Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human CaV3.3 Channel.

Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, CaV3.1, CaV3.2, and CaV3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human CaV3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the CaV3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the CaV3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the CaV3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of in vitro and in silico techniques, we have identified interacting determinants relevant to the CaV3.3/genistein complex formation and propose that genistein directly blocks the function of the human CaV3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rational development of improved and novel low-voltage activated T-type calcium channel inhibitors.

Functional marriage in plasma membrane: Critical cholesterol level-optimal protein activity.

In physiology, homeostasis refers to the condition where a system exhibits an optimum functional level. In contrast, any variation from this optimum is considered as a dysfunctional or pathological state. In this review, we address the proposal that a critical cholesterol level in the plasma membrane is required for the proper functioning of transmembrane proteins. Thus, membrane cholesterol depletion or enrichment produces a loss or gain of direct cholesterol-protein interaction and/or changes in the physical properties of the plasma membrane, which affect the basal or optimum activity of transmembrane proteins. Whether or not this functional switching is a generalized mechanism exhibited for all transmembrane proteins, or if it works just for an exclusive group of them, is an open question and an attractive subject to explore at a basic, pharmacological and clinical level.

Inhibition of CaV2.3 channels by NK1 receptors is sensitive to membrane cholesterol but insensitive to caveolin-1.

Voltage-gated, CaV2.3 calcium channels and neurokinin-1 (NK1) receptors are both present in nuclei of the central nervous system. When transiently coexpressed in human embryonic kidney (HEK) 293 cells, CaV2.3 is primarily inhibited during strong, agonist-dependent activation of NK1 receptors. NK1 receptors localize to plasma membrane rafts, and their modulation by Gq/11 protein-coupled signaling is sensitive to plasma membrane cholesterol. Here, we show that inhibition of CaV2.3 by NK1 receptors is attenuated following methyl-ß-cyclodextrin (MBCD)-mediated depletion of membrane cholesterol. By contrast, inhibition of CaV2.3 was unaffected by intracellular diffusion of caveolin-1 scaffolding peptide or by overexpression of caveolin-1. Interestingly, MΒCD treatment had no effect on the macroscopic biophysical properties of CaV2.3, though it significantly decreased whole-cell membrane capacitance. Our data indicate that (1) cholesterol supports at least one component of the NK1 receptor-linked signaling pathway that inhibits CaV2.3 and (2) caveolin-1 is dispensable within this pathway. Our findings suggest that NK1 receptors reside within non-caveolar membrane rafts and that CaV2.3 resides nearby but outside the rafts. Raft-dependent modulation of CaV2.3 could be important in the physiological and pathophysiological processes in which these channels participate, including neuronal excitability, synaptic plasticity, epilepsy, and chronic pain.

Role of extracellular Na+, Ca2+-activated Cl- channels and BK channels in the contraction of Ca2+ store-depleted tracheal smooth muscle.

1. In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re-addition of Ca(2+) in an in vitro experimental model in which Ca(2+) stores had been depleted and their refilling had been blocked by thapsigargin. 2. Mean (+/-SEM) contraction was diminished by: (i) inhibitors of store-operated calcium channels (SOCC), namely 100 micromol/L SKF-96365 and 100 micromol/L 1-(2-trifluoromethylphenyl) imidazole (to 66.3 +/- 4.4 and 41.3 +/- 5.2% of control, respectively); (ii) inhibitors of voltage-gated Ca(2+) channels Ca(V)1.2 channels, namely 1 micromol/L nifedipine and 10 micromol/L verapamil (to 86.2 +/- 3.4 and 76.9 +/- 5.9% of control, respectively); and (iii) 20 micromol/L niflumic acid, a non-selective inhibitor of Ca(2+)-dependent Cl(-) channels (to 41.1 +/- 9.8% of control). In contrast, contraction was increased 2.3-fold by 100 nmol/L iberiotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) (BK) channels. 3. Furthermore, contraction was significantly inhibited when Na(+) in the bathing solution was replaced by N-methyl-D-glucamine (NMDG(+)) to 39.9 +/- 7.2% of control, but not when it was replaced by Li(+) (114.5 +/- 24.4% of control). In addition, when Na(+) had been replaced by NMDG(+), contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 +/- 1.8 and 24.4 +/- 8.1% of control, respectively). Nifedipine also reduced contractions when Na(+) had been replaced by Li(+) (to 10.7 +/- 3.4% to control), the niflumic acid had no effect (116.0 +/- 4.5% of control). 4. In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca(V)1.2 channels in the contractions induced by the re-addition of Ca(2+) to the solution bathing guinea-pig tracheal rings under conditions of Ca(2+)-depleted sarcoplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na(+), suggesting a role for SOCC in mediating the Na(+) influx.

Functional coupling between the Na+/Ca2+ exchanger and nonselective cation channels during histamine stimulation in guinea pig tracheal smooth muscle.

Airway smooth muscle (ASM) contracts partly due to an increase in cytosolic Ca(2+). In this work, we found that the contraction caused by histamine depends on external Na(+), possibly involving nonselective cationic channels (NSCC) and the Na(+)/Ca(2+) exchanger (NCX). We performed various protocols using isometric force measurement of guinea pig tracheal rings stimulated by histamine. We observed that force reached 53 +/- 1% of control during external Na(+) substitution by N-methyl-D-glucamine(+), whereas substitution by Li(+) led to no significant change (91 +/- 1%). Preincubation with KB-R7943 decreased the maximal force developed (52.3 +/- 5.6%), whereas preincubation with nifedipine did not (89.7 +/- 1.8%). Also, application of the nonspecific NCX blocker KB-R7943 and nifedipine on histamine-precontracted tracheal rings reduced force to 1 +/- 3%, significantly different from nifedipine alone (49 +/- 6%). Moreover, nonspecific NSCC inhibitors SKF-96365 and 2-aminoethyldiphenyl borate reduced force to 1 +/- 1% and 19 +/- 7%, respectively. Intracellular Ca(2+) measurements in isolated ASM cells showed that KB-R7943 and SKF-96365 reduced the peak and sustained response to histamine (0.20 +/- 0.1 and 0.19 +/- 0.09 for KB-R, 0.43 +/- 0.16 and 0.47 +/- 0.18 for SKF, expressed as mean of differences). Moreover, Na(+)-free solution only inhibited the sustained response (0.54 +/- 0.25). These data support an important role for NSCC and NCX during histamine stimulation. We speculate that histamine induces Na(+) influx through NSCC that promotes the Ca(2+) entry mode of NCX and Ca(V)1.2 channel activation, thereby causing contraction.