Identification of Intracellular and Plasma Membrane Calcium Channel Homologues in Pathogenic Parasites

Ca2+ channels regulate many crucial processes within cells and their abnormal activity can be damaging to cell survival, suggesting that they might represent attractive therapeutic targets in pathogenic organisms. Parasitic diseases such as malaria, leishmaniasis, trypanosomiasis and schistosomiasis are responsible for millions of deaths each year worldwide. The genomes of many pathogenic parasites have recently been sequenced, opening the way for rational design of targeted therapies. We analyzed genomes of pathogenic protozoan parasites as well as the genome of Schistosoma mansoni, and show the existence within them of genes encoding homologues of mammalian intracellular Ca2+ release channels: inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs), two-pore Ca2+ channels (TPCs) and intracellular transient receptor potential (Trp) channels. The genomes of Trypanosoma, Leishmania and S. mansoni parasites encode IP3R/RyR and Trp channel homologues, and that of S. mansoni additionally encodes a TPC homologue. In contrast, apicomplexan parasites lack genes encoding IP3R/RyR homologues and possess only genes encoding TPC and Trp channel homologues (Toxoplasma gondii) or Trp channel homologues alone. The genomes of parasites also encode homologues of mammalian Ca2+ influx channels, including voltage-gated Ca2+ channels and plasma membrane Trp channels. The genome of S. mansoni also encodes Orai Ca2+ channel and STIM Ca2+ sensor homologues, suggesting that store-operated Ca2+ entry may occur in this parasite. Many anti-parasitic agents alter parasite Ca2+ homeostasis and some are known modulators of mammalian Ca2+ channels, suggesting that parasite Ca2+ channel homologues might be the targets of some current anti-parasitic drugs. Differences between human and parasite Ca2+ channels suggest that pathogen-specific targeting of these channels may be an attractive therapeutic prospect

Chloride-dependent sarcoplasmic reticulum Ca2+ release correlates with increased Ca2+ activation of ryanodine receptors.

The mechanism by which chloride increases sarcoplasmic reticulum (SR) Ca2+ permeability was investigated. In the presence of 3 microM Ca2+, Ca2+ release from 45Ca(2+)-loaded SR vesicles prepared from procine skeletal muscle was increased approximately 4-fold when the media contained 150 mM chloride versus 150 mM propionate, whereas in the presence of 30 nM Ca2+, Ca2+ release was similar in the chloride- and the propionate-containing media. Ca(2+)-activated [3H]ryanodine binding to skeletal muscle SR was also increased (2- to 10-fold) in media in which propionate or other organic anions were replaced with chloride; however, chloride had little or no effect on cardiac muscle SR 45Ca2+ release or [3H]ryanodine binding. Ca(2+)-activated [3H]ryanodine binding was increased approximately 4.5-fold after reconstitution of skeletal muscle RYR protein into liposomes, and [3H]ryanodine binding to reconstituted RYR protein was similar in chloride- and propionate-containing media, suggesting that the sensitivity of the RYR protein to changes in the anionic composition of the media may be diminished upon reconstitution. Together, our results demonstrate a close correlation between chloride-dependent increases in SR Ca2+ permeability and increased Ca2+ activation of skeletal muscle RYR channels. We postulate that media containing supraphysiological concentrations of chloride or other inorganic anions may enhance skeletal muscle RYR activity by favoring a conformational state of the channel that exhibits increased activation by Ca2+ in comparison to the Ca2+ activation exhibited by this channel in native membranes in the presence of physiological chloride (< or = 10 mM). Transitions to this putative Ca(2+)-activatable state may thus provide a mechanism for controlling the activation of RYR channels in skeletal muscle

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1.

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site

Role of phosphate and calcium stores in muscle fatigue

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (Pi) in the myoplasm ([Pi]myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca2+ release contributes to fatigue and in this review we consider how raised [Pi]myo contributes to this process. Initial evidence came from the observation that increasing [Pi]myo causes reduced SR Ca2+ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca2+ in the SR declines mirroring the decline in SR Ca2+ release. In muscle fibres with inoperative creatine kinase the rise of [Pi]myo is absent during fatigue and the failure of SR Ca2+ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaPi within the SR. The relevance of this mechanism in different types of fatigue in humans is considered

Kinetics of FKBP12.6 Binding to Ryanodine Receptors in Permeabilized Cardiac Myocytes and Effects on Ca Sparks

The Inquisition took place in Mexico from 1571 to 1820. The Antiguo Palacio de la Inquisicion was built in the first third of the 18th century by Pedro de Arrieta who introduced such innovations as the octagonal entrance and the interlaced arches with unsupported junctures located in the building's courtyard. The current building houses a museum devoted to history of medicine Mexico. When the Federal District was created in 1824, the State government needed to move from the building (to Texcoco), to be outside the Federal District.; The Inquisition took place in Mexico from 1571 to 1820. The Antiguo Palacio de la Inquisicion was built in the first third of the 18th century by Pedro de Arrieta who introduced such innovations as the octagonal entrance and the interlaced arches with unsupported junctures located in the building's courtyard. The current building houses a museum devoted to history of medicine Mexico. When the Federal District was created in 1824, the State government needed to move from the building (to Texcoco), to be outside the Federal District

Phosphate ion channels in sarcoplasmic reticulum of rabbit skeletal muscle

Phosphate ions (Pi) enter intracellular Ca2+ stores and precipitate Ca2+. Since transport pathways for Pi across the membrane of intracellular calcium stores have not been identified and anion channels could provide such a pathway, we have examined the Pi conductance of single anion channels from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle using the lipid bilayer technique.Two anion channels in skeletal muscle SR, the small conductance (SCl) and big conductance (BCl) chloride channels, were both found to have a Pi conductance of 10 pS in 50 mm Pi. The SCl channel is a divalent anion channel which can pass HPO42− as well as SO42− (60 pS in 100 mm free SO42−). The BCl channel is primarily a monovalent anion channel. The SCl and BCl channels are permeable to a number of small monovalent anions, showing minor selectivity between Cl−, I− and Br− (Cl− > I− > Br−) and relative impermeability to cations and large polyatomic anions (Cs+, Na+, choline+, Tris+, Hepes− and CH3O3S−).The Pi conductance of SCl and BCl channels suggests that both channel types could sustain the observed Pi fluxes across the SR membrane. Comparison of the blocking effects of the phosphonocarboxylic acids, ATP and DIDS, on the anion channels with their effects on Pi transport suggests that the SCl channel is the more likely candidate for the SR Pi transport mechanism.The SCl channel, with previously unknown function, provides a regulated pathway for Pi across the SR membrane which would promote Pi entry and thereby changes in the rapidly releasable Ca2+ store during onset and recovery from muscle fatigue. Anion channels may provide a pathway for Pi movement into and out of Ca2+ stores in general