled as a single cable (data from Pannabecker et al., 1992). The radial resistance is the transepithelial resistance consisting of the resistances of the apical membrane Ra, basolateral membrane Rbl and the shunt Rsh. The axial resistance of the tubule is the core resistance Rco. (B) The tubule modeled as a double cable. Here the axial resistance consists of the lumen resistance Rlu and the gap-junction resistance Rgap. In both models the transepithelial voltage Vt and the basolateral (Vbl) and apical (Va) membrane voltages are the same. E, electromotive force; De is the electrical diameter of the tubule lumen calculated from the lumen resistance Rlu (Pannabecker et al., 1992).