Arachidonic acid reversibly enhances N-type calcium current at an extracellular site

2001 ◽  
Vol 280 (5) ◽  
pp. C1306-C1318 ◽  
Author(s):  
Curtis F. Barrett ◽  
Liwang Liu ◽  
Ann R. Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Companion Paper ◽  
Current Amplitude ◽  
Voltage Sensitivity ◽  
Channel Activity ◽  
Activation Kinetics ◽  
Bath Application ◽  
Voltage Dependent ◽  
Cervical Ganglion ◽  
Ganglion Neurons

We examined the effects of arachidonic acid (AA) on whole cell Ca2+ channel activity in rat superior cervical ganglion neurons. Our companion paper (Liu L, Barrett CF, and Rittenhouse AR. Am J Physiol Cell Physiol 280: C1293–C1305, 2001) demonstrates that AA induces several effects, including enhancement of current amplitude at negative voltages, and increased activation kinetics. This study examines the mechanisms underlying these effects. First, enhancement is rapidly reversible by bath application of BSA. Second, enhancement appears to occur extracellularly, since intracellular albumin was without effect on enhancement, and bath-applied arachidonoyl coenzyme A, an amphiphilic AA analog that cannot cross the cell membrane, mimicked enhancement. In addition, enhancement is voltage dependent, in that currents were enhanced to the greatest degree at −10 mV, whereas virtually no enhancement occurred positive of +30 mV. We also demonstrate that AA-induced increases in activation kinetics are correlated with enhancement of current amplitude. An observed increase in the voltage sensitivity may underlie these effects. Finally, the majority of enhancement is mediated through N-type current, thus providing the first demonstration that this current type can be enhanced by AA.

scholarly journals Modulation of N-Type Calcium Channel Activity by G-Proteins and Protein Kinase C

2000 ◽  
Vol 115 (3) ◽  
pp. 277-286 ◽  
Author(s):  
Curtis F. Barrett ◽  
Ann R. Rittenhouse
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Current Amplitude ◽  
Whole Cell ◽  
Protein Activation ◽  
Kinase C ◽  
Bath Application ◽  
G Protein Activation ◽  
Cervical Ganglion ◽  
Ganglion Neurons

N-type voltage-gated calcium channel activity in rat superior cervical ganglion neurons is modulated by a variety of pathways. Activation of heterotrimeric G-proteins reduces whole-cell current amplitude, whereas phosphorylation by protein kinase C leads to an increase in current amplitude. It has been proposed that these two distinct pathways converge on the channel's pore-forming α1B subunit, such that the actions of one pathway can preclude those of the other. In this study, we have characterized further the actions of PKC on whole-cell barium currents in neonatal rat superior cervical ganglion neurons. We first examined whether the effects of G-protein–mediated inhibition and phosphorylation by PKC are mutually exclusive. G-proteins were activated by including 0.4 mM GTP or 0.1 mM GTP-γ-S in the pipette, and PKC was activated by bath application of 500 nM phorbol 12-myristate 13-acetate (PMA). We found that activated PKC was unable to reverse GTP-γ-S–induced inhibition unless prepulses were applied, indicating that reversal of inhibition by phosphorylation appears to occur only after dissociation of the G-protein from the channel. Once inhibition was relieved, activation of PKC was sufficient to prevent reinhibition of current by G-proteins, indicating that under phosphorylating conditions, channels are resistant to G-protein–mediated modulation. We then examined what effect, if any, phosphorylation by PKC has on N-type barium currents beyond antagonizing G-protein–mediated inhibition. We found that, although G-protein activation significantly affected peak current amplitude, fast inactivation, holding-potential–dependent inactivation, and voltage-dependent activation, when G-protein activation was minimized by dialysis of the cytoplasm with 0.1 mM GDP-β-S, these parameters were not affected by bath application of PMA. These results indicate that, under our recording conditions, phosphorylation by PKC has no effect on whole-cell N-type currents, other than preventing inhibition by G-proteins.

Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons

2001 ◽  
Vol 280 (5) ◽  
pp. C1293-C1305 ◽  
Author(s):  
Liwang Liu ◽  
Curtis F. Barrett ◽  
Ann R. Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Sympathetic Neurons ◽  
Current Amplitude ◽  
Calcium Currents ◽  
Neonatal Rat ◽  
Protein Activity ◽  
Whole Cell ◽  
Cervical Ganglion ◽  
Ganglion Neurons ◽  
Sites Of Action

We recently reported that arachidonic acid (AA) inhibits L- and N-type Ca2+ currents at positive test potentials in the presence of the dihydropyridine L-type Ca2+ channel agonist (+)-202-791 in dissociated neonatal rat superior cervical ganglion neurons [Liu L and Rittenhouse AR. J Physiol (Lond) 525: 291–404, 2000]. In this first of two companion papers, we characterized the mechanism of inhibition by AA at the whole cell level. In the presence of either ω-conotoxin GVIA or nimodipine, AA decreased current amplitude, confirming that L- and N-type currents, respectively, were inhibited. AA-induced inhibition was concentration dependent and reversible with an albumin-containing wash solution, but appears independent of AA metabolism and G protein activity. In characterizing inhibition, an AA-induced enhancement of current amplitude was revealed that occurred primarily at negative test potentials. Cell dialysis with albumin minimized inhibition but had little effect on enhancement, suggesting that AA has distinct sites of action. We examined AA's actions on current kinetics and found that AA increased holding potential-dependent inactivation. AA also enhanced the rate of N-type current activation. These findings indicate that AA causes multiple changes in sympathetic Ca2+ currents.

scholarly journals Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K+ Current through a Lipoxygenase Pathway in Molluscan Neurones

1997 ◽  
Vol 110 (5) ◽  
pp. 611-628 ◽  
Author(s):  
K.S. Kits ◽  
J.C. Lodder ◽  
M.J. Veerman
Keyword(s):  
Arachidonic Acid ◽  
G Protein ◽  
Lymnaea Stagnalis ◽  
Reversal Potential ◽  
Outward Current ◽  
Egg Laying ◽  
Voltage Sensitivity ◽  
Lipoxygenase Pathway ◽  
Voltage Dependent ◽  
K Current

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED50 = 23 nM) activated a K+ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K+]o = 1.7 mM), only outward current responses occurred. In high K+ salines ([K+]o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K+. In both salines, no responses were measured below −120 mV. Between −120 mV and the K+ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K+ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K+]o. Since this result was also obtained in nearly symmetrical K+ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K+ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba2+, and, partially, Cd2+ and Cs+. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A2 and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K+ current distinguish it from other receptor-driven K+ currents such as the S-current- and G-protein-dependent inward rectifiers.

scholarly journals Hyperpolarization-activated chloride currents in Xenopus oocytes.

1994 ◽  
Vol 103 (2) ◽  
pp. 217-230 ◽  
Author(s):  
G C Kowdley ◽  
S J Ackerman ◽  
J E John ◽  
L R Jones ◽  
J R Moorman
Keyword(s):  
Chloride Channel ◽  
Xenopus Oocytes ◽  
Voltage Sensitivity ◽  
Channel Blockers ◽  
Biological Chemistry ◽  
Activation Kinetics ◽  
Chloride Currents ◽  
Voltage Dependent ◽  
Pharmacologic Profile ◽  
Lower Voltage

During hyperpolarizing pulses, defolliculated Xenopus oocytes have time- and voltage-dependent inward chloride currents. The currents vary greatly in amplitude from batch to batch; activate slowly and, in general, do not decay; have a selectivity sequence of I- > NO3- > Br- > Cl- > propionate > acetate; are insensitive to Ca2+ and pH; are blocked by Ba2+ and some chloride channel blockers; and have a gating valence of approximately 1.3 charges. In contrast to hyperpolarization-activated chloride currents induced after expression of phospholemman (Palmer, C. J., B. T. Scott, and L. R. Jones. 1991. Journal of Biological Chemistry. 266:11126; Moorman, J. R., C. J. Palmer, J. E. John, J. E. Durieux, and L. R. Jones. 1992. 267:14551), these endogenous currents are smaller; have a different pharmacologic profile; have a lower threshold for activation and lower voltage-sensitivity of activation; have different activation kinetics; and are insensitive to pH. Nonetheless, the endogenous and expressed current share striking similarities. Recordings of macroscopic oocyte currents may be inadequate to determine whether phospholemman is itself an ion channel and not a channel-modulating molecule.

scholarly journals Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

2021 ◽  
Author(s):  
Bethan A Cole ◽  
Nadia Pilati ◽  
Jonathan D Lippiat
Keyword(s):  
Intracellular Sodium ◽  
Selectivity Filter ◽  
Channel Structure ◽  
Channel Activity ◽  
Gain Of Function ◽  
Activation Kinetics ◽  
Pathogenic Variants ◽  
Activation Gate ◽  
Sodium Dependent ◽  
Voltage Dependent

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

Sign in / Sign up

Export Citation Format

Share Document