Modulation Of Channel Currents Research Articles

Intracellular ATP and GTP are both required to preserve modulation of N-type calcium channel current by norepinephrine.

Norepinephrine (NE) inhibits voltage-dependent calcium channels of sympathetic neurons. We investigated the role of intracellular nucleotides in this inhibition for clues to receptor-channel coupling mechanisms. Both ATP and GTP are required to preserve NE responsiveness during whole-cell dialysis. The response to NE was gradually lost in bullfrog sympathetic neurons dialyzed with GTP as the only nucleotide, ATP only, or no nucleotides. Replacing ATP with ATP[gamma-S] resulted in spontaneous modulation of calcium channel current, possibly because of production of GTP[gamma-S]. The nonhydrolyzable ATP analog p[NH]ppA could substitute for ATP to preserve NE responsiveness. The protein phosphatase inhibitors okadaic acid and calyculin-A did not affect NE inhibition of calcium channel current, or recovery from that inhibition. These results suggest protein phosphorylation is not involved in the inhibition of calcium channel current, but binding of ATP to some intracellular site is required for the coupling of adrenergic receptors to calcium channels.

Modulation of voltage-dependent Ca channel current by arachidonic acid and other long-chain fatty acids in rabbit intestinal smooth muscle.

The effects of arachidonic acid (AA) and other long-chain fatty acids on voltage-dependent Ca channel current (ICa) were investigated, with the whole cell patch clamp method, in longitudinal smooth muscle cells of rabbit ileum. 10-30 microM AA caused a gradual depression of ICa. The inhibitory effect of AA was not prevented by indomethacin (10 microM) (an inhibitor of cyclooxygenase) or nordihydroguaiaretic acid (10 microM) (an inhibitor of lipoxygenase). 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H7; 25-50 microM) or staurosporine (2 microM) (inhibitors of protein kinase C) did not block the AA-induced inhibition of ICa, and application of phorbol ester (a protein kinase C activator) (phorbol-12,13-dibutyrate, 0.2 microM) did not mimic the AA action. Some other cis-unsaturated fatty acids (palmitoleic, linoleic, and oleic acids) were also found to depress ICa, while a trans-unsaturated fatty acid (linolelaidic acid) and saturated fatty acids (capric, lauric, myristic, and palmitic acids) had no inhibitory effects on ICa. Myristic acid consistently increased the amplitude of ICa at negative membrane potentials. The present results suggest the possible role of AA, and perhaps other fatty acids, in the physiological and/or pathological modulation of ICa in smooth muscle.

Kinetic modulation of guinea-pig cardiac L-type calcium channels by fendiline and reversal of the effects of Bay K 8644.

1. The modulation of L-type calcium channel current (ICa) by fendiline, a diphenylalkylamine type of calcium channel blocker was investigated on guinea-pig ventricular myocytes by use of the whole-cell patch-clamp technique. 2. Fendiline-induced block of ICa is accompanied by modulation of the channel kinetics in a complex manner. The time course of ICa inactivation is significantly faster and the channel availability (f infinity) curve is shifted considerably to more negative potentials by fendiline. These findings can be interpreted qualitatively in terms of a modulated receptor. 3. When the 1,4-dihydropyridine agonist (4R, 4S)-Bay K 8644 was added in presence of 30 microM fendiline a further reduction of ICa instead of the expected stimulatory effect was observed. 4. A similar 'paradoxical' inhibition of ICa was produced by the pure agonist enantiomer (4S)-Bay K 8644. Thus this novel effect of Bay K 8644 cannot be attributed to changes in affinity of the 1,4-dihydropyridine receptor site for (4R)-Bay K 8644 during fendiline action. 5. The IC50 for fendiline was reduced to 3.0 +/- 0.1 microM (control value: 17.0 +/- 2.4 microM) and the Hill slope in its presence was increased to 1.90 +/- 0.1 (control value: 1.39 +/- 0.23) by 1 microM (4R, 4S)-Bay K 8644. 6. (4R,4S)-Bay K 8644 caused the expected stimulation of ICa in the presence of verapamil, diltiazem and nifedipine, overcoming the inhibitory effect of these calcium channel blockers. 7. The 'paradoxical' inhibitory effect of the agonist Bay K 8644 can be explained in terms of an allosteric interaction between fendiline and the dihydropyridine agonist.

Voltage‐dependent modulation of rat sensory neurone calcium channel currents by G protein activation: effect of a dihydropyridine antagonist

The ability of a depolarizing prepulse to increase the rate of activation of IBa has been examined in cultured sensory neurones of the rat. Both in control neurones and in the presence internally of the guanine nucleotide analogue, guanosine 5'-O-3-thiotriphosphate (GTP gamma S) which markedly slows the rate of activation of IBa and reduces its amplitude, a depolarizing prepulse increased the rate of activation of IBa, but did not increase its amplitude measured at the end of the 100 ms voltage step. The calcium channel antagonist (-)-202-791, which we have previously shown to increase the amplitude of IBa in the presence of GTP gamma S, did not occlude the response to a depolarizing prepulse, suggesting that the mechanism of action of (-)-202-791 is not to disrupt the interaction of the channels with activated G proteins.

Modulation of neuronal T-type calcium channel currents by photoactivation of intracellular guanosine 5′-0(3-thio) triphosphate

Low voltage-activated T-type Ca 2+ channel currents were recorded from cultured rat dorsal root ganglion neurons using the whole-cell clamp technique with Ba 2+ as the charge carrier. The T-type Ca 2+ channel current was identified by its low threshold of activation ( V c−50 to −20 mV from v h−90mV), its kinetics of inactivation and its sensitivity to NiCl 2; (100 μM). It was also sensitive to 1-octanol (1 μM). ω-Conotoxin (1 μM) markedly reduced the high threshold voltage-activated Ca 2+ channel currents but did not inhibit the T-type Ca 2+ channel current. Photorelease of intracellular guanosine 5′-0(3-thio) triphosphate from a photolabile “caged” precursor had dose-dependent effects on the T-type Ca 2+ channel current. At a concentration of 6μM, guanosine5'- O(3-thio) triphosphate enhanced the current, but further photorelease of guanosine5'- O(3-thio) triphosphate (up to 20 μM) inhibited the current. Only the inhibitory response was sensitive to pertussis toxin. These data suggest that more than one G-protein is involved in T-type Ca 2+ channel current modulation. Inclusion of guanosine5'- O(2-thio) diphosphate (1 mM) in the patch solution prevented guanosine5'- O(3-thio) triphosphate from potentiating the current, and greatly attenuated the inhibitory effects observed when larger amounts of guanosine5'- O(3-thio) triphosphate were photoreleased. Photorelease of guanosine5'- O(2-thio) diphosphate had no effect on T-type current but did significantly increase the high voltage-activated current. A low concentration of (−)-baclofen (2μM), potentiated T-type current, while 100μM(−)-baclofen inhibited T-type current.

Gamma-aminobutyrate type B receptor modulation of L-type calcium channel current at bipolar cell terminals in the retina of the tiger salamander.

Bipolar-cell axon terminals receive direct synaptic input from amacrine-cell processes, suggesting a possible pathway for modulation of transmitter release. In retinal slices, bath-applied baclofen, a gamma-aminobutyrate type B (GABAB) receptor agonist, reduced a patch-clamp-recorded L-type calcium channel current in a population of bipolar cells with axon terminals that ramify along the midline of the inner plexiform layer. Lucifer yellow staining revealed that this current was found only in bipolar cells that retain axon terminals and their associated telodendria, suggesting that the current is generated at the terminal and also possibly modulated there. T-type calcium currents were found in all bipolar cells, including those without axon terminals, but were not modulated by baclofen. The baclofen-induced reduction of calcium current was enhanced by guanosine 5'-[gamma-thio]triphosphate and eliminated by guanosine 5'-[beta-thio]diphosphate added to the cytoplasm by the patch recording electrode, suggesting that the GABAB receptors act through a guanine nucleotide-binding regulatory protein (G protein). Baclofen also reduced an excitatory synaptic input to a population of amacrine cells with processes that ramify along the midline of the inner plexiform layer--cells probably postsynaptic to the bipolar terminals. This suggests that GABAB receptors modulate not only the calcium current but also transmitter release by a pathway involving G proteins and L-type calcium channels.

Rapid β-Adrenergic Modulation of Cardiac Calcium Channel Currents by a Fast G Protein Pathway

beta-Adrenergic agonists activate the G protein, Gs, which stimulates cardiac calcium currents by both cytoplasmic, indirect and membrane-delimited, direct pathways. To test whether beta-adrenergic agonists might use both pathways in the heart, isoproterenol was rapidly applied to cardiac myocytes, resulting in a biphasic increase in cardiac calcium channel currents that had time constants of 150 milliseconds and 36 seconds. beta-Adrenergic antagonists of a G protein inhibitor blocked both the fast and slow responses, whereas the adenylyl cyclase activator forskolin produced only the slow response. The presence of a fast pathway in the heart can explain what the slow pathway cannot account for: the ability of cardiac sympathetic nerves to change heart rate within a single beat.

Influence of pHo on calcium channel block by amlodipine, a charged dihydropyridine compound. Implications for location of the dihydropyridine receptor.

We have investigated the modulation of L-type calcium channel currents in isolated ventricular cells by the dihydropyridine derivative amlodipine, a weak base with a pKa of 8.6. Under conditions that favor neutral drug molecules, amlodipine block resembles other, previously described, neutral dihydropyridine derivatives: block is more pronounced at depolarized voltages, repetitive pulsing is not needed to promote block, and recovery is complete at hyperpolarized voltages. When the drug is ionized, depolarized voltages still enhance block, however, the time course is slow and speeded by repetitive pulses that open channels. Recovery from block by ionized drug molecules is very slow and incomplete, but can be rapidly modified by changes in external hydrogen ion concentration. We conclude from these observations that the degree of ionization of the drug molecule can affect access to the dihydropyridine receptor and that external protons can interact with the drug-receptor complex even if channels are blocked and closed. These observations place limitations on the location of this receptor in the ventricular cell membrane.

Hydrogen ion modulation of Ca channel current in cardiac ventricular cells. Evidence for multiple mechanisms.

We have investigated the effects of H ions on (L-type) Ca channel current in isolated ventricular cells. We find that the current amplitude is enhanced in solutions that are alkaline relative to pH 7.4 and reduced in solutions acidic to this pH. We measured pH0-induced shifts in channel gating and analyzed our results in terms of surface potential theory. The shifts are well described by changes in surface potential caused by the binding of H ions to negative charges on the cell surface. The theory predicts a pK of 5.8 for this binding. Gating shifts alone cannot explain all of our observations on modulation of current amplitude. Our results suggest that an additional mechanism contributes to modification of the current amplitude.

Modulation of calcium channel currents in guinea‐pig single ventricular heart cells by the dihydropyridine Bay K 8644.

1. A single glass micropipette voltage clamp technique with intracellular dialysis was used to study Ba2+ currents in isolated ventricular cells from guinea-pig hearts. Effects of the 1,4-dihydropyridine Bay K 8644 on whole-cell currents were evaluated at 37 degrees C. 2. Bay K 8644 increased the Ba2+ peak currents at test potentials between -50 and +20 mV and shifted the current-voltage relationships towards hyperpolarizing potentials (leftward shift for Ca2+ channel activation, 13.8 +/- 4.1 mV; n = 9; Bay K 8644, 5 mumol/l). 3. The peak times of the Ba2+ currents were diminished over the voltage range tested between -40 and +20 mV after Bay K 8644 in parallel with a shortening of the time constant of activation that was estimated from fits of the recorded currents with a d2f model. 4. The decay of the Ba2+ currents was fitted with two exponentials including a pedestal. The compound Bay K 8644 accelerated the fast decay over the whole voltage range. The amplitude of the rapidly inactivated component of the Ba2+ currents was strikingly increased after application of Bay K 8644. 5. The steady-state inactivation using a 0.5 or 5 s pre-pulse was shifted towards hyperpolarizing potentials (leftward shift 10.3 +/- 5.2 mV; n = 4; Bay K 8644, 5 mumol/l). 6. The change in the time course of Bay K 8644-modified Ba2+ currents cannot be described solely by a decrease of the backward rate coefficient from an open to a closed state of the Ca2+ channel (Sanguinetti, Krafte & Kass, 1986). The described effects of Bay K 8644 on the inactivation can be both qualitatively and quantitatively described by a model of current-dependent inactivation (Standen & Stanfield, 1982), assuming a lower affinity of an internal binding site for Ba2+ than for Ca2+.

Voltage-dependent modulation of Ca channel current in heart cells by Bay K8644.

We have investigated the voltage-dependent effects of the dihydropyridine Bay K8644 on Ca channel currents in calf Purkinje fibers and enzymatically dispersed rat ventricular myocytes. Bay K8644 increases the apparent rate of inactivation of these currents, measured during depolarizing voltage pulses, and shifts both channel activation and inactivation in the hyperpolarizing direction. Consequently, currents measured after hyperpolarizing conditioning pulses are larger in the presence of drug compared with control conditions, but are smaller than control if they are measured after positive conditioning pulses. Most of our experimental observations on macroscopic currents can be explained by a single drug-induced change in one rate constant of a simple kinetic model. The rate constant change is consistent with results obtained by others with single channel recordings.