Christian Erxleben

Orai proteins interact with TRPC channels and confer responsiveness to store depletion

The TRPC (C-type transient receptor potential) class of ion channels has been hypothesized to participate in store-operated Ca2+ entry (SOCE). Recently, however, STIM1 and Orai1 proteins have been proposed to form SOCE channels. Whether TRPCs participate in SOCE that is dependent on or regulated by Orai has not been explored. Here we show that Orai1 physically interacts with the N and C termini of TRPC3 and TRPC6, and that in cells overexpressing either TRPC3 or TRPC6 in a store-depletion insensitive manner, these TRPCs become sensitive to store depletion upon expression of an exogenous Orai. Thus, Orai-1, -2, and -3 enhanced thapsigargin-induced calcium entry by 50–150% in cells stably overexpressing either TRPC3 or TRPC6. Orai1 expression had no significant effect on endogenous, thapsigargin-induced calcium entry in wild-type cells (HEK-293, COS1), in HEK cells expressing a thapsigargin-sensitive variant of TRPC3 (TRPC3a), or in HEK cells overexpressing another membrane protein, V1aR. Single-channel cation currents present in membrane patches of TRPC3-overexpressing cells were suppressed by expression of Orai1. We propose that Orai proteins by interacting with TRPCs act as regulatory subunits that confer STIM1-mediated store depletion sensitivity to these channels.

Functional interactions among Orai1, TRPCs, and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels

Receptor-operated Ca2+ entry (ROCE) and store-operated Ca2+ entry (SOCE) into cells are functions performed by all higher eukaryotic cells, and their impairment is life-threatening. The main molecular components of this pathway appear to be known. However, the molecular make-up of channels mediating ROCE and SOCE is largely unknown. One hypothesis proposes SOCE channels to be formed solely by Orai proteins. Another proposes SOCE channels to be composed of both Orai and C-type transient receptor potential (TRPC) proteins. Both hypotheses propose that the channels are activated by STIM1, a sensor of the filling state of the Ca2+ stores that activates Ca2+ entry when stores are depleted. The role of Orai in SOCE has been proven. Here we show the TRPC-dependent reconstitution of Icrac, the electrophysiological correlate to SOCE, by expression of Orai1; we also show that R91W-Orai1 can inhibit SOCE and ROCE and that Orai1 and STIM1 expression leads to functional expression of Gd-resistant ROCE. Because channels that mediate ROCE are accepted to be formed with the participation of TRPCs, our data show functional interaction between ROCE and SOCE components. We propose that SOCE/Icrac channels are composed of heteromeric complexes that include TRPCs and Orai proteins.

The human ERG1 channel polymorphism, K897T, creates a phosphorylation site that inhibits channel activity

Single-nucleotide polymorphisms (SNPs) in the human ether-a-go-go-related gene 1, hERG1, are associated with cardiac arrhythmias. The Kv11.1 channels encoded by hERG1 are also essential for rhythmic excitability of the pituitary, where they are regulated by thyroid hormone through a signal transduction cascade involving the phosphatidylinositol 3-kinase (PI3K) and the Ser/Thr-directed protein phosphatase, PP5. Here, we show that the hERG1 polymorphism at codon 897, which is read as a Thr instead of a Lys, creates a phosphorylation site for the Akt protein kinase on the Kv11.1 channel protein. Consequently, hormonal signaling through the PI3K signaling cascade, which normally stimulates K897 channels through PP5-mediated dephosphorylation, inhibits T897 channels through Akt-mediated phosphorylation. Thus, hormonal regulation of Kv11.1 in humans with the T897 polymorphism is predicted to prolong the QT interval of cardiac myocytes. A systematic bioinformatics search for SNPs in human ion channel genes identified 15 additional candidates for such “phosphorylopathies,” which are predicted to create or destroy putative phosphorylation sites. Changes in protein phosphorylation might represent a general mechanism for the interaction of genetic variation and environment on human health.

Modulation of cardiac CaV1.2 channels by dihydropyridine and phosphatase inhibitor requires Ser-1142 in the domain III pore loop

Dihydropyridine-sensitive, voltage-activated calcium channels respond to membrane depolarization with two distinct modes of activity: short bursts of very short openings (mode 1) or repetitive openings of much longer duration (mode 2). Here we show that both the dihydropyridine, BayK8644 (BayK), and the inhibitor of Ser/Thr protein phosphatases, okadaic acid, have identical effects on the gating of the recombinant cardiac calcium channel, CaV1.2 (α1C). Each produced identical mode 2 gating in cell-attached patches, and each prevented rundown of channel activity when the membrane patch was excised into ATP-free solutions. These effects required Ser or Thr at position 1142 in the domain III pore loop between transmembrane segments S5 and S6, where dihydropyridines bind to the channel. Mutation of Ser-1142 to Ala or Cys produced channels with very low activity that could not be modulated by either BayK or okadaic acid. A molecular model of CaV1.2 indicates that Ser-1142 is unlikely to be phosphorylated, and thus we conclude that BayK binding stabilizes mode 2 gating allosterically by either protecting a phospho Ser/Thr on the α1C subunit or mimicking phosphorylation at that site.

Antagonistic Modulation of Neuromuscular Parameters in Crustaceans by the Peptides Proctolin and Allatostatin, Contained in Identified Motor Neurons

Recent work on the effects of two peptides, proctolin and allatostatin, on neuromuscular parameters in an isopod crustacean (Idotea) and the crab Eriphia spinfrons is reviewed. In Idotea, both peptides are present in identified motor neurons which supply a number of muscles with peptidergic innervation. Both peptides exert pre- and postsynaptic effects which are synergistic for a given peptide, but opposite for the two peptides. Proctolin enhances muscle contractions by at least three mechanisms. Postsynaptically, proctolin increases the input resistance of muscle fibres by closing voltage-independent K channels and it increases the inward current through L-type Ca channels. Presynaptically, it increases the mean quantal content of evoked transmitter release of slow and fast excitatory axons. Allatostatin decreases muscle contractions by at least two mechanisms: postsynaptically, it reduces the voltage dependent Ca current and presynaptically, it reduces the mean quantal content of transmitter release. The presynaptic inhibitory effect is also present at neuromuscular endings where GABA effects are absent.

Patch clamp methods for studying calcium channels.

The patch clamp technique, which was introduced by Neher and Sakmann and their colleagues in 1981, has allowed electrophysiologists to record ion channel activity from most mammalian cell types. When well-established precautions are taken to minimize electrical and mechanical fluctuations, current transients as small as 0.5pA and as brief as 0.5ms can be measured reliably in cell-attached patches of plasma membrane with a polished glass pipette when it forms a giga-ohm seal with the membrane. In many cases, this is sufficient to watch individual channel proteins open and close repeatedly in real time on metabolically intact cells. No other technique currently provides a more precise or detailed view of the function and regulation of calcium channel gating. If antibiotics are added to the pipette to permeabilize the membrane underneath to small monovalent cations, thereby allowing the entire cell to be voltage-clamped without disrupting its contents, the integrated activity of all the calcium channels in the surface membrane can be measured.