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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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DOE OSTI2020

Kv2.1 channels play opposing roles in regulating membrane potential, Ca 2+ channel function, and myogenic tone in arterial smooth muscle

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K + channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type Ca V 1.2 channels, lowering intracellular Ca 2+ ([Ca 2+ ] i ) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of Ca V 1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of Ca V 1.2–Ca V 1.2 interactions within these clusters, Kv2.1 increases Ca 2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger Ca V 1.2 clusters, larger [Ca 2+ ] i , and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not Ca V 1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and Ca V 1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca 2+ ] i , and myogenic tone in arterial myocytes.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1994

Contemplating the plasmalemmal control center model

An abundant epidermal mechanosensory calcium-selective ion channel appears able not only to detect mechanical stimuli such as those that initiate gravitropism but also to detect thermal, electrical, and various chemical stimuli. Because it responds to multimodal input with a second messenger output, this channel system seems likely to be an integrator that can engage in feedbacks with many other systems of the cell--and feedback is the hallmark of regulation. In general, the mechanical tension required for channel activation is likely transmitted from the relatively rigid cell wall to the plasma membrane system via linkage or adhesion sites that display antigenicities recognized by antibodies to animal beta-1 integrin, vitronectin, and fibronectin and which have mechanical connections to the cytoskeleton. Thus, functionally, leverage exerted against any given adhesion site will tend to control channels within a surrounding domain. Reactions initiated by passage of calcium ions through the channels could presumably be more effectively regulated if channels within the domains were somewhat clustered and if appropriate receptors, kinases, porters, pumps, and some key cytoskeletal anchoring sites were in turn clustered about them. Accumulating evidence suggests not only that activity of clusters of channels may contribute to control of cytoskeletal architecture and of regulatory protein function within their domain, but also that both a variety of regulatory proteins and components of the cortical cytoskeleton may contribute to control of channel activity. The emerging capabilities of electronic optical microscopy are well suited for resolving the spatial distributions of many of these cytoskeletal and regulatory molecules in living cells, and for following some of their behaviors as channels are stimulated to open and cytosolic calcium builds in their vicinity. Such microscopy, coupled with biochemical and physiological probing, should help to establish the nature of the feedback loops putatively controlled by the linkage sites and their channel domains.

Review↗

DOE OSTI2025

Probing substrate water access through the O1 channel of Photosystem II by single site mutations and membrane inlet mass spectrometry

Abstract Light-driven water oxidation by photosystem II sustains life on Earth by providing the electrons and protons for the reduction of CO 2 to carbohydrates and the molecular oxygen we breathe. The inorganic core of the oxygen evolving complex is made of the earth-abundant elements manganese, calcium and oxygen (Mn 4 CaO 5 cluster), and is situated in a binding pocket that is connected to the aqueous surrounding via water-filled channels that allow water intake and proton egress. Recent serial crystallography and infrared spectroscopy studies performed with PSII isolated fromThermosynechococcus vestitus(T. vestitus) support that one of these channels, the O1 channel, facilitates water access to the Mn 4 CaO 5 cluster during its S 2 →S 3 and S 3 →S 4 →S 0 state transitions, while a subsequent CryoEM study concluded that this channel is blocked in the cyanobacteriumSynechocystis sp.PCC 6803, questioning the role of the O1 channel in water delivery. Employing site-directed mutagenesis we modified the two O1 channel bottleneck residues D1-E329 and CP43-V410 (T. vestitusnumbering) and probed water access and substrate exchange via time resolved membrane inlet mass spectrometry. Our data demonstrates that water reaches the Mn 4 CaO 5 cluster via the O1 channel in both wildtype and mutant PSII. In addition, the detailed analysis provides functional insight into the intricate protein-water-cofactor network near the Mn 4 CaO 5 cluster that includes the pentameric, near planar ‘water wheel’ of the O1 channel.

Plant Sciences↗

NASA NTRS1993

The mechanosensory calcium-selective ion channel: key component of a plasmalemmal control centre?

Mechanosensory calcium-selective ion channels probably serve to detect not only mechanical stress but also electrical, thermal, and diverse chemical stimuli. Because all stimuli result in a common output, most notably a shift in second messenger calcium concentration, the channels are presumed to serve as signal integrators. Further, insofar as second messenger calcium in turn gives rise to mechanical, electrical, and diverse chemical changes, the channels are postulated to initiate regulatory feedbacks. It is proposed that the channels and the feedback loops play a wide range of roles in regulating normal plant function, as well as in mediating disturbance of normal function by environmental stressors and various pathogens. In developing evidence for the physiological performance of the channel, a model for a cluster of regulatory plasmalemmal proteins and cytoskeletal elements grouped around a set of wall-to-membrane and transmembrane linkers has proved useful. An illustration of how the model might operate is presented. It is founded on the demonstration that several xenobiotics interfere both with normal channel behaviour and with gravitropic reception. Accordingly, the first part of the illustration deals with how the channels and the control system within which they putatively operate might initiate gravitropism. Assuming that gravitropism is an asymmetric expression of growth, the activities of the channels and the plasmalemmal control system are extrapolated to account for regulation of both rate and allometry of cell expansion. Finally, it is discussed how light, hormones, redox agents and herbicides could in principle affect growth via the putative plasmalemmal control cluster or centre.

NASA Program Space Biology↗

NASA NTRS2003

Physiological Maturation of Regenerating Hair Cells

The bullfrog saccule, a sensor of gravity and substrate-borne vibration, is a model system for hair cell transduction. Saccular hair cells also increase in number throughout adult life and rapidly recover after hair cell damage, making this organ an ideal system for studying hair cell development, repair, and regeneration. We have used of hair cell and supporting cell immunocytochemical markers to identify damaged hair cells and hair cell precursors in organotypic cultures of the bullfrog saccule. We then used an innovative combination of confocal, electron, and time-lapse microscopy to study the fate of damaged hair cells and the origin of new hair cells after gentamicin ototoxicity in normal and mitotically blocked saccular cultures. These studies have shown that gentamicin ototoxicity produces both lethal and sublethal hair cell damage. They have also shown that hair cell recovery in this organ takes place by both the repair of sublethally damaged hair cells and by the replacement of lost hair cells by mitotic regeneration. In parallel studies, we have used biophysical and molecular biological techniques to study the differentiation and innervation of developing, repairing, and regenerating hair cells. More specifically, we have used RT-PCR to obtain the bullfrog homologues of L-type voltage- gated calcium (L-VGCC) and large-conductance Ca(2+)-activated potassium (BK) channel genes. We have then obtained probes for these genes and, using in situ hybridization, begun to examine their expression in the bullfrog saccule and amphibian papilla. We have also used fluorescent-labeled channel toxins and channel toxin derivatives to determine the time of appearance of L-type voltage-gated calcium (L-VGCC) and Ca(2+)-activated potassium (BK) channels and to study dynamic changes in the number, distribution, and co-localization of these proteins in developing, repairing, and regenerating hair cells. Using time-lapse microscopy, we are also studying the dynamic relationship between ion channel clustering and synaptic formation in hair cells and afferent neurons. In future studies, we will determine when hair cell precursors acquire electrical tuning, and, using whole-cell patch-clamp techniques, identify and characterize their L-VGCC and BK currents. We will also use biophysical techniques to determine the number of L-VGCC and BK channels and the size and gating kinetics of their underlying L-VGCC and BK conductances, correlating these variables with the amplitude and frequency of membrane oscillations produced by intracellular current steps. We expect these studies to determine how hair cells regulate ion channel expression to achieve specific physiological responses.

Baird, Richard A.↗