Mechanism of Ca2+-dependent desensitization in TRP channels

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca²⁺. The mechanism by which Ca²⁺ inhibits TRP channels is currently a matter of intense debate, with Ca²⁺-regulated kinases, phosphatases, phospholipases, and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca²⁺-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca²⁺-dependent activation of phospholipase C and Ca²⁺ binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.     

Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Role ofDrosophila TRP in inositide-mediated Ca2+ entry

Inositol lipid signaling relies on an InsP₃-induced Ca²⁺ release from intracellular stores and on extracellular Ca²⁺ entry, which takes place when the Ca²⁺ stores become depleted of Ca²⁺. This interplay between Ca²⁺ release and Ca²⁺ entry has been termed capacitative Ca²⁺ entry and the inward current calcium release activated current (CRAC) to indicate gating of Ca²⁺ entry by Ca²⁺-store depletion. The signaling pathway and the gating mechanism of capacitative Ca²⁺ entry, however, are largely unknown and the molecular participants in this process have not been identified. In this article we review genetic, molecular, and functional studies of wild-type and mutantDrosophila photoreceptors, suggesting that thetransient receptor potential mutant (trp) is the first putative capacitative Ca²⁺ entry mutant. Furthermore, several lines of evidence suggest that thetrp gene product TRP is a candidate subunit of the plasma membrane channel that is activated by Ca²⁺ store depletion.     

Mechanism of Ca(2+)-dependent desensitization in TRP channels

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca(2+). The mechanism by which Ca(2+) inhibits TRP channels is currently a matter of intense debate, with Ca(2+)-regulated kinases, phosphatases, phospholipases and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca(2+)-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca(2+)-dependent activation of phospholipase C and Ca(2+) binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.     

Regulation of Ca2+ release-activated Ca2+ channels by INAD and Ca2+ influx factor

Thecoupling mechanism between depletion of Ca²⁺ stores in theendoplasmic reticulum and plasma membrane store-operated ion channelsis fundamental to Ca²⁺ signaling in many cell types and hasyet to be completely elucidated. Using Ca²⁺release-activated Ca²⁺ (CRAC) channels in RBL-2H3 cells asa model system, we have shown that CRAC channels are maintained in theclosed state by an inhibitory factor rather than being opened by theinositol 1,4,5-trisphosphate receptor. This inhibitory role can befulfilled by the Drosophila protein INAD (inactivation-noafter potential D). The action of INAD requires Ca²⁺ andcan be reversed by a diffusible Ca²⁺ influx factor. Thusthe coupling between the depletion of Ca²⁺ stores and theactivation of CRAC channels may involve a mammalian homologue of INADand a low-molecular-weight, diffusible store-depletion signal.

     

Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release

Ca2+ release from Ca2+ stores is a 'quantal' process; it terminates after a rapid release of stored Ca2+. To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a 'brake' on store release. However, the mechanism for the attenuation of Ca2+ efflux remains unknown. We show that Ca2+ release is controlled by voltage- and Ca2+-activated potassium channels in the Ca2+ store. The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope. The blockage or closure of the store BK channel due to Ca2+ efflux developed lumen-negative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3'-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release. The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the luminal potential oscillated bistably by approximately 45 mV in amplitude. Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+.     

Ontogenesis of transmembrane signaling systems for control of cardiac Ca2+ channels

Cardiac ICa is primarily controlled by the Ca2+ influx across the cell membrane during the action potential. Neurotransmitters and hormones are known to play an important role in controlling the availability of cardiac Ca2+ slow channels and [Ca]i through the transmembrane signaling systems, such as the receptor-adenylate cyclase system and the PI hydrolysis system. This article has reviewed some of the important properties of the transmembrane signaling systems for the control of Ca2+ channels and how they changed during development, especially in the rat heart. Since the phosphorylation hypothesis was first proposed over 15 years ago, considerable advances have been made in understanding the mechanisms for regulation of Ca2+ slow channels at the molecular level. During development, the properties of the cardiac Ca2+ channels, as well as the transmembrane signaling systems, change electrophysiologically, pharmacologically, and biochemically. Because conflicting data have been reported on the developmental changes in these properties, it is still not clear how the complex physiological function of the Ca2+ channels is gradually integrated during development. To answer this question, more data, new concepts, and new approaches are required.     

The spatial relationship between Ca2+ channels and Ca2+-activated channels and the function of Ca2+-buffering in avian sensory neurons

In order to learn about the endogenous Ca2+-buffering in the cytoplasm of chick dorsal root ganglion (DRG) neurons and the distance separating the ryanodine receptor Ca2+ release channels (RyRs) from the plasma membrane, we monitored the amplitude and time course of Ca2+-activated Cl- currents (I(ClCa)) in protocols that manipulated Ca2+-buffering. I(ClCa)was activated by Ca2+ influx via voltage-gated Ca2+ channels or by Ca2+ release via RyRs activated by 10 mM caffeine. I(ClCa)was measured in neurons at 20 degrees C and 35 degrees C using the amphotericin perforated patch technique that preserves endogenous Ca2+-buffering, or at 20 degrees C in neurons dialyzed with pipette solutions designed to replace the endogenous Ca2+ buffers. The amplitude of I(ClCa)activated by Ca2+ influx or Ca2+ at 20 degrees C was similar in the amphotericin neurons and neurons dialyzed with an 'unbuffered' pipette solution containing 10 mM citrate and 3 mM ATP as the only Ca2+ binding molecules. Thus, endogenous mobile Ca2+ buffers are relatively unimportant in chick DRG neurons. Warming the neurons from 20 degrees C to 35 degrees C increased the amplitude and the rate of deactivation of I(ClCa)consistent with an increased rate of Ca2+ buffering by fixed endogenous Ca2+-buffers. Dialysis with 2 mM EGTA/0.1 microM free Ca2+ reduced the amplitude and increased the rate of deactivation of I(ClCa)activated by Ca2+ influx and abolished I(ClCa)activated by Ca2+ release. Dialysis with 2 mM BAPTA/0.1 microM free Ca2+ abolished I(ClCa)activated by Ca2+ influx or release. Dialysis with 42 mM HEEDTA/0.5 microM free Ca2+ caused the persistent activation of I(ClCa). Calculations using a Ca2+-diffusion model suggest that the voltage-gated Ca2+ channels and the Ca2+-activated Cl- channels are separated by 50-400 nm and that the RyRs are more than 600 nm from the plasma membrane.