Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.

The role of small, hydrophobic peptides that are associated with ion pumps or channels is still poorly understood. By using the Xenopus oocyte as an expression system, we have characterized the structural and functional properties of the γ peptide which co‐purifies with Na,K‐ATPase. Immuno‐radiolabeling of epitope‐tagged γ subunits in intact oocytes and protease protection assays show that the γ peptide is a type I membrane protein lacking a signal sequence and exposing the N‐terminus to the extracytoplasmic side. Co‐expression of the rat or Xenopus γ subunit with various proteins in the oocyte reveals that it specifically associates only with isozymes of Na,K‐ATPase. The γ peptide does not influence the formation and cell surface expression of functional Na,K‐ATPase α–β complexes. On the other hand, the γ peptide itself needs association with Na,K‐ATPase in order to be stably expressed in the oocyte and to be transported efficiently to the plasma membrane. γ subunits do not associate with individual α or β subunits but only interact with assembled, transport‐competent α–β complexes. Finally, electrophysiological measurements indicate that the γ peptide modulates the K+ activation of Na,K pumps. These data document for the first time the membrane topology, the specificity of association and a potential functional role for the γ subunit of Na,K‐ATPase.

The biological role of small membrane proteins of the new FXYD family is largely unknown. The best characterized FXYD protein is the γ‐subunit of the Na,K‐ATPase (NKA) that modulates the Na,K‐pump function in the kidney. Here, we report that, similarly to γa and γb splice variants, the FXYD protein CHIF (corticosteroid‐induced factor) is a type I membrane protein which is associated with NKA in renal tissue, and modulates the Na,K‐pump transport when expressed in Xenopus oocytes. In contrast to γa and γb, which both decrease the apparent Na+ affinity of the Na,K‐pump, CHIF significantly increases the Na+ affinity and decreases the apparent K+ affinity due to an increased Na+ competition at external binding sites. The extracytoplasmic FXYD motif is required for stable γ‐subunit and CHIF interaction with NKA, while cytoplasmic, positively charged residues are necessary for the γ‐subunit's association efficiency and for CHIF's functional effects. These data document that CHIF is a new tissue‐specific regulator of NKA which probably plays a crucial role in aldosterone‐responsive tissues responsible for the maintenance of body Na+ and K+ homeostasis.

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