Molecular mechanism of light-driven sodium pumping

ABSTRACTMicrobial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience.An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters.KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies.The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release.Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

INSECT MALPIGHIAN TUBULES: V-ATPase ACTION IN ION AND FLUID TRANSPORT.

Insect Malpighian tubules secrete fluid into the lumen as part of their function as excretory organs. The underlying ion transport is, when stimulated, faster than in any other known tissue. It is driven by the activity of an H+-transporting V-ATPase situated on the luminal cell membranes. This ATPase, together with cation/H+ antiporter(s), constitutes a common cation pump which can transport sodium ions, potassium ions or both. Treatments that selectively slow cation transport across the epithelium cause the secreted fluid to become alkaline, whereas those that selectively reduce the rate of anion passage lead to secretion of acid fluid.

Membrane dynamics in insect malpighian tubules

Urine formation in insects occurs in the Malpighian tubules by means of active ion transport and osmotically coupled water flow. The rates of urine formation can vary with time and can be modulated by diuretic hormones, developmental events, and intracellular parasitism. This paper reviews a number of recent studies in which it has been demonstrated that variations in transport rate are associated with substantial changes in tubule ultrastructure in the form of membrane insertion into and deletion from the apical microvilli. The principal driving force for fluid movement in Malpighian tubules is thought to be a common cation pump located in the apical membranes. It is proposed that modulation of the apical microvillar membrane may reflect regulation by the cells of the number of common cation pump units involved in fluid secretion.