Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 μM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 μM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself

Novel genetically encoded fluorescent probes enable real-time detection of potassium in vitro and in vivo

Changes in intra-and extracellular potassium ion (K+) concentrations control many important cellular processes and related biological functions. However, our current understanding of the spatiotemporal patterns of physiological and pathological K+ changes is severely limited by the lack of practicable detection methods. We developed K+-sensitive genetically encoded, Forster resonance energy transfer-(FRET) based probes, called GEPIIs, which enable quantitative real-time imaging of K+ dynamics. GEPIIs as purified biosensors are suitable to directly and precisely quantify K+ levels in different body fluids and cell growth media. GEPIIs expressed in cells enable time-lapse and real-time recordings of global and local intracellular K+ signals. Hitherto unknown Ca2+-triggered, organelle-specific K+ changes were detected in pancreatic beta cells. Recombinant GEPIIs also enabled visualization of extracellular K+ fluctuations in vivo with 2-photon microscopy. Therefore, GEPIIs are relevant for diverse K+ assays and open new avenues for live-cell K+ imaging

Protein kinase-C mediates dual modulation of L-type Ca2+ channels in human vascular smooth muscle

AbstractThe role of protein kinase C (PKC) in cellular regulation of L-type Ca2+ channels was investigated in human umbilical vein smooth muscle. Activation of PKC, by low concentrations (< 30 nM) of 12-O-tetradecanoyl-phorbol-13-acetate (TEA) caused inhibition of Ca2+ channels, while higher concentrations of TPA (>100 nM) elicited a transient rise, followed by sustained inhibition of Ca2+ channel activity in cell-attached patches. Low TPA concentrations predominantly reduced channel availability, while high concentrations of TPA (100 nM) transiently increased channel availability and, in addition, prolonged mean open time. The inactive 4-α-phorbol-12,13-didecanoate failed to affect channel activity, and pretreatment of the cells with PKC inhibitors (H-7, chelerythrine) antagonized inhibitory and stimulatory effects of TPA. Our results provide evidence for two distinct PKC-dependent mechanisms of L-type Ca2+ channel regulation in smooth muscle

Interdisciplinary Concepts in Cardiovascular HealthVolume II: Secondary Risk Factors /

XIII, 137 p. 22 illus., 18 illus. in color.onlin

Exploring TRPC3 Interaction with Cholesterol through Coarse-Grained Molecular Dynamics Simulations

Transient receptor potential canonical 3 (TRPC3) channel belongs to the superfamily of transient receptor potential (TRP) channels which mediate Ca2+ influx into the cell. These channels constitute essential elements of cellular signalling and have been implicated in a wide range of diseases. TRPC3 is primarily gated by lipids and its surface expression has been shown to be dependent on cholesterol, yet a comprehensive exploration of its interaction with this lipid has thus far not emerged. Here, through 80 µs of coarse-grained molecular dynamics simulations, we show that cholesterol interacts with multiple elements of the transmembrane machinery of TRPC3. Through our approach, we identify an annular binding site for cholesterol on the pre-S1 helix and a non-annular site at the interface between the voltage-sensor-like domain and pore domains. Here, cholesterol interacts with exposed polar residues and possibly acts to stabilise the domain interface