Voltage-activated H+ currents will be studied in rat alveolar epithelial cells in primary culture, using the whole-cell and excised patch configurations of the gigohm-seal technique. Alveolar epithelial cells produce and secrete pulmonary surfactant, which keeps alveoli from collapsing during expiration. The alveolar epithelium also prevents edema by absorbing fluid, and regulates the pH and electrolyte composition of the alveolar subphase. H+ currents, described recently in mammalian cells, serve to extrude acid from cells. This project will explore the nature of the H+ conductance mechanism (gH) and the cooperative regulation of H+ channels gating by pH-O, PH-i, and voltage. Is the voltage-dependence set by a """"""""proton well"""""""" mechanism, in which membrane voltage and H+ concentration have kinetically equivalent effects? Are there separate allosteric protonation sites on the inner and outer sides of the channel? The results will help define the conditions under which the gH is activated in intact cells. It is not known whether H+ currents are mediated by ion channels, or how protons permeate the putative channels. A fundamental hypothesis proposed is that voltage-activated H+ channels are NOT simple water-filled pores, but operate by a """"""""proton-wire"""""""" mechanism in which protons jump across a hydrogen-bonded chain formed at least partially of side groups within an integral membrane protein. This mechanism differs from a """"""""water-wire"""""""" mechanism, protons hopping from one water molecule to the next, by which H+ permeates other kinds of ion channels which ARE water-filled pores. Single-channel currents will be recorded directly if possible, taking advantage of recent improvements in patch-clamp circuitry and quartz pipettes. The behavior of single H+ channels will also be studied using stationary H+ current noise (variance) analysis at varying temperatures, pH-O and PH-i and in the presence of deuterium, to define the nature of the proton conductance. The behavior of H+ channels at physiological temperatures will be determined. The gH, whose voltage dependence is set by pH-O and pH-i will be used as a biological sensor to study antiport in alveolar epithelium. Fundamental properties of this important pH-regulating transporter, including its dependence on internal and external [H+] and [Na+], and its possible regulation by phosphorylation will be examined using this novel approach. The possibility that Na+-H+ antiport has a rate-determining voltage- dependent step in its reaction cycle will be tested.
Showing the most recent 10 out of 32 publications
