I. Origin of the voltage dependence of G protein regulation of P/Q-type calcium channels. II. A new tail of Pax6 and its interaction with calcium channel beta subunits

Part I. Origin of the Voltage Dependence of G Protein Regulation of P/Q-Type Ca2+ Channels. G protein (Gbetagamma)-mediated voltage-dependent inhibition of N- and P/Q-type Ca2+ channels contributes to presynaptic inhibition and short-term synaptic plasticity. It slows down channel activation and shifts the activation voltage to a more depolarized potential. These effects are the opposite of beta subunit (Ca vbeta) modulation. Intrigued by this contrariety, researchers have widely studied the interplay between Gbetagamma and Cavbeta in channel regulation. However, some critical issues, including whether Ca vbeta is necessary for Gbetagamma inhibition and whether Gbetagamma dislodges Cavbeta, remain controversial. In Chapter 3 of Part I, I addressed these questions. Gbetagamma inhibition of P/Q-type channels was reconstituted in inside-out membrane patches from Xenopus oocytes. Channels devoid of Cavbeta [produced by removing a mutant Cavbeta with a reduced affinity for the alpha-interacting domain (AID)] were still inhibited by Gbetagamma, but without any voltage dependence. Furthermore, a truncated Cavbeta containing only the AID-binding guanylate kinase (GK) domain could fully confer the voltage dependence. On the other hand, removal of Gbetagamma shifted the activation curve back to normal. In addition, under Gbetagamma treatment, channels with or without Cavbeta exhibited different inactivation speed. My results demonstrate an indispensable role of Cavbeta in voltage-dependent Gbetagamma inhibition and favor the scenario that Gbetagamma does not dislodge Ca vbeta from the channel complex.;The voltage dependence of Gbetagamma-mediated inhibition of N- and P/Q-type Ca2+ channels derives from the dissociation of Gbetagamma, from the Gbetagamma-binding pocket in the inhibited channels, but the molecular and biophysical mechanisms underlying Gbetagamma unbinding remain largely unclear. After showing that Cavbeta is required for this voltage dependence, in Chapter 4 of Part I, I report that the molecular pivot underlying the essential role of Cavbeta is a rigid alpha-helical structure between the AID, the primary Cavbeta docking site on the channel alpha1 subunit, and the pore-lining IS6 segment. When this rigid alpha-helical structure was disrupted by insertion of multiple glycines between the AID and IS6 segment, Cavbeta failed to confer voltage-dependence onto Gbetagamma regulation. However, another alpha 1 mutant containing 7 alanines (which support the formation of alpha-helical structures) in the same place exhibited all the main features of voltage-dependent Gbetagamma inhibition. These results suggest that depolarization-triggered movement of IS6, coupled to the subsequent conformational change of the Gbetagamma-binding pocket through a rigid a-helix induced by Cavbeta, causes the dissociation of Gbetagamma, and is fundamental to voltage-dependent Gbetagamma inhibition.;Part II. A New Tail of Pax6 and Its Interaction with Ca2+ Channel beta Subunits. Pax6 is a transcriptional factor that plays important roles in the development of the eye, nose, brain and pancreas. It contains two DNA-binding domains (paired domain and homeodomain), a glycine-rich linker connecting these two domains, and a carboxyl-terminal proline, serine and threonine-rich (PST) domain. Both the sequence and function of Pax6 are highly conserved from invertebrates to human. Here I report a new splicing isoform of Pax6, Pax6(S), fished out from a human adult brain cDNA library. Pax6(S) retained the Paired domain and Homeodomain of Pax6. However, its carboxyl-terminus was composed of a truncated classic PST domain and a unique S tail. The sequence of Pax6(S) was highly conserved only among primates. Its spatial-temporal tissue expression profiles also seemed to be different from those of classic Pax6. These divergences suggest a yet-to-be-defined noncanonical role of Pax6(S) during development.;I next report the interaction between Pax6(S) and Ca2+ channel beta subunits revealed by a yeast two-hybrid library screen. This interaction was mainly endowed by the unique S tail of Pax6(S). Co-expression of a fragment of Pax6(S) containing the S tail with the channel complex in Xenopus oocytes did not affect the surface expression or biophysical properties of the channels. However, Cavbeta was translocated from the cytoplasm into the nuclei in the presence of the S tail, which led to a decrease in the transcriptional activity of Pax6(S). My results imply that Cavbeta acts as a transcriptional suppressor in regulating Pax6(S) activity.