Search

Canonical transient receptor potential (TRPC) channels control influxes of [Ca.sup.2+] and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the [Ca.sup.2+] influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced [Ca.sup.2+] response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a [Ca.sup.2+]-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy. [Ca.sup.2+] signaling | pyrazole compounds | TRPC channels | TRPC3

Cation channels of the TRP superfamily are widely expressed in the nervous system, and important progress has been made in elucidating the gating properties and physiological roles of neuronal TRPs. Recent studies have firmly established the role of temperature-sensitive TRPs (thermoTRPs) as the principal molecular thermometers in the peripheral sensory system, and provided the first molecular insight into the mechanisms underlying the exquisite thermo- and chemosensitivity of these channels. Moreover, accumulating evidence implicates TRP channels in the development of the central nervous system. In particular, [Ca.sup.2+] influx via TRPC channels appears to be a critical component of the signalling cascade that mediates the guidance of growth cones and survival of neurons in response to chemical cues such as neurotrophins or Netrin-1.

We present a high-throughput method to quantify the functional activity of potassium ([K.sup.+]) ion channels in primary human lymphocytes. This method is rapid, automated, specific (here for the voltage-gated Kvl.3 ion channel), and capable of measuring, in parallel, the electrical currents of over 200 individual lymphocytes isolated from freshly drawn blood. The statistics afforded by high-throughput measurements allowed direct comparison of Kvl.3 activity in different subsets of lymphocytes, including [CD4.sup.+] and [CD8.sup.+] T cells, [gamma][delta] T cells, and B cells. High-throughput measurements made it possible to quantify the heterogeneous, functional response of Kvl.3 ion channel activity upon stimulation of [CD4.sup.+] and [CD8.sup.+] T cells with mitogen. These experiments enabled elucidation of time-courses of functional Kvl.3 activity upon stimulation as well as studies of the effects of the concentration of mitogenic antibodies on Kvl.3 levels. The results presented here suggest that Kvl.3 ion channel activity can be used as a functional activation marker in T cells and that it correlates to cell size and levels of a surface antigen, CD25. Moreover, this work presents an enabling methodology that can be applied widely, allowing high-throughput screening of specific voltage-gated ion channels in a variety of primary cells.

Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3-4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL. To this end, we characterized the function and structure of wild-type MscL and a mutant form of the protein (G22N MscL) that spontaneously adopts an open substate. Patchclamp analysis of MscL that had been reconstituted into liposomes revealed that wild-type MscL was activated only by mechanical stimuli, whereas G22N MscL displayed spontaneous opening to the open substate. In accord with these results, [Ca.sup.2+] influx into G22N MscL-containing liposomes occurred in the absence of mechanical stimulation. The electrophoretic migration of chemically cross-linked G22N MscL was slower than that of cross-linked wild-type MscL, suggesting that G22N MscL is in an expanded form. Finally, electron microscopy using low-angle rotary shadowing revealed the presence of a pore at the center of G22N MscL. No pore could be detected in wild-type MscL. However, wild-type MscL possessed a protrusion at one end, which was absent in G22N MscL. The deletion of carboxyl-terminal 27 residues resulted in the loss of protrusion and proper multimerization. The structures of wild-type and G22N MscL reveal that the opening of MscL is accompanied by the dissociation of a carboxyl-terminal protrusion and pore formation. electon microscopy | patch clamp | liposome | low-angle rotary shadowing | bacterial ion channel

Posterior body wall muscle contraction (pBoc) in the nematode Caenorhabditis elegans occurs rhythmically every 45-50 s and mediates defecation, pBoc is controlled by inositol-1,4,5-trisphosphate ([IP.sub.3])-dependent [Ca.sup.2+] oscillations in the intestine. The intestinal epithelium can be studied by patch clamp electrophysiology, [Ca.sup.2+] imaging, genome-wide reverse genetic analysis, forward genetics, and molecular biology and thus provides a powerful model to develop an integrated systems level understanding of a nonexcitable cell oscillatory [Ca.sup.2+] signaling pathway. Intestinal cells express an outwardly rectifying [Ca.sup.2+] (ORCa) current with biophysical properties resembling those of TRPM channels. Two TRPM homologues, GON-2 and GTL-1, are expressed in the intestine. Using deletion and severe loss-of-function alleles of the gtl-1 and gon-2 genes, we demonstrate here that GON-2 and GTL-1 are both required for maintaining rhythmic pBoc and intestinal [Ca.sup.2+] oscillations. Loss of GTL-1 and GON-2 function inhibits [I.sub.ORCa] ~70% and ~90%, respectively. [I.sub.ORCa] is undetectable in gon-2;gtl-1 double mutant cells. These results demonstrate that (a) both gon-2 and gtl-1 are required for ORCa channel function, and (b) GON-2 and GTL-1 can function independently as ion channels, but that their functions in mediating [I.sub.ORCa] are interdependent. [I.sub.ORCa], [I.sub.GON-2], and [I.sub.GTL-1] have nearly identical biophysical properties. Importantly, all three channels are at least 60-fold more permeable to [Ca.sup.2+] than [Na.sup.+]. Epistasis analysis suggests that GON-2 and GTL-1 function in the I[P.sub.3] signaling pathway to regulate intestinal [Ca.sup.2+] oscillations. We postulate that GON-2 and GTL-1 form heteromeric ORCa channels that mediate selective [Ca.sup.2+] influx and function to regulate I[P.sub.3] receptor activity and possibly to refill ER [Ca.sub.2+] stores.

We have previously reported that the hEAG [K.sup.+] channels are responsible for the potential membrane hyperpolarization that induces human breast cancer cell progression into the G1 phase of the cell cycle. In the present study, we evaluate the role and functional expression of the intermediate-conductance [Ca.sup.2+.sub.]-activated [K.sup.+] channel, hIK1-like, in controlling cell cycle progression. Our results demonstrate that hIK1 current density increased in cells synchronized at the end of the G1 or S phase compared with those in the early GI phase. This increased current density paralleled the enhancement in hIK1 mRNA levels and the highly negative membrane potential. Furthermore, in cells synchronized at the end of G1 or S phases, basal cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) was also higher than in cells arrested in early G 1. Blocking hIK1 channels with a specific blocker, clotrimazole, induced both membrane potential depolarization and a decrease in the [[Ca.sup.2+].sub.i] in cells arrested at the end of G1 and S phases but not in cells arrested early in the G1 phase. Blocking hIK1 with clotrimazole also induced cell proliferation inhibition but to a lesser degree than blocking hEAG with astemizole. The two drugs were essentially additive, inhibiting MCF-7 cell proliferation by 82% and arresting >90% of cells in the GI phase. Thus, although the progression of MCF-7 cells through the early GI phase is dependent on the activation of hEAG [K.sup.+] channels, when it comes to G1 and checkpoint G1/S transition, the membrane potential appears to be primarily dependent on the hIK1-activity level. breast cancer; calcium-activated potassium channels; proliferation

We examined changes in electrical and morphological properties of rat osteoclasts in response to prostaglandin (PG)[E.sub.2]. PG[E.sub.2] (> 10 nM) stimulated an outwardly rectifying [Cl.sup.-] current in a concentration-dependent manner and caused a long-lasting depolarization of cell membrane. This PG[E.sub.2]-induced [Cl.sup.-] current was reversibly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), and tamoxifen. The anion permeability sequence of this current was [I.sup.-] > [Br.sup.-][approximately equal to] [Cl.sup.-] > [gluconate.sup.-]. When outwardly rectifying [Cl.sup.-] current was induced by hyposmotic extracellular solution, no further stimulatory effect of PG[E.sub.2] was seen. Forsknlin and dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) mimicked the effect of PG[E.sub.2]. The PG[E.sub.2]-induced [Cl.sup-] current was inhibited by pretreatment with guanosine 5'-O-2-(thiodiphosphate) (GDP[beta]S), Rp-adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS), N-(2-[p-bromocinnmnylamino]ethyl)-5-isoquinolinesulfonamide dihydrochloride (H-89), and protein kinase A inhibitors. Even in the absence of nonosteoclastic cells, PG[E.sub.2] (1 [micro]M) reduced cell surface area and suppressed motility of osteoclasts, and these effects were abolished by Rp-cAMPS or H-89. PG[E.sub.2] is known to exert its effects through four subtypes of PGE receptors (EP1-EP4). EP2 and EP4 agonists (ONO-AE1-259 and ONO-AE1-329, respectively), but not EP1 and EP3 agonists (ONO-DI-004 and ONO-AE-248, respectively), mimicked the electrical and morphological actions of PG[E.sub.2] on osteoclasts. Our results show that PG[E.sub.2] stimulates rat osteoclast [Cl.sup.-] current by activation of a cAMP-dependent pathway through EP2 and, to a lesser degree, EP4 receptors and reduces osteoclast motility. This effect is likely to reduce bone resorption. prostanoid receptor agonists; electrophysiology; motile activity; bone resorption