UC Davis

Ion channels and transporters are membrane proteins that play important roles in many physiological functions. This dissertation will focus on CLC-0 a Cl¬- channel from the CLC channel/transporter family. The CLC family members are expressed ubiquitously in various tissues and are critical for many physiological processes, such as acidification of intracellular vesicles, regulation of muscle excitability, and the control of the secretion and reabsorption of salts in renal tubules, etc. Of all the members of the CLC family, CLC-0 is the prototype channel of the family. CLC-0 is expressed in the electric organ of Torpedo electric rays. The channel exhibits two distinct gating mechanisms: the fast and the slow gating (also known as “inactivation” gating). The fast-gating mechanism is better understood, while the slow gating mechanism is still murky. In this dissertation, I examine the role of intracellular H+ in the slow gating of CLC-0. Our inquiry began with an observation that intracellular H+ inhibits the current of CLC-0. This inhibition of CLC-0 by intracellular H+ results from the closure of the slow gate, as supported by similar voltage-dependence and temperature-dependence between intracellular H+-induced inhibition and the slow-gate closure. Furthermore, the mutants of CLC-0 that show less slow-gate closure are more difficult to be inhibited by intracellular H+. Further studies show that after H+ inhibition the current recovery kinetics of these mutants at -40 mV membrane potential are faster than that of the WT channel. Detailed analyses of the current recovery kinetics of these mutants reveal multiple inactivation (slow-gate closed) states of CLC-0. I have also studied the effects of membrane voltage and ion permeation on the channel recovery from inactivation. Finally, since protonation initiates the channel inactivation, I have also attempted to search for the proton-binding site(s). Although, no definitive protonation site(s) have been identified, the overall results from this study have provided insights into the slow-gating mechanism of CLC-0.