Rapid agonist-evoked coupling of type II Ins(1,4,5)P3 receptor with human transient receptor potential (hTRPC1) channels in human platelets.

Depletion of intracellular Ca2+ stores results in the activation of SMCE (store-mediated Ca2+ entry) in many cells. The mechanism of activation of SMCE is poorly understood. In human platelets, a secretion-like coupling model may be involved. This proposes that store depletion results in trafficking of portions of the endoplasmic reticulum to the plasma membrane, enabling coupling between proteins in the two membranes. In support of this, we have shown that, in human platelets, agonist-evoked Ca2+ store depletion results in de novo and reversible coupling of the Ins P3RII [type II inositol (1,4,5)trisphosphate receptor] with the putative Ca2+ entry channel hTRPC1 [human canonical transient receptor potential 1 (protein); Rosado, Brownlow and Sage (2002) J. Biol. Chem. 277, 42157-42163]. A crucial test of the hypothesis that this coupling activates SMCE is that it should occur rapidly enough to account for agonist-evoked Ca2+ entry. In the present study, we have used quenched- and stopped-flow approaches to determine the latencies of thrombin-evoked coupling of Ins P3RII with hTRPC1 and of thrombin-evoked bivalent cation entry using Mn2+ quenching of fura 2 fluorescence. Thrombin-evoked Mn2+ entry was detected with a latency of 0.81+/-0.07 s (S.E.M., n =7) or 1.36+/-0.09 s (S.E.M., n =7) at a concentration of 1.0 or 0.1 unit/ml respectively. Coupling between Ins P3RII and hTRPC1, assessed at 100 ms intervals, was first detected with a latency of 0.9 or 1.4 s after stimulation with thrombin at a concentration of 1.0 or 0.1 unit/ml respectively. These results support the hypothesis that de novo coupling of Ins P3RII with hTRPC1 could activate SMCE in human platelets

Acetylcholine receptor δ subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific ε subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR δ subunit gene, δ756ins2 and δE59K. Expression studies demonstrate that δ756ins2 is a null mutation. By contrast, both fetal and adult AChR containing δE59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, δE59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases