Conformational changes in a mammalian voltage-dependent potassium channel inactivation peptide

Fast inactivation is restored in inactivation deletion mutant voltage-gated potassium (Kv) channels by application of synthetic inactivation 'ball' peptide. Using Fourier transform infrared and circular dichroism spectroscopy, we have investigated the structure of synthetic Kv3.4 channel ball peptide, in a range of environments relevant to the function of the ball domain. The ball peptide contains no alpha-helix or beta-sheet in reducing conditions in aqueous solution, but when cosolubilized with anionic lipid or detergent in order to mimic the environment which the ball domain encounters during channel inactivation, the ball peptide adopts a partial beta-sheet structure. Oxidation of the Kv3.4 ball peptide facilitates formation of a disulfide bond between Cys6 and Cys24 and adoption of a partial beta-sheet structure in aqueous solution; the tendency of the oxidized ball peptide to adopt beta-sheet is generally greater than that of the reduced ball peptide in a given environment. THREADER modeling of the Kv3.4 ball peptide structure predicts a beta-hairpin-like conformation which corresponds well to the structure suggested by spectroscopic analysis of the ball peptide in its cyclic arrangement. A V7E mutant Kv3.4 ball peptide analogue of the noninactivating Shaker B L7E mutant ball peptide cannot adopt beta-structure whatever the environment, and regardless of oxidation state. The results suggest that the Kv3.4 ball domain undergoes a conformational change during channel inactivation and may implicate a novel regulatory role for intramolecular disulfide bond formation in the Kv3.4 ball domain in vivo.     

Musing of the feline hepatic voltage-dependent delayed-rectifier potassium channel gating

SETTING: Measures known to improve adherence such as short course chemoprophylaxis and directly observed therapy can be enhanced to a significant extent/by the use of incentives. Adherence to tuberculosis therapy is influenced by several factors, including the health care system, complexity of therapeutic regimens and patient's characteristics. Individual factors that negatively influence patient's adherence are the most difficult to counter. Preventive tuberculosis therapy is doubly challenging because the benefit of treatment is not felt, while toxicity from the medication, when it occurs, is experienced immediately. Ingenious incentives therefore have to make it worth the patient's while. During a study on preventive regimens, a request for an incentive, Sustacal, was observed to help completion of preventive regimens. Components of individual TB programs may help in patient adherence; it is important for health care staff to identify these aspects and, if they are successful, utilize these as an incentive to complete treatment.     

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.     

Mechanism of cloned ATP-sensitive potassium channel activation by oleoyl-CoA

Insulin secretion from pancreatic beta cells is coupled to cell metabolism through closure of ATP-sensitive potassium (KATP) channels, which comprise Kir6.2 and sulfonylurea receptor (SUR1) subunits. Although metabolic regulation of KATP channel activity is believed to be mediated principally by the adenine nucleotides, other metabolic intermediates, including long chain acyl-CoA esters, may also be involved. We recorded macroscopic and single-channel currents from Xenopus oocytes expressing either Kir6.2/SUR1 or Kir6. 2DeltaC36 (which forms channels in the absence of SUR1). Oleoyl-CoA (1 microM) activated both wild-type Kir6.2/SUR1 and Kir6.2DeltaC36 macroscopic currents, approximately 2-fold, by increasing the number and open probability of Kir6.2/SUR1 and Kir6.2DeltaC36 channels. It was ineffective on the related Kir subunit Kir1.1a. Oleoyl-CoA also impaired channel inhibition by ATP, increasing the Ki values for both Kir6.2/SUR1 and Kir6.2DeltaC36 currents by approximately 3-fold. Our results indicate that activation of KATP channels by oleoyl-CoA results from an interaction with the Kir6.2 subunit, unlike the stimulatory effects of MgADP and diazoxide which are mediated through SUR1. The increased activity and reduced ATP sensitivity of KATP channels by oleoyl-CoA might contribute to the impaired insulin secretion observed in non-insulin-dependent diabetes mellitus.     

Shaker potassium channel gating. II: Transitions in the activation pathway

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.     

Extracellular Mg2+ regulates activation of rat eag potassium channel

The rat homologue of Drosophila ether à gogo cDNA (rat eag) encodes voltage-activated potassium (K) channels with distinct activation properties. Using the Xenopus expression system, we examined the importance of extracellular Mg2+ on the activation of rat eag. Extracellular Mg2+ at physiological concentrations dramatically slowed the activation in a dose- and voltage-dependent manner. Other divalent cations exerted similar effects on the activation kinetics that correlated with their enthalpy of hydration. Lowering the external pH also resulted in a slowing of the activation. Protons competed with Mg2+ as the effect of Mg2+ was abolished at low pH. A kinetic model for rat eag activation was derived from the data indicating that all four channel subunits undergo a Mg2+-dependent conformational transition prior to final channel activation. The strong dependence of rat eag activation on both the resting potential and the extracellular Mg2+ concentration constitutes a system for fine-tuning K channel availability in neuronal cells.     

Dopamine inhibition: enhancement of GABA activity and potassium channel activation in hypothalamic and arcuate nucleus neurons

Dopamine (DA) decreases activity in many hypothalamic neurons. To determine the mechanisms of DA's inhibitory effect, whole cell voltage- and current-clamp recordings were made from primary cultures of rat hypothalamic and arcuate nucleus neurons (n = 186; 15-39 days in vitro). In normal buffer, DA (usually 10 microM; n = 23) decreased activity in 56% of current-clamped cells and enhanced activity in 22% of the neurons. In neurons tested in the presence of glutamate receptor antagonists D,L-2-amino-5-phosphonovalerate (AP5; 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM), DA application (10 microM) revealed heterogeneous effects on electrical activity of cells, either hyperpolarization and decrease in activity (53% of 125) or depolarization and increase in spontaneous activity (22% of 125). The DA-mediated hyperpolarization of membrane potential was associated with a decrease in the input resistance. The reversal potential for the DA-mediated hyperpolarization was -97 mV, and it shifted in a positive direction when the concentration of K+ in the incubating medium was increased, suggesting DA activation of K+ channels. Because DA did not have a significant effect on the amplitude of voltage-dependent K+ currents, activation of voltage-independent K+ currents may account for most of the hyperpolarizing actions of DA. DA-mediated hyperpolarization and depolarization of neurons were found during application of the Na+ channel blocker tetrodotoxin (1 microM). The hyperpolarization was blocked by the application of DA D2 receptor antagonist eticlopride (1-20 microM; n = 7). In the presence of AP5 and CNQX, DA (10 microM) increased (by 250%) the frequency of spontaneous inhibitory postsynaptic currents (IPSCs) in 11 of 19 neurons and evoked IPSCs in 7 of 9 cells that had not previously shown any IPSCs. DA also increased the regularity and the amplitude (by 240%) of spontaneous IPSCs in 9 and 4 of 19 cells, respectively. Spontaneous and DA-evoked IPSCs and inhibitory postsynaptic potentials were blocked by the gamma-aminobutyrate A (GABA(A)) antagonist bicuculline (50 microM), verifying their GABAergic origin. Pertussis toxin pretreatment (200 ng/ml; n = 15) blocked the DA-mediated hyperpolarizations, but did not prevent depolarizations (n = 3 of 15) or increases in IPSCs (n = 6 of 10) elicited by DA. Intracellular neurobiotin injections (n = 21) revealed no morphological differences between cells that showed depolarizing or hyperpolarizing responses to DA. Immunolabeling neurobiotin-filled neurons that responded to DA (n = 13) showed that GABA immunoreactive neurons (n = 4) showed depolarizing responses to DA, whereas nonimmunoreactive neurons (n = 9) showed both hyperpolarizing (n = 6) and depolarizing (n = 3) responses. DA-mediated hyperpolarization, depolarization, and increases in frequency of postsynaptic activity could be detected in embryonic hypothalamic or arcuate nucleus neurons after only 5 days in vitro, suggesting that DA could play a modulatory role in early development. These findings suggest that DA inhibition in hypothalamic and arcuate nucleus neurons is achieved in part through the direct inhibition of excitatory neurons, probably via DA D2 receptors acting through a Gi/Go protein on K+ channels, and in part through the enhancement of GABAergic neurotransmission.     

Ca2+ channel beta3 subunit enhances voltage-dependent relief of G-protein inhibition induced by muscarinic receptor activation and Gbetagamma

The Ca2+ channel beta subunit has been shown to reduce the magnitude of G-protein inhibition of Ca2+ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca2+ channel beta3 subunit causes M2 muscarinic receptor-mediated inhibition of alpha1B Ca2+ currents to become more voltage-dependent. We report here that the beta3 subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of alpha1B channels to G-protein-mediated inhibition seen after coexpression of the Ca2+ channel beta3 subunit. Additionally, the beta3 subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed Gbeta1gamma1, in a manner similar to the changes it produces in relief of M2 receptor-induced inhibition. We conclude that the Ca2+ channel beta3 subunit reduces the magnitude of G-protein inhibition of alpha1B Ca2+ channels by enhancing the rate of dissociation of the G-protein betagamma subunit from the Ca2+ channel alpha1B subunit.     

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.     

Transmembrane movement of the shaker K+ channel S4

We have probed internal and external accessibility of S4 residues to the membrane-impermeant thiol reagent methanethiosulfonate-ethyltrimethlammonium (MTSET) in both open and closed, cysteine-substituted Shaker K+ channels. Our results indicate that S4 traverses the membrane with no more than 5 amino acids in the closed state, and that the distribution of buried residues changes when channels open. This change argues for a displacement of S4 through the plane of the membrane in which an initially intracellular residue moves to within 3 amino acids of the extracellular solution. These results demonstrate that the putative voltage-sensing charges of S4 actually reside in the membrane and that they move outward when channels open. We consider constraints placed on channel structure by these results.     

Patch-clamp and amperometric recordings from norepinephrine transporters: channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.     

M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons

Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.     

Parallel dose-response studies of the voltage-dependent Na+ channel antagonist BW619C89, and the voltage-dependent Ca2+ channel antagonist nimodipine, in rat transient focal cerebral ischaemia

We have compared two classes of putative neuroprotectants, the voltage-dependent Na+ channel antagonist BW619C87 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl) pyrimidine], and the voltage-dependent Ca2+ channel antagonist nimodipine, in a rat model of transient focal cerebral ischaemia. BW619C87 (10-50 mg/kg) or nimodipine (10-100 microg/kg) were injected intravenously 5 min before induction of 2 h transient focal cerebral ischaemia via intraluminal thread occlusion of the middle cerebral artery. BW619C87 was a potent neuroprotectant over the range tested, maximally reducing the volume of hemispheric ischaemic damage by 51% at the 50 mg/kg dose. Nimodipine maximally reduced ischaemic damage by 33% at the 50 microg/kg dose, although the maximal level of neuroprotection afforded by BW619C89 and nimodipine was not significantly different. This is the first study to compare these two classes of drug directly in a model of middle cerebral artery occlusion with reperfusion, and it supports the effectiveness of both as neuroprotectants.     

Anticonvulsant activity and interactions with neuronal voltage-dependent sodium channel of analogues of ameltolide

Fifteen compounds related to ameltolide (LY 201116) were studied for (i) anticonvulsant potential in the maximal electroshock-induced seizures (MES) and the subcutaneous pentylenetetrazol (sc Ptz) tests in mice and rats and (ii) interactions with neuronal voltage-dependent sodium channels. Compounds were chosen ranging in anticonvulsant activity in mice from very active to inactive. The active compounds were defined as those protecting 50% of the animals at doses between 10 and 50 micromol/kg and inactive compounds as those protecting 50% of the animals at doses greater than 1 mmol/kg. The series studied included three N-(2,6-dimethylphenyl)benzamides (compounds 1, 2 (ameltolide), and 3), three N-(2,2,6, 6-tetramethyl)piperidinyl-4-benzamides (compounds 4, 5, 6), one phenylthiourea (compound 7), five N-(2,6-dimethylphenyl)phthalimides (compounds 8, 9, 10, 13, and 14), two N-phenylphthalimide derivatives (compounds 11 and 12), and one N-(2,2,6, 6-tetramethyl)piperidinyl-4-phthalimide (compound 15). Phenytoin (PHT) was employed as the reference prototype antiepileptic drug. After inital screening in mice, compounds 1, 2, 3, 5, 8, 9, 10, 13, and 14 were selected for further testing in rats. Anticonvulsant ED50s (effective doses in at least 50% of animals tested) of compounds in the MES test were determined in rats dosed orally and amounted to 52 (1), 135 (2), 284 (3), 231 (8), 131 (9), 25 (10), 369 (13), 354 (14), and 121 (PHT) micromol/kg, compound 5 presenting with an ED50 value higher than 650 micromol/kg. In our hands, the apparent IC50s (inhibitory concentrations 50) of compounds toward binding to rat brain synaptosomes of [3H]batrachotoxinin-A-20alpha-benzoate were 0.25 (1), 0.97 (2), 0.35 (3), 25.8 (5), 161.3 (8), 183.5 (9), 0.11 (10), 1.86 (13), 47.8 (14), and 0.86 (PHT) microM. The relationship between the activity in the MES test and the capacity to interact in vitro with neuronal voltage-dependent sodium channels and the fact that the IC50 values obtained in the in vitro test are close to the brain concentrations at which anticonvulsant activities are reported to occur for ameltolide strongly suggest that the anticonvulsant properties of most compounds tested could be a direct result of their interaction with the neuronal voltage-dependent sodium channel.     

Muscarinic activation of a voltage-dependent cation nonselective current in rat association cortex

The ionic mechanism underlying the acetylcholine-induced depolarization of layer V pyramidal neurons of rat prefrontal cortex was examined using whole-cell recording in in vitro rat brain slices. Consistent with previous results, pressure application of acetylcholine to layer V pyramidal neurons elicited a strong depolarization. Pharmacological analysis of this response indicated that it was mediated by the stimulation of muscarinic receptors as it was mimicked by muscarinic agonists, but not by nicotine, and was blocked by atropine. The inward current responsible for the depolarization resulted from the activation of a voltage-dependent, cation nonselective current. Thus, the amplitude of the current was critically dependent on extracellular sodium concentration but not on extracellular potassium or chloride concentration. Examination of the I-V relationship for the muscarinic current using voltage clamp revealed that the current reversed near -15 mV and exhibited a strong voltage dependence, turning off rapidly in the subthreshold range. The voltage dependence of the current led to the appearance of a current associated with a conductance decrease when examined using steady-state voltage- or current-clamp measurements. This might have led to earlier misidentification of this response as mediated by a decrease in potassium conductance. These results question the traditional interpretation that muscarinic depolarization in cortex is mediated by a decrease in potassium conductance. They indicate that the fundamental mechanism responsible for muscarinic depolarization in prefrontal cortex involves the activation of a voltage-dependent, cation nonselective current. This current might represent a previously unsuspected mechanism capable of mediating slow depolarization in the central nervous system.     

Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity

Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases. To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231. In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type. In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities. In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231. To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.     

Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function

Literature review for the years 1997 and 1998 presents new concepts for gastric carcinoma (lymphoma and proximal adenocarcinoma excluded). In the light of 50 papers, this update emphasizes the role of Helicobacter Pylori in gastric carcinogenesis, different staging systems, video-laparoscopic staging, treatment of early and advanced gastric cancer and new biological prognostic factors.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.