Population Of Calcium Channels Research Articles

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd2+ concentration (10–20 μM), while Ni2+ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.

24R,25-(OH)2 Vitamin D3 Inhibits 1α,25-(OH)2 Vitamin D3 and Testosterone Potentiation of Calcium Channels in Osteosarcoma Cells

Calcium influx via L-type calcium channels in osteoblast cells causes a rapid (in seconds) elevation in intracellular calcium initiated by plasma membrane receptors for 1alpha, 25-dihydroxyvitamin D3 (1alpha,25-D3). 24R,25-Dihydroxyvitamin D3 (24,25-D3) alone, in concentrations up to 200 nM, does not cause potentiation of calcium currents in osteoblasts, but it does inhibit the current potentiation by 1alpha,25-D3. To determine how various steroids interact in their potentiation of calcium channels, the action of vitamin D3 analogues and testosterone with calcium channels in the rat osteoblast-like cell line ROS 17/2.8 was investigated. Bath additions of both 1alpha,25-D3 and testosterone at doses below K1/2 (the dose causing 50% left shift in the current-voltage relationship) are additive in their ability to potentiate calcium channels. When 1alpha,25-D3 and testosterone are added together at concentrations that would cause a maximal shift in the current-voltage relationship by each agent alone (Vmax), the effect of these steroids is not additive. Taken together these data suggest one population of calcium channels is activated by 1alpha, 25-D3 or testosterone. The shift in the current-voltage relationship caused by 1alpha,25-D3 is reduced by 1beta,25-dihydroxyvitamin D3 (1beta,25-D3), an agent which is thought to act specifically on the plasma membrane receptor for 1alpha,25-D3, but the potentiation caused by testosterone is not blocked by 1beta,25-D3. However, 24, 25-D3 inhibits the left shift in the peak current-voltage relationship mediated by either 1alpha,25-D3 and testosterone. This result implies that 1) 1beta,25-D3 directly displaces 1alpha,25-D3 but not testosterone from its plasma membrane receptor, and 2) the rapid (in seconds) stimulatory effects of 1alpha,25-D3 and testosterone on calcium channels are mediated by separate plasma membrane receptors for testosterone and 1alpha,25-D3, which are blocked by another receptor for 24,25-D3. The interaction of these three receptors with L-type calcium channels is pertussis toxin-sensitive.

Effects of methoxamine and barium on 45Ca 2+ fluxes in the rat vas deferens

The aim of this study was to determine whether methoxamine and barium stimulate 45Ca 2+ uptake or efflux in the rat vas deferens in a manner that correlates with their contractile activity, and whether 45Ca 2+ movements are inhibited by verapamil or nifedipine. Basal La 3+-resistant (cellular) 45Ca 2+ uptake was significantly greater in the epididymal half (791 ± 27 nmol g −1) than in the prostatic half (654 ± 14 nmol g −1) of the rat vas deferens and was unaffected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) was without effect on 45Ca 2+ uptake in either half but BaCl 2 (1 mM) increased 45Ca 2+ uptake by 31% in the prostatic half and by 22% in the epididymal half. The barium-induced increases in 45Ca 2+ uptake were markedly reduced or abolished by verapamil (2 μM) or nifedipine (0.3 μM), which at these concentrations have no effect on the rhythmic contractions but abolish the initial small phasic contraction induced by barium. The basal rate of 45Ca 2+ efflux from the intact vas deferens (into Ca 2+ containing Krebs-Henseleit solution or into Ca-free Krebs-Henseleit solution ± EGTA 0.05 mM) was not affected by verapamil (61 μM) or nifedipine (14 μM). Methoxamine (8 μM) produced a marked, transient and reversible increase in 45Ca 2+ efflux into 2.5 mM CaCl 2 Krebs-Henseleit in 50% of the intact vasa deferentia examined which was augmented by verapamil (61 μM). BaCl 2 (1 mM) produced a small increase in 45Ca 2+ efflux into Ca 2+-containing and Ca 2+-free Krebs-Henseleit solutions from some intact vasa deferentia and this was not inhibited by nifedipine (14 μM). It is concluded that the BaCl 2-induced increase in 45Ca 2+ uptake is likely to be associated with the initial phase of the contractile response, rather than the secondary rhythmic contractions. Rhythmic contractions initiated by methoxamine and barium may be initiated by ‘trigger Ca’ entering through a population of calcium channels that have low affinity for calcium antagonist drugs.

Dopamine inhibits voltage-activated calcium channel currents in rat pars intermedia pituitary cells

Several lines of evidence suggest that dopamine actsas a neurotransmitter that inhibits both hormone secretion and electrical activity in pituitary intermediate cells (melanotrophs). In this study we examined the effects of exogenously applied dopamine on voltage activated calcium currents recorded with the whole-cell mode of the patch-clamp technique from short-term primary cultures of melanotrophs. Two types of calcium currents were distinguished by their voltage dependence and kinetics of inactivation similar to the low voltage-activated currents (LVA; or T-type) and high voltage-activated currents (HVA; N&L-types) of calcium currents. Exogenously applied dopamine (2–20 μM) reversibly reduced both LVA and HVA types of calcium currents. Evidence for these results came from experiments in which LVA and HVA calcium currents were separated by stepping to different membrane potenials from a fixed holding potential (V h) or by changing V h. These results suggest that dopamine can regulate the entry of calcium into melanotrophs by acting on at least two different populations of calcium channels thereby affecting hormone secretion and electrical activity.

Modulation of a subthreshold calcium current by the neuropeptide FMRFamide in Aplysia neuron R15.

1. The effect of the endogenous neuropeptide FMRFamide (Phe-Met-Arg-Phe-amide) on the Aplysia bursting pacemaker neuron R15 was studied. Brief local applications of FMRFamide, both on R15 somata in situ, and on R15 somata that were isolated and maintained in primary cell culture, cause a hyperpolarization of the membrane potential and a suppression of spontaneous bursting or beating pacemaker activity. 2. Two-electrode voltage-clamp experiments revealed that FMRFamide decreases the amplitude of an inward current, which activates with depolarization starting at a membrane potential less depolarized than the threshold for action potentials. Previous studies have established that this subthreshold inward current is carried by calcium and is essential for the generation of bursting pacemaker activity in Aplysia neurons. The effect of FMRFamide on the subthreshold inward current of R15 is blocked by divalent cation calcium channel blockers, such as cobalt and manganese, and is unaffected by changing the external concentration of potassium or chloride ions, or addition of blockers of the calcium-activated potassium current, such as external tetraethylammonium or internal EGTA. 3. The subthreshold calcium current of R15 is also decreased by dopamine and by an unidentified synaptic neurotransmitter. These substances mimic and occlude the action of FMRFamide on the subthreshold calcium current, suggesting that all three transmitters converge to affect the same population of calcium channels in neuron R15. 4. The subthreshold calcium current is enhanced by neurotransmitters that elevate cyclic AMP in R15, including serotonin, and the Aplysia neuropeptide egg-laying hormone (ELH). Likewise, the effect of FMRFamide on the subthreshold calcium current is enhanced by serotonin, ELH, and a cyclic AMP analog, suggesting that FMRFamide and cyclic AMP have antagonistic actions on the same population of calcium channels in neuron R15. 5. We conclude that the suppression of spontaneous bursting or beating pacemaker activity in neuron R15 by FMRFamide is due to a decrease in the subthreshold calcium current. The subthreshold calcium current in R15 is a common target for modulation by many different transmitters, acting via several distinct molecular mechanisms.

Voltage-Dependent Calcium Channels in Smooth Muscle Cells

Channels highly selective for calcium, which open when the membrane is depolarized, have been found in all smooth muscle cells examined, whether or not they can generate action potentials. The current through these channels can also be carried by barium and is blocked by cadmium, cobalt, or manganese. If single smooth muscle cells of rabbit ear artery are held at -80 mV under voltage clamp, stepping to more positive potentials elicits an inward current which appears at about -60 mV and is well sustained; at more positive potentials, peak current is achieved within 10-20 ms and current then inactivates substantially. Maximum current in solutions with 1.5 mM calcium is found at -10 mV and in solutions with high calcium or barium concentrations the characteristics of the inward current are considerably altered. Low- and high-threshold components of current, possibly due to different populations of calcium channels, can be distinguished. Nifedipine reduces inward current at more positive test and holding potentials but potentiates inward current if the test and holding potentials are in a more negative range.