Excretion

Voltage polarization of the basolateral membrane of principal cells in malpighian tubules of the Yellow Fever mosquito Aedes aegypti: role of the vacuolar H+-ATPase at the apical membrane

R. Masia & K. W. Beyenbach

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

The mechanisms of K + and Na + entry from the peritubular bath into principal cells of Malpighian tubules of the yellow fever mosquito was studied in single principal cells with the methods of two-electrode voltage clamp (TEVC). In the presence of normal Ringer solution in the peritubular bath, the intracellular voltage of principal cells (Vpc) was -86.9 mV and cell resistance (Rpc) was 408 K? (n=23). The addition of 5 mM Ba 2+ to the peritubular Ringer significantly increased Rpc to 716 K?, indicating entry of K + into the cell via Ba 2+ -sensitive K + channels. However, Ba 2+ -block of these K + channels significantly hyperpolarized Vpc to -101.1 mV. This response is opposite to the effect of Ba 2+ in Malpighian tubules of ants (Weltens, Cell Physiol. Biochem. 2, 101, 1992) and most other eucaryotic cells, where diffusion of K + out of the cell is the major source of the membrane voltage, and where the block of K + conductance depolarizes the membrane voltage towards zero. With K + channels blocked by Ba 2+ , the addition of di-butyryl cAMP (db-cAMP, 0.1 mM), significantly depolarized Vpc to -53.2 mV and significantly decreased Rpc to 510 K?, confirming the increase in basolateral membrane Na + conductance (Am. J. Physiol. 248, R339, 1985). This Na + conductance derives from apparently basolateral membrane Na + channels that have low affinity for amiloride, because amiloride at a concentration of 0.1 mM failed to block the effects of db-cAMP. A 10-fold higher amiloride concentration fully reversed the effects of db-cAMP on Rpc (765 K?), and it significantly increased Vpc from -53.2 mV to -71.0 mV (akin to the block of K + channels by Ba 2+ ). The hyperpolarization of Vpc after block of basolateral membrane K + channels indicates that the essential polarizing component of this membrane is not a K + diffusion potential, but rather the electrogenic vacuolar H + -ATPase (proton pump) located in the apical membrane. Active H + transport into the tubule lumen polarizes the apical membrane (cell negative) with respect to the luminal fluid to values in excess of 110 mV. Current returning from the lumen to the cytoplasmic side of the proton pump, via the shunt and the basolateral membrane, couples the electrical potential of the apical membrane to the basolateral membrane, where it provides the electromotive force for the entry of K + and Na + into the cell. (NSF IBN 9604394)

Index terms: vacuolar H + -ATPase, energizing cell membranes, principal cells, electrical coupling, voltage-driven K + and Na + entry.

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