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The dependence of electrical transport pathways in Malpighian tubules on ATP

Daniel S. Wu and Klaus W. Beyenbach^*

Department of Biomedical Sciences, VRT 8014, Cornell University, Ithaca, NY 14853, USA

View larger version (88K): [in a new window]   Fig. 1. Electrophysiological study of a principal cell by the two-electrode voltage-clamp (TEVC) method in isolated Malpighian tubules of Aedes aegypti. (A) Transepithelial secretion of NaCl and KCl under control conditions. The question mark indicates uncertainty as to whether the exchanger transports both Na+ and K+. (B) Electrical equivalent circuit of transepithelial NaCl and KCl secretion consisting of the active transport pathway for Na+ and K+ ions through principal cells and the passive transport of Cl- through the shunt pathway. Ebl and Ea are the electromotive forces at the basolateral and apical membrane, respectively. Rbl, Ra and Rsh are the resistances of the basolateral membrane, the apical membrane, and the shunt, respectively. V is voltage measured across basolateral (bl) and apical (a) membranes of the cell. Vt and Rt are the transepithelial voltage (lumen-positive) and resistance, respectively. Numerical data are from previous studies (Pannabecker et al., 1992). (C) TEVC study of a principal cell in an isolated Malpighian tubule. (D) The measurement circuit in TEVC studies yields the input resistance Rpc, which is the sum of the parallel resistances of the basolateral membrane and the apical membrane in series with the shunt.

View larger version (22K): [in a new window]   Fig. 2. Dose—response curves of the effects of (A) cyanide (KCN) and (B) dinitrophenol (DNP) on the basolateral membrane voltage (Vbl, solid line) and the input resistance (Rpc, broken line) of principal cells of Malpighian tubules of the yellow fever mosquito Aedes aegypti. EC50 is the concentration at half-maximum response. Values from 6-14 principal cells (in parentheses) are shown.

View larger version (19K): [in a new window]   Fig. 3. Time course of the effects of 0.3 mmol l-1 cyanide (KCN) on the basolateral membrane voltage (Vbl), input resistance (Rpc), and the virtual active transport current (Iat(virt)) in a representative principal cell, and on intracellular ATP concentrations [ATP]i in 6-16 sets of five Malpighian tubules each. Values are mean ± S.E.M. (number of tubule sets). The temporal relationship between the electrical variables and [ATP] are approximate because exchanging the peritubular medium to include CN took 43 s in the electrical assay and 10 s in the ATP assay.

View larger version (15K): [in a new window]   Fig. 4. Loss of the K+-conductance of the basolateral membrane of principal cells after metabolic inhibition with CN. K+-conductance was evaluated by the effects of a tenfold increase in bath [K+], from 3.4 mmol l-1 to 34 mmol l-1, on the basolateral membrane voltage Vbl and input resistance Rpc of principal cells of Malpighian tubules isolated from Aedes aegypti. Values are means ± S.E.M. of five cells. NS, not significant.

View larger version (15K): [in a new window]   Fig. 5. Loss of Ba2+-blockade of K+-channels of the basolateral membrane of principal cells after metabolic inhibition with CN. The effect of peritubular BaCl2 (5 mmol l-1), a competitive blocker of K+-channels, was evaluated on basolateral membrane voltage Vbl and input resistance Rpc of principal cells of Malpighian tubules isolated from Aedes aegypti. Values are means ± S.E.M. of 9-50 cells. NS, not significant.

View larger version (97K): [in a new window]   Fig. 6. The apical brush border membrane of principal cells in Malpighian tubules of Aedes aegypti. The apical membrane is characterized by a tall, mitochondrion-rich brush border (A) which brings the site of ATP synthesis in close proximity to the site of utilization (B). ATP is synthesized at the inner mitochondrial membrane by the ATP synthase, consisting of the catalytic F1 segment and the proton-conducting F0 segment. What drives ATP synthesis is the proton gradient generated by the electron transport chain (ETC). ATP4- is transported across the inner membrane in exchange for ADP3-. ATP4- goes on to diffuse across the outer mitochondrial membrane to the apical membrane of the brush border. The hydrolysis of ATP by the V-type H+-ATPase powers H+ transport into the micro-environment of the brush border, thereby generating a large apical membrane voltage (-110.6 mV on average). The H+ electrochemical potential thus established across the apical membrane can drive the extrusion of Na+ and K+ via nH+/cation exchange transporter(s) that have yet to be isolated.

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