Roscovitine, a cyclin-dependent kinase inhibitor, affects several gating mechanisms to inhibit cardiac L-type (Ca(V)1.2) calcium channels.

BACKGROUND AND PURPOSE: L-type calcium channels (Ca((V))1.2) play an important role in cardiac contraction. Roscovitine, a cyclin-dependent kinase inhibitor and promising anticancer drug, has been shown to affect Ca((V))1.2 by inhibiting current amplitude and slowing activation. This research investigates the mechanism by which roscovitine inhibits Ca((V))1.2 channels. EXPERIMENTAL APPROACH: Ca((V))1.2 channels were transfected into HEK 293 cells, using the calcium phosphate precipitation method, and currents were measured using the whole-cell patch clamp technique. KEY RESULTS: Roscovitine slows activation at all voltages, which precludes one previously proposed mechanism. In addition, roscovitine enhances voltage-dependent, but not calcium-dependent inactivation. This enhancement resulted from both an acceleration of inactivation and a slowing of the recovery from inactivation. Internally applied roscovitine failed to affect Ca((V))1.2 currents, which supports a kinase-independent mechanism and extracellular binding site. Unlike the dihydropyridines, closed state inactivation was not affected by roscovitine. Inactivation was enhanced in a dose-dependent manner with an IC(50)=29.5+/-12 microM, which is close to that for slow activation and inhibition. CONCLUSIONS AND IMPLICATIONS: We conclude that roscovitine binds to an extracellular site on Ca((V))1.2 channels to inhibit current by both slowing activation and enhancing inactivation. Purine-based drugs could become a new option for treatment of diseases that benefit from L-channel inhibition such as cardiac arrhythmias and hypertension.

E(f)-current contributes to whole-cell calcium current in low calcium in frog sympathetic neurons.

Because Ca(2+) plays diverse roles in intracellular signaling in neurons, several types of calcium channels are employed to control Ca(2+) influx in these cells. Our experiments focus on resolving the paradox of why whole-cell current has not been observed under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels, referred to as E(f)-channels, are present in the membrane at a density greater than the channels that carry approximately 90% of whole-cell current in low Ba(2+); but, E(f)-current has not been detected in low Ba(2+). Using Ca(2+) instead of Ba(2+) as the charge carrier, we recorded a possible E-type current in frog sympathetic neurons. The current was resistant to specific blockers of N-, L-, and P/Q-type calcium channels but was more sensitive to Ni(2+) block than was N- or L-current. Current amplitude in Ca(2+) is slightly greater than that in Ba(2+). In 3 mM Ca(2+), the current contributed approximately 12% of total current at peak voltage and increased at voltages more hyperpolarized to the peak, reaching approximately 40% at -30 mV, where whole-cell current starts to activate. The presence of E(f)-current in 3 mM Ca(2+) suggests a potential role for E(f)-channels in regulating calcium influx into sympathetic neurons.

Reluctant gating of single N-type calcium channels during neurotransmitter-induced inhibition in bullfrog sympathetic neurons.

Whole-cell recordings have been used to extensively characterize the voltage-dependent inhibition of N-type calcium current induced by various neurotransmitters. Results from these studies have yielded several predictions on the effect of inhibition on N-channel gating, namely delayed channel opening and inhibition-induced reluctant openings. Previous single N-channel studies observed delayed channel opening but failed to find reluctant openings. However, strong depolarizations may be necessary to see reluctant openings, but this was not tested. We have examined N-channel gating at voltages depolarized to those used previously and found a neurotransmitter-induced open state that has properties predicted for the reluctant open state. The openings had lower open probability (P(o)) and brief open times compared to the dominant gating state observed in control (high P(o)). These reluctant events were reduced after strong depolarizing pulses used to reverse inhibition. The threshold voltage for activation of reluctant events was approximately 30 mV depolarized to that of the normal gating state (high P(o)). However, an action potential will provide sufficient depolarization to open reluctant N-channels.

Effect of high Ba(2+) on norepinephrine-induced inhibition of N-type calcium current in bullfrog sympathetic neurons.

The voltage-dependent inhibition of N-type calcium current by neurotransmitters is the best-understood example of neuronal calcium channel inhibition. One of the mechanisms by which this pathway is thought to inhibit the calcium current is by reducing the permeation of divalent cations through the channel. In this study one prediction of this hypothesis was examined, that high concentrations of divalent cations reduce the maximum neurotransmitter-induced inhibition. Norepinephrine (NE)-induced inhibition was compared in external solutions containing either 2 or 100 mM Ba(2+). Initially, NE dose-response curves were generated by averaging data from many neurons, and it was found that the relationship was right shifted in the high-Ba(2+) external solution without an effect on maximum inhibition. The IC(50) was 0.6 and 3 microM in 2 and 100 mM Ba(2+), respectively. This shift was verified by comparing the effect of NE on single neurons exposed to both 2 and 100 mM Ba(2+). The inhibition induced by 1 microM NE was reduced in 100 mM Ba(2+) compared with that in 2 mM Ba(2+). However, the response to 100 microM NE was identical between high and low Ba(2+). Thus, divalent cations appear to act as a competitive inhibitor of NE binding, which likely results from these ions' interacting with negatively charged amino acids that are important for catecholamine binding to adrenergic receptors. Because the maximum inhibition induced by NE was similar in low and high Ba(2+), the effect of inhibition on single N-type calcium channels was not altered by the divalent cation concentration.

Gating of single N-type calcium channels recorded from bullfrog sympathetic neurons.

For many neurons, N-type calcium channels provide the primary pathway for calcium influx during an action potential. We investigated the gating properties of single N-type calcium channels using the cell-attached patch technique. With 100 mM Ba2+ in the pipet, mean N-channel open probability (Po, measured over 100 ms) increased with depolarization, but the range at a single voltage was large (e.g., Po at +40 mV ranged from 0.1 to 0.8). The open dwell time histograms were generally well fit by a single exponential with mean open time (tauo) increasing from 0.7 ms at +10 mV to 3.1 ms at +40 mV. Shut time histograms were well fit by two exponentials. The brief shut time component (taush1 = 0.3 ms) did not vary with the test potential, while the longer shut time component (taush2) decreased with voltage from 18.9 ms at +10 mV to 2.3 ms at +40 mV. Although N-channel Po during individual sweeps at +40 mV was often high ( approximately 0.8), mean Po was reduced by null sweeps, low Po gating, inactivation, and slow activation. The variability in mean Po across patches resulted from differences in the frequency these different gating processes were expressed by the channels. Runs analysis showed that null sweeps tended to be clustered in most patches, but that inactivating and slowly activating sweeps were generally distributed randomly. Low Po gating (Po = 0.2, tauo = 1 ms at +40 mV) could be sustained for approximately 1 min in some patches. The clustering of null sweeps and sweeps with low Po gating is consistent with the idea that they result from different modes of N-channel gating. While Po of the main N-channel gating state is high, the net Po is reduced to a maximum value of close to 0.5 by other gating processes.

Identification of the single channels that underlie the N-type and L-type calcium currents in bullfrog sympathetic neurons.

Most of the whole-cell calcium current of frog sympathetic neurons is an N-type current, blocked by omega-conotoxin GVIA (omegaCGVIA). Thus, these cells should be an excellent system to study the properties of single N-type channels. However, a channel that is active near -10 mV in isotonic Ba2+, originally identified as "N-type," corresponds more closely to a omegaCGVIA-resistant component of the whole-cell current observed in 100 mM Ba2+. That conclusion would imply that the true single-channel correlate of the macroscopic N-current remains to be identified in frog sympathetic neurons. I report here recordings from cell-attached patches of a calcium channel that activates in the appropriate voltage range (>0 mV, in isotonic Ba2+) and is blocked by omegaCGVIA. This channel has a slope conductance of 20 pS (range, 17-25 pS) and a single-channel current of -1.3 pA at 0 mV. Other channels active in the same voltage range (24 pS, -1.3 pA at 0 mV) were identified as L-type channels because they exhibited long openings after repolarization in the presence of 1 microM Bay K 8644 and were resistant to omegaCGVIA. A third channel type (13-19 pS) was distinguished by current amplitude (-0.6 pA at 0 mV) and strong inactivation at -40 mV. The similarity in slope conductance among these channels demonstrates that distinguishing them requires the consideration of additional properties. The omegaCGVIA-sensitive channel can be identified as an N-type calcium channel.

Neurotransmitters acting via different G proteins inhibit N-type calcium current by an identical mechanism in rat sympathetic neurons.

1. We studied the mechanism of voltage-dependent inhibition of N-type calcium current by norepinephrine (NE) and vasoactive intestinal peptide (VIP) in adult rat superior cervical ganglion (SCG) neurons using the whole cell patch-clamp technique. 2. The voltage dependence of inhibition is manifest in the reversal of inhibition by strong depolarization. We tested the hypothesis that this voltage dependence results from disruption of G proteins binding to calcium channels. According to this hypothesis, the kinetics of calcium current reinhibition following a strong depolarization should become faster for higher concentrations of active G proteins. 3. Assuming that larger inhibitions result from higher concentrations of active G proteins, we used different concentrations of NE to alter the amplitude of inhibition and, thus, the active G protein concentration. We found that the kinetics of reinhibition at -80 mV following a depolarizing pulse to +80 mV were faster for larger inhibitions. 4. VIP induces voltage-dependent inhibition of N-current via a different G protein (Gs) than that of NE (Go). We found that the effect of VIP on reinhibition kinetics was identical to that produced by NE. 5. Combined application of NE and VIP did not greatly increase the amplitude of the inhibition but significantly increased the rate of reinhibition. Thus NE plus VIP appear to greatly increase the concentration of the molecule binding to the channel (G protein according to the hypothesis). 6. The kinetics of calcium current disinhibition during strong depolarization (step to +80 mV) did not change with the size of the inhibition induced by NE, VIP or application of NE and VIP together. 7. Both the concentration-dependent reinhibition kinetics and concentration-independent disinhibition kinetics are consistent with the hypothesis that active G proteins bind directly to N-type calcium channels to modulate their activity in rat sympathetic neurons.

Concentration dependence of neurotransmitter effects on calcium current kinetics in frog sympathetic neurones.

1. Noradrenaline (NA) slows the activation kinetics of N-type calcium channels, via G proteins. It has been suggested that the G proteins act by binding directly to the calcium channels. If the slow kinetics reflect binding and unbinding of G proteins, the rates should depend on the concentration of activated G protein. 2. We used different concentrations of NA, and increasing durations of intracellular dialysis with GTP-gamma-S, to vary the concentration of activated G protein. 3. At depolarized potentials (-20 or -10 mV), the slow activation kinetics showed no detectable concentration dependence. This analysis required correction for effects of inactivation on the measured time constants. 4. At -80 mV, reinhibition of calcium channel current was more rapid for larger responses. Thus, the effect appears to be concentration dependent at -80 mV, but not at more depolarized voltages. 5. This voltage dependence is actually expected from kinetic principles: the binding step is rate limiting when the position of equilibrium is toward the bound state (at -80 mV), but not when equilibrium favours unbinding (when the channel is open). 6. During inhibition, the channel appears to 'sense' directly the concentration of the modulator, possibly active G proteins.

Reevaluation of Ca2+ channel types and their modulation in bullfrog sympathetic neurons.

With 90 mM Ba2+, the main Ca2+ current in frog sympathetic neurons peaks near +30 mV and is blocked by omega-conotoxin GVIA (omega-CgTx). It is modulated by norepinephrine (NE) in a voltage-dependent manner via a membrane-delimited mechanism. Surprisingly, a different current dominates at more negative voltages (-30 to +10 mV). That novel current is not sensitive to selective blockers of L- or N-type channels (respectively, dihydropyridines or omega-CgTx) and is inhibited weakly if at all by NE. It is selectively inactivated at -40 mV and is selectively blocked by Ni2+, whereas Cd2+ is slightly more potent against the main current. The novel current is associated with a 19 pS channel (0.6 pA at 0 mV). This channel may have been misidentified as the single-channel correlate of the whole-cell N-type Ca2+ current in some previous studies.

Phosphorylation enhances inactivation of N-type calcium channel current in bullfrog sympathetic neurons.

We have investigated the effects of phosphatase and protein kinase inhibitors on calcium channel currents of bullfrog sympathetic neurons using the whole cell configuration of the patch clamp technique. Intracellular dialysis with the phosphatase inhibitors okadaic acid and calyculin A markedly enhanced the decline of inward current during a depolarizing voltage step. Tail current analysis demonstrated that this was genuine inactivation of calcium channel current, not activation of an outward current. The rapidly inactivating current is N-type calcium current (blocked by omega-conotoxin and resistant to nifedipine). Staurosporine, a nonselective protein kinase inhibitor, prevented the action of okadaic acid, suggesting that protein phosphorylation is involved. Under control conditions, the time course of inactivation could be described by the sum of two exponentials (tau = 150 ms and 1200 ms), plus a constant (apparently noninactivating) component, during depolarizations lasting 2 s. Okadaic acid induced a rapid inactivation process (tau = 15 ms) that was absent or negligible under control conditions, without obvious effect on the two slower time constants. As in control cells, inactivation in okadaic-acid-treated cells was strongest near -20 mV, with less inactivation at more positive voltages. However, inactivation did not depend on calcium influx. Modulation of calcium channel activity by phosphorylation may underly the spontaneous shift between inactivating and noninactivating modes recently observed for N-type calcium channels. Differences in basal phosphorylation levels could also explain why N-type calcium channels, originally described as rapidly and completely inactivating, inactivate slowly and incompletely in many neurons.

Effects of temperature on calcium current of bullfrog sympathetic neurons.

1. The temperature dependence of whole-cell calcium current was studied in bullfrog sympathetic neurons. 2. The factor by which the peak calcium current increased upon a 10 degrees C increase in temperature (Q10) was 1.6 +/- 0.1 (mean +/- S.E.M., n = 9), for a change from 20 to 30 degrees C. 3. Activation and deactivation were more rapid at a higher temperature. The Q10 was approximately 7 in the middle of the voltage range (near -10 mV), where the kinetics were slowest. Time constants were less temperature dependent at more positive or negative voltages (Q10 approximately 2 at -70 mV). 4. Near -10 mV, activation and deactivation were associated with a large enthalpy and a large positive entropy change. Deactivation at -70 mV reflected a smaller enthalpy change, and almost no change in entropy. 5. Activation is only slightly more temperature dependent than deactivation, when both are measured at the same voltage. 6. The peak current shifts slightly (approximately 5 mV) to more negative voltages upon a change from 20 to 30 degrees C. 7. Inactivation has a Q10 of approximately 2 at -10 mV. 8. Changes in the kinetics of activation or inactivation could be observed during recording at a constant temperature. These changes were generally small, especially for activation kinetics, and could be distinguished from temperature-dependent changes. 9. The changes in entropy and Q10 with voltage suggest that the rate-limiting steps for activation and deactivation are different at extreme voltages vs. the middle of the voltage range.

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.

Calcium current modulation in frog sympathetic neurones: L-current is relatively insensitive to neurotransmitters.

1. Neurotransmitters (noradrenaline, NA; chicken II luteinizing hormone-releasing hormone, LHRH) and activators of G proteins (GTP-gamma-S and AlF3) partially inhibit calcium current in bullfrog sympathetic neruones. Activation of the remaining current is slowed and shifted to more positive voltages. 2. The N-type calcium current appears to be the type modulated, since approximately 90% of peak current is blocked by omega-conotoxin (omega CgTx) and modulation is not affected by nisoldipine. 3. Calcium current at relatively negative voltages (-30 to -50 mV) is resistant to transmitter modulation. The current at such voltages is also resistant to omega CgTx, suggesting that it results from a different type of calcium channel. 4. The omega CgTx-resistant current includes dihydropyridine (DHP)-sensitive and DHP-resistant components. The omega CgTx- and DHP-resistant current is inhibited by transmitter agonist, but the DHP-sensitive (L-type) current is not. 5. In cells dialysed with a low concentration of calcium buffer (0.1 mM-BAPTA), transmitters still inhibit N-current incompletely. However, L-current was partially inhibited (approximately 10%) by LHRH, NA and the muscarinic agonist oxotremorine-M (OXO-M).

How many persons in Canada have been infected with human immunodeficiency virus? An exploration using backcalculation methods.

We have used the technique of backcalculation to estimate the number of persons in Canada who have been infected with human immunodeficiency virus type 1 (HIV) as of July 1989. We first corrected national AIDS surveillance data in Canada for reporting delay and for underreporting. We then used standard Weibull natural history models as well as an alternative progression model in which the hazard of AIDS was dampened in keeping with observed data from large cohorts. Maximum likelihood techniques were then used to derive the infection curve most consistent with these data. Our best estimate based on this alternative progression model, an underreporting rate of 20%, and a logistic infection curve, was that approximately 29,000 persons in Canada had ever had HIV infection as of July 1989. In a sensitivity analysis utilizing less likely assumptions, the estimates ranged from 17,243 to 48,277. Restricting the same backcalculation process to females under the same assumptions, we estimated that approximately 2,900 females in Canada had ever had HIV infection as of July 1989. The best fitting step function infection curve in females appears to be continuing to rise. Given these estimates, it follows that approximately one in ten infected persons in Canada is female. However, females have only accounted for about one in seventeen AIDS cases. These data are in accord with the widespread impression that transmission of HIV to women has occurred more recently and is on the rise across Canada. The current estimate is lower than previous estimates which placed the number of infected Canadians in the 50,000 to 100,000 range. This lowering should not be taken to mean the situation is improving; rather, the early estimates were simply too high having been based on inadequate data and a rudimentary understanding of the natural history of HIV. Backcalculation is an excellent technique for modelling the incidence of HIV infection several years in the past, but it is not reliable for the most recent few years. A significant increase in HIV infection rates may have occurred in the past few years and it would be beyond the capacity of backcalculation to detect. Backcalculation and unlinked cross-sectional specimen surveys together have the potential to provide an effective means of monitoring the HIV epidemic.

Calcium current modulation in frog sympathetic neurones: multiple neurotransmitters and G proteins.

1. Whole-cell calcium currents of bullfrog sympathetic neurones were partially inhibited by noradrenaline (NA), chicken-II-luteinizing hormone-releasing hormone (LHRH), muscarine, ATP, substance P, or intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate)(GTP-gamma-S) or aluminium fluoride. These agents had similar effects on the activation kinetics of calcium current. 2. The amplitude of the LHRH effect varied from cell to cell. This did not correlate with cell size or the time of whole-cell dialysis. 3. The response to LHRH desensitized rapidly. Desensitization to LHRH did not affect inhibition by NA, ATP or substance P. 4. The effects of LHRH and NA were partially additive. 5. Cells dialysed with GTP-gamma-S still responded to NA or LHRH. However, NA or LHRH inhibited a smaller fraction of the calcium current than usual, and second applications of the same transmitter to GTP-gamma-S-dialysed cells were ineffective. 6. In GTP-gamma-S-dialysed cells, application of LHRH occluded the response to NA, but LHRH was still effective after application of NA. 7. The effect of GTP-gamma-S decreased during prolonged dialysis. 8. The effect of NA was selectively reduced by intracellular dialysis with the A-protomer of pertussis toxin (PTX), or extracellular pretreatment with high concentrations of whole PTX at room temperature. These treatments had little or no effect on the action of LHRH or ATP. 9. It is concluded that multiple G proteins can produce identical changes in calcium channel gating. The adrenergic receptor preferentially couples to a PTX-sensitive G protein.

Separation and modulation of calcium currents in bullfrog sympathetic neurons.

The calcium current of frog sympathetic neurons has relatively rapid activation kinetics (tau < 3 ms) in response to changes in voltage. Pharmacologically, the current is blocked approximately 90% of omega-conotoxin, but < 10% by dihydropyridine antagonists. This suggests that nearly all of the current is N type. However, inactivation is slow and incomplete even for depolarizations lasting > 1 s, consistent with recent evidence that N-type channels do not always inactivate rapidly. The calcium current is partially inhibited via receptors for acetylcholine, luteinizing hormone releasing hormone, substance P, ATP, and norepinephrine. These effects are mimicked by internal dialysis with GTP-gamma-S, suggesting involvement of a G protein. The transmitters affect the activation kinetics of the calcium current in a voltage-dependent manner, which can be modeled as a reversible shift of some channels to "reluctant" states in which strong depolarization is needed to produce channel opening. The effects of transmitters develop and recover with t1/2 approximately 1-2 s, so if a second messenger is involved in receptor-calcium channel coupling, it must act rapidly.

National surveillance of occupational exposure to the human immunodeficiency virus.

In September 1985, a prospective study was initiated to monitor the occurrence of occupational exposures to human immunodeficiency virus (HIV)-infected blood and body fluids in Canada. This program was coordinated by the Federal Centre for acquired immune deficiency syndrome (aids) (now the Division of HIV/aids Epidemiology at the Laboratory Centre for Disease Control). The objective was to determine the risk to workers of acquiring HIV infection as a result of exposure to HIV-infected blood and other body fluids. To be eligible, a worker must have sustained a documented parenteral, mucous membrane or skin contact exposure to blood or body fluids from an HIV-infected person. A baseline specimen was collected within a week of the exposure and then at six weeks, 12 weeks, six months and 12 months. Information concerning the type of exposure, precautions used and post exposure treatment was submitted to the Federal Centre for aids on standard data collection forms. All information was anonymous, identified only by a code number. Guidelines for counselling an exposed employee were provided with enrollment material. As of July 29, 1991, 414 employees have been included in the study. Two hundred and thirty-seven of the 414 exposures (57%) were needlestick injuries of which 167 (70%) were sustained by nurses. Other exposures consisted of open wound contamination, eye splashes, scalpel wounds and skin contact with blood and body fluids. To date, there have been no seroconversions among workers enrolled in the surveillance program.

Seroprevalence and demographic characteristics of injection drugs users among individuals at risk for HIV infection in Winnipeg, Manitoba, Canada.

From June 1986 to March 1990, a prospective seroprevalence survey and questionnaire of individuals at risk for HIV infection was conducted with volunteers in Winnipeg. Of 610 individuals enrolled, 146 were injected drug users (IDU). Fifteen IDU were in a methadone treatment program and all were seronegative. Three of 131 remaining IDU were HIV-1 seropositive (2.3%), a rate similar to 2.2% positive (20+ of 927) in diagnostic specimens from IDU tested in the province. Demographics and behaviour of 131 IDU were compared with 335 individuals, of whom 112 were gay/bisexual [24 of whom also had sexually transmitted diseases (STD)] and 223 heterosexuals who had STD. Males enrolled were significantly older than females. Multivariate analysis showed that factors independently associated with IDU were: a younger age, less education, mental health counselling, unemployment, and a history of jaundice or hepatitis. Drugs most commonly used were Ritalin/Talwin, cocaine, and heroin. Over 90% of individuals admitted to sharing needles. In spite of the low seroprevalence of HIV-1 infections, these individuals are important for the potential spread of HIV because of multiple means of acquiring and transmitting HIV and a high rate of needle sharing.

Geographic and birth cohort associations of Kaposi's sarcoma among homosexual men in Canada.

The authors conducted an analysis of all 677 cases of Kaposi's sarcoma among the 3,047 cases of acquired immunodeficiency syndrome diagnosed in homosexual/bisexual men in Canada between 1980 and 1989. The proportion with Kaposi's sarcoma declined from 32.2% during 1980-1985 to 15.0% in 1989. The proportion with Kaposi's sarcoma was significantly higher in primary epidemic centers (Vancouver, Toronto, and Montreal) and in men in the 1945-1954 birth cohort independent of year of diagnosis. These data are consistent with an environmental cofactor for Kaposi's sarcoma which is likely to be a sexually transmitted agent.