Ulises Meza

RGS2 blocks slow muscarinic inhibition of N‐type Ca2+ channels reconstituted in a human cell line

1 Native N‐type Ca2+ channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Gαq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N‐type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned α1B (CaV2.2) Ca2+ channel subunits and M1 receptors. Expressed Ca2+ currents were recorded using standard whole‐cell, ruptured‐patch techniques. 2 Rapid application of carbachol produced two kinetically distinct components of Ca2+ channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5‐40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. 3 The fast component of inhibition was selectively blocked by the Gβγ‐binding region of β‐adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gβγ released from Gαq/11. 4 The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Gαq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non‐hydrolysable GTPγS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase‐accelerating protein (GAP). 5 These experiments demonstrate that slow muscarinic inhibition of N‐type Ca2+ channels can be reconstituted in non‐neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.

Neurokinin 1 receptors trigger overlapping stimulation and inhibition of CaV2.3 (R-type) calcium channels.

Neurokinin (NK) 1 receptors and CaV2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of CaV2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of CaV2.3. In particular, weak activation of these receptors evokes mainly stimulation of CaV2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by CaV2.3, L-type (CaV1.3), N-type (CaV2.2), and P/Q-type (CaV2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of CaV2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either Gαq/11 (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-β1) or Gβγ subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering Gβγ, whereas a slow component of inhibition was reduced by buffering Gαq/11. When both Gβγ and Gαq/11 were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of CaV2.3. We also report that NK1 receptors accelerate the inactivation kinetics of CaV2.3 currents. Altogether, our results indicate that NK1 receptors modulate CaV2.3 using three different signaling mechanisms: a fast inhibition mediated by Gβγ, a slow inhibition mediated by Gαq/11, and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.

Protein kinase C-mediated inhibition of recombinant T-type Cav3.2 channels by neurokinin 1 receptors.

The voltage-activated T-type calcium channel (CaV3.2) and the G protein-coupled neurokinin 1 (NK1) receptor are expressed in peripheral tissues and in central neurons, in which they participate in diverse physiological processes, including neurogenic inflammation and nociception. In the present report, we demonstrate that recombinant CaV3.2 channels are reversibly inhibited by NK1 receptors when both proteins are transiently coexpressed in human embryonic kidney 293 cells. We found that the voltage-dependent macroscopic properties of CaV3.2 currents were unaffected during NK1 receptor-mediated inhibition. However, inhibition was attenuated in cells coexpressing either the dominant-negative Gαq Q209L/D277N or the regulator of G protein signaling (RGS) proteins 2 (RGS2) and 3T (RGS3T), which are effective antagonists of Gαq/11. By contrast, inhibition was unaffected in cells coexpressing human rod transducin (Gαt), which buffers Gβγ. Channel inhibition was blocked by 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) and bisindolylmaleimide I, selective inhibitors of phospholipase Cβ and protein kinase C (PKC), respectively. Inhibition was occluded by application of the PKC activator phorbol-12-myristate-13-acetate. Altogether, these data indicate that NK1 receptors inhibit CaV3.2 channels through a voltage-independent signaling pathway that involves Gαq/11, phospholipase Cβ, and PKC. Our results provide novel evidence regarding the mechanisms underlying T-type calcium channel modulation by G protein-coupled receptors. Functional coupling between CaV3.2 channels and NK1 receptors may be relevant in neurogenic inflammation, neuronal rhythmogenesis, nociception, and other physiological processes.

Novel outwardly rectifying anion conductance in Xenopus oocytes

We describe a novel, strongly outwardly rectifying anion current in Xenopus laevis oocytes, that we have named ICl,Or. The properties of ICl,Or are different from those of any other anion conductance previously described in these cells. Typically, ICl,Or amplitude was small when extracellular Cl− (Cle) was the permeant anion. However, when Cle was replaced by lyotropic anions ICl,Or became evident as a time-independent current. ICl,Or was voltage dependent and showed a remarkable outwards rectification with little or no inwards tail current. The relative selectivity sequence determined from current amplitudes was: SCN−≥ClO4−>I−>Br−≥NO3−>Cl−. ICl,Or was insensitive to Gd3+ but was blocked by micromolar concentrations of niflumic acid, DIDS or Zn2+. Furthermore, ICl,Or was not affected by buffering intracellular Ca2+ with BAPTA. Low extracellular pH inhibited ICl,Or with a pK of 5.8. We propose that ICl,Or might result from activation of endogenous ClC-5-like Cl− channels present in Xenopus oocytes.

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 Ca2+ in an in vitro experimental model in which Ca2+ 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 µmol/L SKF‐96365 and 100 µmol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca2+ channels CaV1.2 channels, namely 1 µmol/L nifedipine and 10 µmol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20 µmol/L niflumic acid, a non‐selective inhibitor of Ca2+‐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 Ca2+‐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 CaV1.2 channels in the contractions induced by the re‐addition of Ca2+ to the solution bathing guinea‐pig tracheal rings under conditions of Ca2+‐depleted sacroplasmic 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.

Regulation of Kv7.2/Kv7.3 channels by cholesterol: Relevance of an optimum plasma membrane cholesterol content

Kv7.2/Kv7.3 channels are the molecular correlate of the M-current, which stabilizes the membrane potential and controls neuronal excitability. Previous studies have shown the relevance of plasma membrane lipids on both M-currents and Kv7.2/Kv7.3 channels. Here, we report the sensitive modulation of Kv7.2/Kv7.3 channels by membrane cholesterol level. Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells were significantly inhibited by decreasing the cholesterol level in the plasma membrane by three different pharmacological strategies: methyl-β-cyclodextrin (MβCD), Filipin III, and cholesterol oxidase treatment. Surprisingly, Kv7.2/Kv7.3 channels were also inhibited by membrane cholesterol loading with the MβCD/cholesterol complex. Depletion or enrichment of plasma membrane cholesterol differentially affected the biophysical parameters of the macroscopic Kv7.2/Kv7.3 currents. These results indicate a complex mechanism of Kv7.2/Kv7.3 channels modulation by membrane cholesterol. We propose that inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol depletion involves a loss of a direct cholesterol-channel interaction. However, the inhibition of Kv7.2/Kv7.3 channels by membrane cholesterol enrichment could include an additional direct cholesterol-channel interaction, or changes in the physical properties of the plasma membrane. In summary, our results indicate that an optimum cholesterol level in the plasma membrane is required for the proper functioning of Kv7.2/Kv7.3 channels.

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-β-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.

Phosphorylation-Dependent Regulation of Voltage-Gated Ca2+ Channels

Overview N eurotransmitters, hormones, growth factors and extracellular matrix proteins bind to cell membrane receptors that, in turn, activate intracellular signaling cascades. Often, these cascades involve signaling by protein or lipid kinases and protein or lipid phosphatases. Protein kinases add phosphate groups to serine, threonine or tyrosine residues within proteins, and lipid kinases add phosphates to the inositol rings of certain lipids; phosphatases reverse this process. Phosphorylation or dephosphorylation can switch-on** or switch-ofF* protein or lipid activity. It has long been known that voltage-gated Câ * channels are regulated through signaling events involving phosphorylation. In this chapter, we summarize recent findings in this field. We have attempted to minimize overlap with other recent reviews. Abbreviations AC= adenylyl cyclase; Akt/PKB= protein kinase B; AKAP= A kinase anchoring protein; p2AR= beta 2 adrenergic receptor; CaM= calmodulin; CaMKII= Ca^*/calmodulin-dependent protein kinase II; GPCR= G-protein-coupled receptor, NCS-1= neuronal calcium sensor-1; nNOS= neuronal nitric oxide synthase; PDE= phosphodiesterase; PI3K= phosphoinositide 3-kinase; PIP2= phosphatidylinositol 4,5-bisphosphate; PIP3= phosphatidylinositol 3,4,5-trisphosphate; PKA= protein kinase A; PKC= protein kinase C; PKG= protein kinase G; PLC= phospholipase C; PP2A= protein phosphatase 2A; RTK= receptor tyrosine kinase; sGC= soluble guanylyl cyclase.

Molecular mechanisms and physiological relevance of RGK proteins in the heart

The primary route of Ca2+ entry into cardiac myocytes is via 1,4‐dihydropyridine‐sensitive, voltage‐gated L‐type Ca2+ channels. Ca2+ influx through these channels influences duration of action potential and engages excitation‐contraction (EC) coupling in both the atria and the myocardium. Members of the RGK (Rad, Rem, Rem2 and Gem/Kir) family of small GTP‐binding proteins are potent, endogenously expressed inhibitors of cardiac L‐type channels. Although much work has focused on the molecular mechanisms by which RGK proteins inhibit the CaV1.2 and CaV1.3 L‐type channel isoforms that expressed in the heart, their impact on greater cardiac function is only beginning to come into focus. In this review, we summarize recent findings regarding the influence of RGK proteins on normal cardiac physiology and the pathological consequences of aberrant RGK activity.