Biomedical subjects

C J Lingle

Publications and source records attributed to C J Lingle.

At least 19 recordsLinked to original sources

PubMed2001

Inactivation of BK channels mediated by the NH(2) terminus of the beta3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade.

A family of auxiliary beta subunits coassemble with Slo alpha subunit to form Ca(2)+-regulated, voltage-activated BK-type K(+) channels. The beta subunits play an important role in regulating the functional properties of the resulting channel protein, including apparent Ca(2)+ dependence and inactivation. The beta3b auxiliary subunit, when coexpressed with the Slo alpha subunit, results in a particularly rapid ( approximately 1 ms), but incomplete inactivation, mediated by the cytosolic NH(2) terminus of the beta3b subunit (Xia et al. 2000). Here, we evaluate whether a simple block of the open channel by the NH(2)-terminal domain accounts for the inactivation mechanism. Analysis of the onset of block, recovery from block, time-dependent changes in the shape of instantaneous current-voltage curves, and properties of deactivation tails suggest that a simple, one step blocking reaction is insufficient to explain the observed currents. Rather, blockade can be largely accounted for by a two-step blocking mechanism (C(n) <---> O(n) <---> O(*)(n) <---> I(n)) in which preblocked open states (O*(n)) precede blocked states (I(n)). The transitions between O* and I are exceedingly rapid accounting for an almost instantaneous block or unblock of open channels observed with changes in potential. However, the macroscopic current relaxations are determined primarily by slower transitions between O and O*. We propose that the O to O* transition corresponds to binding of the NH(2)-terminal inactivation domain to a receptor site. Blockade of current subsequently reflects either additional movement of the NH(2)-terminal domain into a position that hinders ion permeation or a gating transition to a closed state induced by binding of the NH(2) terminus.

Animals↗

PubMed2001

Gating properties conferred on BK channels by the beta3b auxiliary subunit in the absence of its NH(2)- and COOH termini.

Both beta1 and beta2 auxiliary subunits of the BK-type K(+) channel family profoundly regulate the apparent Ca(2)+ sensitivity of BK-type Ca(2)+-activated K(+) channels. Each produces a pronounced leftward shift in the voltage of half-activation (V(0.5)) at a given Ca(2)+ concentration, particularly at Ca(2)+ above 1 microM. In contrast, the rapidly inactivating beta3b auxiliary produces a leftward shift in activation at Ca(2)+ below 1 microM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583-605, this issue), we have shown that some of the apparent beta3b-mediated shift in activation at low Ca(2)+ arises from rapid unblocking of inactivated channels, unlike the actions of the beta1 and beta2 subunits. Here, we compare effects of the beta3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the beta3b subunit and compare it to beta3b constructs lacking either the NH(2)- or COOH terminus or both. The results demonstrate that, although the NH(2) terminus appears to be the primary determinant of the beta3b-mediated shift in V(0.5) at low Ca(2)+, removal of the NH(2) terminus reveals two other interesting aspects of the action of the beta3b subunit. First, the conductance-voltage curves for activation of channels containing the beta3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the beta3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between beta and alpha subunits can affect BK channel function. The COOH terminus of the beta3b subunit produces no discernible functional effects.

Animals↗

PubMed2001

Allosteric regulation of BK channel gating by Ca(2+) and Mg(2+) through a nonselective, low affinity divalent cation site.

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K(+) channels is enhanced by cytosolic calcium (Ca(2+)). Activation is sensitive to a range of [Ca(2+)] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 alpha subunits at [Ca(2+)] and [Mg(2+)] up to 100 mM. The half-activation voltage (V(0.5)) is steeply dependent on [Ca(2+)] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 microM Ca(2+). As [Ca(2+)] is increased to millimolar levels, the V(0.5) is strongly shifted again to more negative potentials. When channels are activated by 300 microM Ca(2+), further addition of either mM Ca(2+) or mM Mg(2+) produces similar negative shifts in steady-state activation. Millimolar Mg(2+) also produces shifts of similar magnitude in the complete absence of Ca(2+). The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca(2+)] increase activation rates, addition of 10 mM Mg(2+) to 0 Ca(2+) produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg(2+) does not participate in Ca(2+)-dependent steps that influence current activation rate. We conclude that millimolar Mg(2+) and Ca(2+) concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca(2+)-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca(2+) binding steps, four voltage sensors, and four independent lower affinity Ca(2+)/Mg(2+) binding steps describes well the behavior of G-V curves over a range of Ca(2+) and Mg(2+). The ability of a broad range of [Ca(2+)] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

Action Potentials↗

PubMed2000

Rectification and rapid activation at low Ca2+ of Ca2+-activated, voltage-dependent BK currents: consequences of rapid inactivation by a novel beta subunit.

A family of accessory beta subunits significantly contributes to the functional diversity of large-conductance, Ca(2+)- and voltage-dependent potassium (BK) channels in native cells. Here we describe the functional properties of one variant of the beta subunit family, which confers properties on BK channels totally unlike any that have as yet been observed. Coexpression of this subunit (termed beta3) with Slo alpha subunits results in rectifying outward currents and, at more positive potentials, rapidly inactivating ( approximately 1 msec) currents. The underlying rapid inactivation process results in an increase in the apparent activation rate of macroscopic currents, which is coupled with a shift in the activation range of the currents at low Ca(2+). As a consequence, the currents exhibit more rapid activation at low Ca(2+) relative to any other BK channel subunit combinations that have been examined. In part because of the rapid inactivation process, single channel openings are exceedingly brief. Although variance analysis suggests a conductance in excess of 160 pS, fully resolved single channel openings are not observed. The inactivation process results from a cytosolic N-terminal domain of the beta3 subunit, whereas an extended C-terminal domain does not participate in the inactivation process. Thus, the beta3 subunit appears to use a rapid inactivation mechanism to produce a current with a relatively rapid apparent activation time course at low Ca(2+). The beta3 subunit is a compelling example of how the beta subunit family can finely tune the gating properties of Ca(2+)- and voltage-dependent BK channels.

Amino Acid Sequence↗

PubMed2000

Anticonvulsants but not general anesthetics have differential blocking effects on different T-type current variants.

The sensitivity to anticonvulsants and anesthetics of Ca(2+) currents arising from alpha1G and alpha1H subunits was examined in stably transfected HEK293 cells. For comparison, in some cases blocking effects on dorsal root ganglion (DRG) T currents were also examined under identical ionic conditions. The anticonvulsant, phenytoin, which partially blocks DRG T current, blocked alpha1G current completely but with weaker affinity ( approximately 140 microM). Among different cells, alpha1H current exhibited either of two responses to phenytoin. In one subpopulation of cells, phenytoin produced a partial, higher affinity block (IC(50) approximately 7.2 microM, maximum block approximately 43%) similar to that in DRG neurons. In other cells, phenytoin produced complete, but lower affinity, blockade (IC(50) approximately 138 microM, maximum block approximately 89%). Another anticonvulsant, alpha-methyl-alpha-phenylsuccinimide (MPS), blocked DRG current partially, but blocked both alpha1G and alpha1H currents completely with weaker affinity ( approximately 1.7 mM). These data suggest that higher affinity blockade of T-type currents by phenytoin and MPS may require additional regulatory factors that can contribute to native T-type channels. In contrast, anesthetics blocked all T current variants similarly and completely. Block of alpha1G current by anesthetics had the following order of potency: propofol (IC(50) approximately 20.5 microM) > etomidate ( approximately 161 microM) = octanol ( approximately 160 microM) > isoflurane ( approximately 277 microM) > ketamine ( approximately 1.2 mM), comparable with results on DRG T currents. Barbiturates completly blocked alpha1G currents with potency [thiopental ( approximately 280 microM), pentobarbital ( approximately 310 microM), phenobarbital ( approximately 1.54 mM)] similar to that in DRG cells. The effects of propofol, octanol, and pentobarbital on alpha1H currents were indistinguishable from effects on alpha1G currents.

Anesthetics, General↗

PubMed2000

Activation of BK channels in rat chromaffin cells requires summation of Ca(2+) influx from multiple Ca(2+) channels.

Large-conductance Ca(2+) and voltage-dependent K(+) channels (BK channels) in many tissues require high Ca(2+) concentrations for activation and therefore might be expected to be tightly coupled to Ca(2+) channels. However, in most cases, little is known about the relative organization of the BK channels and the Ca(2+) channels involved in their activation. We probed the nature of the organization of BK and Ca(2+) channels in rat chromaffin cells by manipulating Ca(2+) influx through Ca(2+) channels and by altering cellular Ca(2+) buffering using EGTA and bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA). The results were analyzed to determine the distance between Ca(2+) and BK channels that would be most consistent with the experimental data. Most BK channels are close enough to Ca(2+) channels to be resistant to the buffering action of millimolar of EGTA, but are far enough to be inhibited by BAPTA. Analysis of the EGTA/BAPTA results suggests that BK channels are at a distance of 50 to 160 nm from Ca(2+) channels. A model that assumes random distribution of Ca(2+) and BK channels fails to account for the observed [Ca(2+)](i) detected by BK channels, suggesting that a specific mechanism may exist to mediate the functional coupling between these channels. Importantly, the effects of EGTA and BAPTA cannot be explained by assuming a one-to-one coupling between Ca(2+) and BK channels. Rather, Ca(2+) influx through a number of Ca(2+) channels appears to act in concert to regulate the behavior of any individual BK channel. Thus differences in BK channel open probabilities may be explained by differences in the extent of Ca(2+) domain overlap at the sites of individual BK channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

Molecular basis for the inactivation of Ca2+- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells.

Large-conductance Ca2+- and voltage-dependent potassium (BK) channels exhibit functional diversity not explained by known splice variants of the single Slo alpha-subunit. Here we describe an accessory subunit (beta3) with homology to other beta-subunits of BK channels that confers inactivation when it is coexpressed with Slo. Message encoding the beta3 subunit is found in rat insulinoma tumor (RINm5f) cells and adrenal chromaffin cells, both of which express inactivating BK channels. Channels resulting from coexpression of Slo alpha and beta3 subunits exhibit properties characteristic of native inactivating BK channels. Inactivation involves multiple cytosolic, trypsin-sensitive domains. The time constant of inactivation reaches a limiting value approximately 25-30 msec at Ca2+ of 10 microM and positive activation potentials. Unlike Shaker N-terminal inactivation, but like native inactivating BK channels, a cytosolic channel blocker does not compete with the native inactivation process. Finally, the beta3 subunit confers a reduced sensitivity to charybdotoxin, as seen with native inactivating BK channels. Inactivation arises from the N terminal of the beta3 subunit. Removal of the beta3 N terminal (33 amino acids) abolishes inactivation, whereas the addition of the beta3 N terminal onto the beta1 subunit confers inactivation. The beta3 subunit shares with the beta1 subunit an ability to shift the range of voltages over which channels are activated at a given Ca2+. Thus, the beta-subunit family of BK channels regulates a number of critical aspects of BK channel phenotype, including inactivation and apparent Ca2+ sensitivity.

Adrenal Glands↗

PubMed1999

Blockade of Ba2+ current through human alpha1E channels by two steroid analogs, (+)-ACN and (+)-ECN.

Previous work suggests that different neuroactive steroids may exhibit some selectivity in their blocking effects on different high-voltage activated (HVA) Ca2+ currents. At least some of these effects appear to involve direct blocking actions on Ca2+ channels. Thus, direct investigation of the effects of various steroids on cloned Ca2+ channel variants may lead to the development of potent and selective small-molecular weight Ca2+ channel blockers. Here we examine the effects of two steroids on a cloned human alpha1E Ca2+ channel both with and without a beta3 subunit, when expressed in HEK293 cells. One compound, (+)-ACN, has been previously shown to block N-, Q-, and R-subtypes of HVA current without affecting L- and P-type current. The second compound, (+)-ECN, weakly blocks total HVA current in hippocampal neurons. (+)-ECN differs from (+)-ACN in lacking effects on GABA receptors, but shares with (+)-ACN an ability to partially inhibit T current in DRG neurons (Todorovic, S.M., Prakriya, M., Nakashima, Y.M. et al., 1998. Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid lacking GABA-mimetic activity. Mol. Pharmacol. 54, 918-927). (+)-ACN can block 100% of Ba2+ current in HEK cells arising either from the alpha1E subunit (IC50 approximate to 10 microM) or the alpha1Ebeta3 combination (IC50 approximate to 5 microM), while (+)-ECN maximally blocks only about 80% of the alpha1E (10 microM) or alpha1Ebeta3 (16 microM) current. Blockade by (+)-ACN exhibits several differences from blockade by (+)-ECN. (+)-ACN increases the apparent rate of onset of inactivation, particularly for the alpha1E variant, slows recovery from inactivation, and more profoundly shifts the voltage-dependence of current availability for both alpha1E and alpha1Ebeta3 variants than does (+)-ECN. Although the complexity of the normal inactivation kinetics of alpha1E variants makes interpretation of the (+)-ACN-induced kinetic alterations difficult, the results suggest that the two steroids are to some extent acting by distinct mechanisms, and perhaps at different sites.

Animals↗

PubMed1999

RINm5f cells express inactivating BK channels whereas HIT cells express noninactivating BK channels.

Large-conductance Ca2+- and voltage-activated BK-type K+ channels are expressed abundantly in normal rat pancreatic islet cells and in the clonal rat insulinoma tumor (RINm5f) and hamster insulinoma tumor (HIT) beta cell lines. Previous work has suggested that the Ca2+ sensitivity of BK channels in RIN cells is substantially less than that in HIT cells, perhaps contributing to differences between the cell lines in responsiveness to glucose in mediating insulin secretion. In both RIN cells and normal pancreatic beta cells, BK channels are thought to play a limited role in responses of beta cells to secretagogues and in the electrical activity of beta cells. Here we examine in detail the properties of BK channels in RIN and HIT cells using inside-out patches and whole cell recordings. BK channels in RIN cells exhibit rapid inactivation that results in an anomalous steady-state Ca2+ dependence of activation. In contrast, BK channels in HIT cells exhibit the more usual noninactivating behavior. When BK inactivation is taken into account, the Ca2+ and voltage dependence of activation of BK channels in RIN and HIT cells is essentially indistinguishable. The properties of BK channel inactivation in RIN cells are similar to those of inactivating BK channels (termed BKi channels) previously identified in rat chromaffin cells. Inactivation involves multiple, trypsin-sensitive cytosolic domains and exhibits a dependence on Ca2+ and voltage that appears to arise from coupling to channel activation. In addition, the rates of inactivation onset and recovery are similar to that of BKi channels in chromaffin cells. The charybdotoxin (CTX) sensitivity of BKi currents is somewhat less than that of the noninactivating BK variant. Action potential voltage-clamp waveforms indicate that BK current is activated only weakly by Ca2+ influx in RIN cells but more strongly activated in HIT cells even when Ca2+ current magnitude is comparable. Concentrations of CTX sufficient to block BKi current in RIN cells have no effect on action potential activity initiated by glucose or DC injection. Despite its abundant expression in RIN cells, BKi current appears to play little role in action potential activity initiated by glucose or DC injection in RIN cells, but BK current may play an important role in action potential repolarization in HIT cells.

Action Potentials↗

PubMed1999

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells.

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 microM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.

Action Potentials↗

PubMed1998

Inactivating BK channels in rat chromaffin cells may arise from heteromultimeric assembly of distinct inactivation-competent and noninactivating subunits.

Inactivating and noninactivating variants of large-conductance, Ca2+-dependent, voltage-dependent BK-type channels are found in rat chromaffin cells and are largely segregated into different cells. Here we test the hypothesis that, within the population of cells that express inactivating BK current (BKi current), the BKi channels are largely heteromultimers composed of inactivation-competent subunits (bk(i)) and noninactivating subunits (bk(s)). Several independent types of evidence support this view. The gradual removal of inactivation by trypsin is consistent with the idea that in most cells and patches there are, on average, about two to three inactivation domains per channel. In addition, several aspects of blockade of BKi current by charybdotoxin (CTX) are consistent with the idea that BKi channels contain differing numbers (one to four) of relatively CTX-resistant bk(i) subunits. Finally, the frequency of occurrence of noninactivating BKs channels in patches with predominantly inactivating BKi channels is consistent with the binomial expectations of random, independent assembly of two distinct subunits, if most cells have, on average, about two to three bk(i) subunits per channel. These results suggest that the phenotypic properties of BKi currents and the resulting cellular electrical excitability may exhibit a continuum of behavior that arises simply from the differential expression of two distinct subunits.

Animals↗

PubMed1998

Properties of Ba2+ currents arising from human alpha1E and alpha1Ebeta3 constructs expressed in HEK293 cells: physiology, pharmacology, and comparison to native T-type Ba2+ currents.

Currents arising from human alpha1E and alpha1Ebeta3 Ca2+ channel subunits expressed in HEK-293 cells were examined with whole-cell recording methods and compared to properties of T-current in DRG neurons studied under identical ionic conditions. Coexpression of alpha1E subunit with the beta3 subunit shifted activation to more negative potentials. Activation and deactivation of both variants were comparable at most voltages, with deactivation becoming faster, but less voltage-dependent, at more negative potentials. The inactivation time course for alpha1E and alpha1Ebeta3 currents was best described by at least two exponential components. Recovery from inactivation was markedly voltage-dependent and similar for both constructs. In comparison to alpha1E and alpha1Ebeta3 constructs, T current activation was shifted to more negative potentials, activation was typically slower, deactivation exhibited a steeper voltage-dependence, and recovery from inactivation was less voltage-dependent. Over most of the activation range, native T current inactivated more completely and in a single exponential fashion. Despite some pharmacological similarities (e.g. octanol, barbiturates) between alpha1E and T-type currents, aspects of blockade by amiloride and phenytoin appear to distinguish alpha1E current from T-type currents. The results define several distinguishing features of alpha1E currents that distinguish them from native T-type currents.

Amiloride↗

PubMed1998

The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocks N-, Q-, and R-type, but not L- and P-type, high voltage-activated Ca2+ current in hippocampal and dorsal root ganglion neurons of the rat.

High voltage-activated (HVA) Ca2+ current (ICa) was recorded from neonatal rat hippocampal and adult rat dorsal root ganglion neurons. In both cell types, (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile [(+)-ACN], a neuroactive steroid, had no effect on nifedipine- (L-type) or omega-agatoxin IVA- (P-type) sensitive ICa. Selective blockade of N-type current with omega-conotoxin GVIA and of Q-type current with omega-conotoxin MVIIC indicated that (+)-ACN inhibits both N- and Q-type current components in both cell types. Current persisting after blockade of all other current components (R-type) was also sensitive to (+)-ACN. Half-blockade of (+)-ACN-sensitive HVA current occurred in the range of 3-25 microM, with N-type current somewhat more sensitive than Q- or R-type. The (+)-ACN enantiomer, (-)-ACN, and pregnanolone were somewhat less effective at inhibiting total HVA current than (+)-ACN, whereas several steroid analogs, including alfaxalone, were relatively ineffective at inhibiting total HVA current. Neither guanosine-5'-O-(2-thio)diphosphate nor guanosine-5'-O-(3-thio)triphosphate altered the ability of (+)-ACN to inhibit HVA current in dorsal root ganglion neurons, indicating that (+)-ACN acts directly on Ca2+ channels. The partial selectivity exhibited by (+)-ACN among different HVA current components suggests that manipulations of steroid analogues may be a useful strategy in the generation of more selective, more potent, small-molecular-weight HVA channel blockers.

Androstanols↗

PubMed1998

Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid that lacks gamma-aminobutyric acid-modulatory activity.

A number of steroids seem to have anesthetic effects resulting primarily from their ability to potentiate currents gated by gamma-aminobutyric acidA (GABAA) receptor activation. One such compound is (3alpha,5alpha, 17beta)-3-hydroxyandrostane-17-carbonitrile [(+)-ACN]. We were interested in whether carbonitrile substitution at other ring positions might result in other pharmacological consequences. Here we examine effects of (3beta,5alpha, 17beta)-17-hydroxyestrane-3-carbonitrile [(+)-ECN] on GABAA receptors and Ca2+ channels. In contrast to (+)-ACN, (+)-ECN does not potentiate GABAA-receptor activated currents, nor does it directly gate GABAA-receptor mediated currents. However, both steroids produce an enantioselective reduction of T-type current. (+)-ECN blocked T current with an IC50 value of 0.3 microM with a maximal block of 41%. (+)-ACN produced a partial block of T current (44% maximal block) with an IC50 value of 0.4 microM. Block of T current showed mild use- and voltage-dependence. The (-)-ECN enantiomer was about 33 times less potent than (+)-ECN, with an IC50 value of 10 microM and an amount of maximal block comparable to (+)-ECN. (+)-ECN was less effective at blocking high-voltage-activated Ca2+ current in DRG neurons (IC50 value of 9. 3 microM with maximal block of about 27%) and hippocampal neurons. (+)-ECN (10 microM) had minimal effects on voltage-gated sodium and potassium currents in rat chromaffin cells. The results identify a steroid with no effects on GABAA receptors that produces a partial inhibition of T-type Ca2+ current with reasonably high affinity and selectivity. Further study of steroid actions on T currents may lead to even more selective and potent agents.

Anesthetics↗

PubMed1998

Pharmacological properties of T-type Ca2+ current in adult rat sensory neurons: effects of anticonvulsant and anesthetic agents.

We have used the whole cell patch-clamp method to study pharmacological properties of low-voltage-activated (LVA) Ca2+ current in freshly dissociated neurons from dorsal root ganglia of adult rats. Inward barium current [in the presence of internal fluoride to reduce L-type high-voltage-activated (HVA) and external 1 microM omega-conotoxin GVIA to block N-type HVA current- was evoked from negative holding potentials of -90 mV to test potentials of -25 mV and showed complete inactivation during 200-ms test pulses. Amiloride blocked approximately 90% of current with half-maximal block (EC50) of 75 microM and a Hill coefficient (n) of 0.99. LVA current was blocked completely by inorganic Ca2+ channel blockers: lanthanum (EC50 = 0. 53 microM) > zinc (EC50 = 11.3 microM) > cadmium (EC50 = 20 microM)> nickel (EC50 = 51 microM). The antiepileptics, ethosuximide (EC50 = 23.7 mM, n = 1.4), phenytoin (EC50 = 7.3 microM, n = 1.3), alpha-methyl-alpha-phenylsuccinimide (EC50 = 170 microM, n = 2.1), and valproic acid (EC50 = 330 microM, n = 1.9) maximally blocked approximately 100, 60, 26, and 17% of T current, respectively. Another antiepileptic, carbamazepine (</=100 microM), and convulsants such as pentylenetetrazole (1 mM) and tert-butyl-bicyclo [2.2.2] phosphorothionate (50 microM) had no effect on T current. Barbiturates completely blocked T current: thiopental (EC50 = 153 microM, n =1.2) > pentobarbital (EC50 = 334 microM, n = 1.2) > methohexital (EC50 = 502 microM, n = 1.3) > phenobarbital (EC50 = 1. 7 mM, n = 1.2). Blockade by thiopental and pentobarbital did not show voltage or use dependence. General anesthetics blocked T current completely and reversibly: propofol (EC50 = 12.9 microM, n = 1.3) > octanol(EC50 = 122 microM, n = 1.2) > etomidate (EC50 = 205 microM, n =1.3) > isoflurane (EC50 = 303 microM, n = 2.3) > halothane (EC50 = 655 microM, n = 2.0) > ketamine (EC50 = 2.5 mM, n = 1.1). Mibefradil, a novel Ca2+ channel blocker, blocked dorsal root ganglion T current in a voltage- and use-dependent fashion with an EC50 of approximately 3 microM (n = 1.3). When compared with results on other T currents, these data indicate that significant differences exist among different T currents in terms of pharmacological sensitivities. Furthermore, differences in pharmacological sensitivity of T currents among peripheral neurons, CNS, and neuroendocrine cells may contribute to the spectrum of effects of particular analgesic, anticonvulsant, and anesthetic drugs.

Amiloride↗

PubMed1997

A cysteine-rich domain defined by a novel exon in a slo variant in rat adrenal chromaffin cells and PC12 cells.

cDNA libraries from rat chromaffin cells and PC12 cells were screened for homologs to the mouse mSlo gene that encodes a large conductance, calcium (Ca2+)- and voltage-activated potassium channel (BK channel). One Slo variant contained sequence encoding a cysteine-rich, 59-amino acid insert for a previously described site of alternative splicing. This insert is reminiscent of zinc-finger domains. The exon was found in RNA from pancreas, anterior pituitary, cerebellum, and hippocampus. Expression in Xenopus oocytes of a Slo construct containing this exon conferred a -30 to -20 mV shift of the conductance-voltage curve. A previously uncharacterized alternative splice junction near the C-terminal end of Slo was also identified. In contrast to BK channels in rat chromaffin cells, none of the Slo variants exhibited inactivation when expressed in Xenopus oocytes. PCR screening of chromaffin cell RNA failed to reveal a homolog of an accessory beta subunit known to influence Slo channel function. Furthermore, a beta-subunit-dependent Slo channel activator, dehydrosoyasaponin I, was without effect on chromaffin cell BK current. The results argue that an accessory subunit may not be a required component of the native chromaffin cell BK channel.

Adrenal Medulla↗

PubMed1997

The cytosolic inactivation domains of BKi channels in rat chromaffin cells do not behave like simple, open-channel blockers.

Most BK-type voltage- and Ca(2+)-dependent K+ channels in rat chromaffin cells exhibit rapid inactivation. This inactivation is abolished by brief trypsin application to the cytosolic face of membrane patches. Here we examine the effects of cytosolic channel blockade and pore occupancy on this inactivation process, using inside-out patches and whole-cell recordings. Occupancy of a superficial pore-blocking site by cytosolic quaternary blockers does not slow inactivation. Occupancy of a deeper pore-blocking site by cytosolic application of Cs+ is also without effect on the onset of inactivation. Although the rate of inactivation is relatively unaffected by changes in extracellular K+, the rate of recovery from inactivation (at -80 and -140 mV with 10 microM Ca2+) is faster with increases in extracellular K+ but is unaffected by the impermeant ion, Na+. When tail currents are compared after repolarization, either while channels are open or after inactivation, no channel reopening is detectable during recovery from inactivation. BK inactivation appears to be mechanistically distinct from that of other inactivating voltage-dependent channels. Although involving a trypsin-sensitive cytosolic structure, the block to permeation does not appear to occur directly at the cytosolic mouth or inner half of the ion permeation pathway.

Adrenal Medulla↗

PubMed1997

Anabaseine is a potent agonist on muscle and neuronal alpha-bungarotoxin-sensitive nicotinic receptors.

We assessed the pharmacological activity of anabaseine, a toxin found in certain animal venoms, relative to nicotine and anabasine on a variety of vertebrate nicotinic receptors, using cultured cells, the Xenopus oocyte expression system, contractility assays with skeletal and smooth muscle strips containing nicotinic receptors and in vivo rat prostration assay involving direct injection into the lateral ventricle of the brain. Anabaseine stimulated every subtype of nicotinic receptor that was tested. It was the most potent frog skeletal muscle nicotinic receptor agonist. At higher concentrations it also blocked the BC3H1 (adult mouse) muscle type receptor ion channel. The affinities of the three nicotinoid compounds for rat brain membrane alpha-bungarotoxin binding sites and their potencies for stimulating Xenopus oocyte homomeric alpha7 receptors, expressed in terms of their active monocation concentrations, displayed the same rank order, anabaseine>anabasine> nicotine. Although the maximum currents generated by anabaseine and anabasine at alpha7 receptors were equivalent to that of acetylcholine, the maximum response to nicotine was only about 65% of the acetylcholine response. At alpha4-beta2 receptors the affinities and apparent efficacies of anabaseine and anabasine were much less than that of nicotine. Anabaseine, nicotine and anabasine were nearly equipotent on sympathetic (PC12) receptors, although parasympathetic (myenteric plexus) receptors were much more sensitive to anabaseine and nicotine but less sensitive to anabasine. These differences suggest that there may be different subunit combinations in these two autonomic nicotinic receptors. The preferential interactions of anabaseine, anabasine and nicotine with different receptor subtypes provides molecular clues that should be helpful in the design of selective nicotinic agonists.

Anabasine↗