Intramembrane charge movements and excitation– contraction coupling expressed by two-domain fragments of the Ca2+ channel

To investigate the molecular basis of the voltage sensor that triggers excitation–contraction (EC) coupling, the four-domain pore subunit of the dihydropyridine receptor (DHPR) was cut in the cytoplasmic linker between domains II and III. cDNAs for the I-II domain (α1S 1–670) and the III-IV domain (α1S 701-1873) were expressed in dysgenic α1S-null myotubes. Coexpression of the two fragments resulted in complete recovery of DHPR intramembrane charge movement and voltage-evoked Ca2+ transients. When fragments were expressed separately, EC coupling was not recovered. However, charge movement was detected in the I-II domain expressed alone. Compared with I-II and III-IV together, the charge movement in the I-II domain accounted for about half of the total charge (Qmax = 3 ± 0.23 vs. 5.4 ± 0.76 fC/pF, respectively), and the half-activation potential for charge movement was significantly more negative (V1/2 = 0.2 ± 3.5 vs. 22 ± 3.4 mV, respectively). Thus, interactions between the four internal domains of the pore subunit in the assembled DHPR profoundly affect the voltage dependence of intramembrane charge movement. We also tested a two-domain I-II construct of the neuronal α1A Ca2+ channel. The neuronal I-II domain recovered charge movements like those of the skeletal I-II domain but could not assist the skeletal III-IV domain in the recovery of EC coupling. The results demonstrate that a functional voltage sensor capable of triggering EC coupling in skeletal myotubes can be recovered by the expression of complementary fragments of the DHPR pore subunit. Furthermore, the intrinsic voltage-sensing properties of the α1A I-II domain suggest that this hemi-Ca2+ channel could be relevant to neuronal function.

Analysis of differential gene expression by display of 3' end restriction fragments of cDNAs.

We have developed an approach to study changes in gene expression by selective PCR amplification and display of 3' end restriction fragments of double-stranded cDNAs. This method produces highly consistent and reproducible patterns, can detect almost all mRNAs in a sample, and can resolve hidden differences such as bands that differ in their sequence but comigrate on a gel. Bands corresponding to known cDNAs move to predictable positions on the gel, making this a powerful approach to correlate gel patterns with cDNA data bases. Applying this method, we have examined differences in gene expression patterns during T-cell activation. Of a total of 700 bands that were evaluated in this study, as many as 3-4% represented mRNAs that are upregulated, while approximately 2% were down-regulated within 4 hr of activation of Jurkat T cells. These and other results suggest that this approach is suitable for the systematic, expeditious, and nearly exhaustive elucidation of subtle changes in the patterns of gene expression in cells with altered physiologic states.

Poems, letters and prose fragments of Kirke White /

Mode of access: Internet.

Biographical sketches of Bath celebrities, ancient and modern : with some fragments of local history /

Includes index.

King Horn : with fragments of Floriz and Blancheflur, and of the Assumption of Our Lady : from a MS (GG. 4.27.2) in the Cambridge University Library : also from MSS. in the British Museum The Assumption of Our Lady (A.D. MSS. 10036), and Fragments of the Floyres and Blanchefleur (Cotton Vitelluis D.111) /

Includes glossarial index.

Fragments of a Plate with Engraved Designs

Glass, Islamic, Abbasid; 19/32 in.x 11 1/32 in.; glass, blown with applied trail, scratch-engraved

How to attract and hold an audience; a practical treatise on the nature, preparation, and delivery of public discourse,

Mode of access: Internet.

Plain speaking : a theory and grammar of spontaneous discourse /

Mode of access: Internet.

Posthumous fragments of Margaret Nicholson.

Cf. Forman, H.B. ... The Shelley library ... London, 1886, p. 13-14.

Fragments of descriptive verse /

"Vol. II."

Fragments of the natural history of Pennsylvania /

No more published; pt. 1 treats of birds, with data regarding vegetation and miscellaneous observations.