Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-l-type Ca2+ channel

The activity of l-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming α1 subunits of l-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive l-type α1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type α1A subunit. The resulting chimeric α1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the l-type α1C subunit and 92% of the binding energy of the l-type α1S subunit for the high-affinity DHP antagonist PN200–110. The activity of chimeric Ca2+ channels containing α1A/DHPS was increased 3.5 ± 0.7-fold by the DHP agonist (−)Bay K8644. The effect of this agonist was stereoselective as in l-type Ca2+ channels since (+) Bay K8644 inhibited the activity of α1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Multiple calcium channel transcripts in rat osteosarcoma cells: selective activation of alpha 1D isoform by parathyroid hormone.

Osteoblasts express calcium channels that are thought to be involved in the transduction of extracellular signals regulating bone metabolism. The molecular identity of the pore-forming subunit (alpha 1) of L-type calcium channel(s) was determined in rat osteosarcoma UMR-106 cells, which express an osteoblast phenotype. A homology-based reverse transcriptase-polymerase chain reaction cloning strategy was employed that used primers spanning the fourth domain. Three types of cDNAs were isolated, corresponding to the alpha 1S (skeletal), alpha 1C (cardiac), and alpha 1D (neuroendocrine) isoforms. In the transmembrane segment IVS3 and the extracellular loop formed by the IVS3-S4 linker, a single pattern of mRNA splicing was found that occurs in all three types of calcium channel transcripts. Northern blot analysis revealed an 8.6-kb mRNA that hybridized to the alpha 1C probe and 4.8- and 11.7-kb mRNAs that hybridized to the alpha 1S and alpha 1D probes. Antisense oligonucleotides directed to the calcium channel alpha 1D transcript, but not those directed to alpha 1S or alpha 1C transcripts, inhibited the rise of intracellular calcium induced by parathyroid hormone. However, alpha 1D antisense oligonucleotides had no effect on the accumulation of cAMP induced by parathyroid hormone. When L-type calcium channels were activated with Bay K 8644, antisense oligonucleotides to each of the three isoforms partially inhibited the rise of intracellular calcium. The present results provide evidence for the expression of three distinct calcium channel alpha 1-subunit isoforms in an osteoblast-like cell line. We conclude that the alpha 1D isoform is selectively activated by parathyroid hormone.

Age determination of sediment cores of the southeastern Weddell Sea, Antarctica

On the continental margin of the southeastern Weddell Sea, Antarctica, several channel-ridge systems can be traced on the eastern side of the Crary Fan. Swath mapping of the bathymetry reveals three southwest-northeast trending ridges up to 300 m high with channels on their southeastern side. The structures occur on a terrace of the continental slope in water depths of 2000 - 3300 m. We carried out sedimentological studies on cores from three sites. Two of the studied cores are from ridges, one is from the northwestern part of the terrace. The stratigraphy of the recovered sediments is based on accelerator mass spectrometer 14C determinations, stable oxygen and carbon isotopes analyses and paleomagnetic measurements. The sediments represent a period from the last glacial maximum (LGM) to recent time. They are composed predominantly of terrigenous components. We distinguish four different sedimentary facies and assign them to processes controlling sedimentation. Microlaminated muds and cross-stratified coarse-silty sediments originated from contour currents. Bioturbated sediments reflect the increasing influence of hemipelagic sedimentation. Structureless sediments with high contents of ice-rafted debris characterize slumps. The inferred contour currents shaping the continental slope during the LGM were canalized within the channels and supplied microlaminated mud to the western sedimentary ridges due to deflection to the left induced by the Coriolis force. The lamination of the sediments is attributed to seasonal variations of current velocities. The thermohaline bottom currents were directed to the northeast and hence opposite to the Weddell Gyre. Cross-stratified coarse-silty contourites on the ridges are intercalated with the muds and indicate spillover of faster thermohaline flows. Average sedimentation rates on the terrace of the continental slope were unusually high (250 cm/ka) during the LGM, indicating active growth phases of the Crary Fan during glacial intervals. A substantial environmental change at 19.5 - 20 ka is documented in the sediments by a gradual change from lamination to bioturbation. During the recent interglacial, bioturbated sediments were deposited in all parts of the terrace. Because of a reduction of the contour current velocities (4-7 cm/s), the water masses of the Weddell Gyre, supplying fine-grained sediments from northeast, gain a greater influence on sedimentation on the continental slope. Higher percentages of microfossils indicate enhanced biogenic productivity. Increased iceberg activity is documented by greater amounts of ice-rafted debris. The interglacial sedimentation rates decrease to a few cm/ka and indicate that the Crary Fan became relatively sediment-starved during interglacial intervals.

Sedimentological and geophysical investigation of sediment cores from the Amundsen Sea

Ice loss from the marine-based, potentially unstable West Antarctic Ice Sheet (WAIS) contributes to current sea-level rise and may raise sea level by up to 3.3 to 5 meters in the future. Over the past few decades, glaciers draining the WAIS into the Amundsen Sea Embayment (ASE) have shown accelerated ice flow, rapid thinning and grounding-line retreat. However, the long-term context of this ice-sheet retreat is poorly constrained, limiting our ability to accurately predict future WAIS behaviour. Here we present a new chronology for WAIS retreat from the inner continental shelf of the eastern ASE based on radiocarbon dates from three marine sediment cores. The ages document a retreat of the grounding line to within ~93 km of its modern position before 11.7±0.7 kyr BP (thousand years before present). This early deglaciation is consistent with ages for grounding-line retreat from the western ASE. Our new data demonstrate that, other than in the Ross Sea, WAIS retreat in the ASE has not continued progressively since the Last Glacial Maximum. Furthermore, our results suggest that the grounding-line position in the ASE was predominantly stable throughout the Holocene, and that any episodes of fast retreat similar to that observed today must have been short-lived. Alternatively, today's rapid retreat was unprecedented during the Holocene. Therefore, the current ice loss must originate in recent changes in regional climate, ocean circulation or ice-sheet dynamics. Incorporation of these results into models is essential to produce robust predictions of future ice-sheet change and its contribution to sea-level rise.

Mineralogical compositions of sediment samples from the San Francisco Bay coastal system

The mineralogical compositions of 119 samples collected from throughout the San Francisco Bay coastal system, including bayfloor and seafloor, area beaches, cliff outcrops, and major drainages, were determined using X-ray diffraction (XRD). Comparison of the mineral concentrations and application of statistical cluster analysis of XRD spectra allowed for the determination of provenances and transport pathways. The use of XRD mineral identifications provides semi-quantitative compositions needed for comparisons of beach and offshore sands with potential cliff and river sources, but the innovative cluster analysis of XRD diffraction spectra provides a unique visualization of how groups of samples within the San Francisco Bay coastal system are related so that sand-sized sediment transport pathways can be inferred. The main vector for sediment transport as defined by the XRD analysis is from San Francisco Bay to the outer coast, where the sand then accumulates on the ebb tidal delta and also moves alongshore. This mineralogical link defines a critical pathway because large volumes of sediment have been removed from the Bay over the last century via channel dredging, aggregate mining, and borrow pit mining, with comparable volumes of erosion from the ebb tidal delta over the same period, in addition to high rates of shoreline retreat along the adjacent, open-coast beaches. Therefore, while previously only a temporal relationship was established, the transport pathway defined by mineralogical and geochemical tracers support the link between anthropogenic activities in the Bay and widespread erosion outside the Bay. The XRD results also establish the regional and local importance of sediment derived from cliff erosion, as well as both proximal and distal fluvial sources. This research is an important contribution to a broader provenance study aimed at identifying the driving forces for widespread geomorphic change in a heavily urbanized coastal-estuarine system.

Stable hydrogen and carbon isotope records and concentration data for four long chain n-alkanes from core GIK16160-3 derived from the Zambezi delta in the Mozambique Channel during the last 37,000 years

Hydrogen isotope values (dD) of sedimentary terrestrial leaf wax such as n-alkanes or n-acids have been used to map and understand past changes in rainfall amount in the tropics because dD of precipitation is commonly assumed as the first order controlling factor of leaf wax dD. Plant functional types and their photosynthetic pathways can also affect leaf wax dD but these biological effects are rarely taken into account in paleo studies relying on this rainfall proxy. To investigate how biological effects may influence dD values we here present a 37,000-year old record of dD and stable carbon isotopes (d13C) measured on four n-alkanes (n-C27, n-C29, n-C31, n-C33) from a marine sediment core collected off the Zambezi River mouth. Our paleo d13C records suggest that each individual n-alkanes had different C3/C4 proportional contributions. n-C29 was mostly derived from a C3 dicots (trees, shrubs and forbs) dominant vegetation throughout the entire record. In contrast, the longer chain n-C33 and n-C31 were mostly contributed by C4 grasses during the Glacial period but shifted to a mixture of C4 grasses and C3 dicots during the Holocene. Strong correlations between dD and d13C values of n-C33 (correlation coefficient R2 = 0.75, n = 58) and n-C31 (R2 = 0.48, n = 58) suggest that their dD values were strongly influenced by changes in the relative contributions of C3/C4 plant types in contrast to n-C29 (R2 = 0.07, n = 58). Within regions with variable C3/C4 input, we conclude that dD values of n-C29 are the most reliable and unbiased indicator for past changes in rainfall, and that dD and d13C values of n-C31 and n-C33 are sensitive to C3/C4 vegetation changes. Our results demonstrate that a robust interpretation of palaeohydrological data using n-alkane dD requires additional knowledge of regional vegetation changes from which nalkanes are synthesized, and that the combination of dD and d13C values of multiple n-alkanes can help to differentiate biological effects from those related to the hydrological cycle.