165 research outputs found
The 61st Symposium of the Society of General Physiologists Membrane Biophysics of Fusion, Fission, and Rafts in Health and Disease
Calcium-Flux-Mediated Modulation of InsP3R Channel Activity by ER Luminal Calcium Concentration
Spontaneous Channel Activity of the Inositol 1,4,5-Trisphosphate (InsP3) Receptor (InsP3R). Application of Allosteric Modeling to Calcium and InsP3 Regulation of InsP3R Single-channel Gating
The InsP3R Ca2+ release channel has a biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing the sensitivity of the channel to inhibition by high [Ca2+]i. To determine if relieving Ca2+ inhibition is sufficient for channel activation, we examined single-channel activities in low [Ca2+]i in the absence of InsP3, by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent channel activities with low open probability Po (βΌ0.03) were observed in [Ca2+]i < 5 nM with the same frequency as in the presence of InsP3, whereas no activities were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i of the channel to be 1.2β4.0 nM in the absence of InsP3, and demonstrate that the channel can be active when all of its ligand-binding sites (including InsP3) are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies of [Ca2+]i and InsP3 regulation of steady-state channel gating behavior of types 1 and 3 InsP3R isoforms, including spontaneous InsP3-independent channel activities, the tetrameric channel can adopt six different conformations, the equilibria among which are controlled by two inhibitory and one activating Ca2+-binding and one InsP3-binding sites in a manner outlined in the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the Ca2+ affinities of the high-affinity inhibitory sites in different conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent low-affinity inhibitory site. The model also suggests that besides the ligand-regulated gating mechanism, the channel has a ligand-independent gating mechanism responsible for maximum channel Po being less than unity. The validity of this model was established by its successful quantitative prediction of channel behavior after it had been exposed to ultra-low bath [Ca2+]
HCO3β Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion
Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Clβ secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Clβ secretion was accompanied by secretion of HCO3β, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 Β± 0.01 in physiological medium (5% CO2β25 mM HCO3β). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 Β± 0.01 U concomitantly with a fall in Clβ content revealed by cell shrinkage, reflecting Clβ secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2βHCO3β-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3β efflux by ion substitution or exposure to the Clβ channel inhibitor niflumic acid (100 ΞΌM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1β4 and 6β9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3β during Ca2+-evoked fluid secretion by a mechanism that involves the apical membrane secretory Clβ channel, with HCO3β secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na+-dependent pHi regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na+-free media
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