Axotomy- and autotomy-induced changes in Ca2+ and K+ channel currents of rat dorsal root ganglion neurons

Sciatic nerve section (axotomy) increases the excitability of rat dorsal root ganglion (DRG) neurons. The changes in Ca2+ currents, K+ currents, Ca2+ sensitive K+ current, and hyperpolarization-activated cation current (I(H)) that may be associated with this effect were examined by whole cell recording. Axotomy affected the same conductances in all types of DRG neuron. In general, the largest changes were seen in "small" cells and the smallest changes were seen in "large" cells. High-voltage-activated Ca2+ channel current (HVA-I(Ba)) was reduced by axotomy. Although currents recorded in axotomized neurons exhibited increased inactivation, this did not account for all of the reduction in HVA-I(Ba). Activation kinetics were unchanged, and experiments with nifedipine and/or omega-conotoxin GVIA showed that there was no change in the percentage contribution of L-type, N-type, or "other" HVA-I(Ba) to the total current after axotomy. T-type (low-voltage-activated) I(Ba) was not affected by axotomy. Ca2+ sensitive K+ conductance (g(K,Ca)) appeared to be reduced, but when voltage protocols were adjusted to elicit similar amounts of Ca2+ influx into control and axotomized cells, I(K,Ca)(s) were unchanged. After axotomy, Cd2+ insensitive, steady-state K+ channel current, which primarily comprised delayed rectifier K+ current (I(K)), was reduced by about 60% in small, medium, and large cells. These data suggest that axotomy-induced increases in excitability are associated with decreases in I(K) and/or decreases in g(K,Ca) that are secondary to decreased Ca2+ influx. Because I(H) was reduced by axotomy, changes in this current do not contribute to increased excitability. The amplitude and inactivation of I(Ba) in all cell types was changed more profoundly in animals that exhibited self-mutilatory behavior (autotomy). The onset of this behavior corresponded with significant reduction in I(Ba) of large neurons. This finding supports the hypothesis that autotomy, that may be related to human neuropathic pain, is associated with changes in the properties of large myelinated sensory neurons.     

Ca2+ channel currents in dorsal root ganglion neurons of P/Q-type voltage-gated Ca2+ channel mutant mouse, rolling mouse Nagoya

The role of the P/Q-type voltage-gated Ca(2+) channels (VGCCs) in release of neurotransmitters involved in nociception is not fully understood. Rolling mouse Nagoya (tg(rol)), a P/Q-type channel mutant mouse, expresses P/Q-type VGCC whose activation curve has a higher half activation potential and a smaller slope factor than the wild type channel. We previously reported that tg(rol) mice showed hypoalgesic responses to noxious stimuli. In this study, we examined the VGCC current in dorsal root ganglion (DRG) neurons by the whole-cell patch-clamp method. Both ω-agatoxin IVA (0.1 μM) and ω-conotoxin GVIA (1 μM) inhibited the VGCC current by about 40-50% in both the homozygous tg(rol) (tg(rol)/tg(rol)) and wild type (+/+) mice. The voltage-activation relationships of the total VGCC current and the ω-agatoxin IVA-sensitive component in the tg(rol)/tg(rol) mice shifted positively compared to the +/+ mice, whereas that sensitive to the ω-conotoxin GVIA was not different between the two genotypes. The time constant of activation of the VGCC current at -20 mV was longer in the tg(rol)/tg(rol) mice than in the +/+ mice. These changes in the properties of the VGCC in the tg(rol)/tg(rol) mouse may reduce the amount of the released neurotransmitters and account for the hypoalgesic responses.     

Melatonin modulates wireless (2.45 GHz)-induced oxidative injury through TRPM2 and voltage gated Ca(2+) channels in brain and dorsal root ganglion in rat

We aimed to investigate the protective effects of melatonin and 2.45 GHz electromagnetic radiation (EMR) on brain and dorsal root ganglion (DRG) neuron antioxidant redox system, Ca(2+) influx, cell viability and electroencephalography (EEG) records in the rat. Thirty two rats were equally divided into four different groups namely group A1: Cage control, group A2: Sham control, group B: 2.45 GHz EMR, group C: 2.45 GHz EMR+melatonin. Groups B and C were exposed to 2.45 GHz EMR during 60 min/day for 30 days. End of the experiments, EEG records and the brain cortex and DRG samples were taken. Lipid peroxidation (LP), cell viability and cytosolic Ca(2+) values in DRG neurons were higher in group B than in groups A1 and A2 although their concentrations were increased by melatonin, 2-aminoethyldiphenyl borinate (2-APB), diltiazem and verapamil supplementation. Spike numbers of EEG records in group C were lower than in group B. Brain cortex vitamin E concentration was higher in group C than in group B. In conclusion, Melatonin supplementation in DRG neurons and brain seems to have protective effects on the 2.45 GHz-induced increase Ca(2+) influx, EEG records and cell viability of the hormone through TRPM2 and voltage gated Ca(2+) channels.     

Rapid inhibitory effects of corticosterone on calcium influx in rat dorsal root ganglion neurons

Corticosterone may nongenomically affect cell functions in addition to its well-characterized effects on gene expression. The purpose of this study is to examine if corticosterone has a rapid nongenomic effect on excitability of dorsal root ganglion neurons by using patch-clamp and single-cell Ca(2+) microfluometry techniques. The results show that corticosterone has a dose-dependent rapid inhibitory effect on the voltage-dependent calcium currents in dorsal root ganglion neurons. Moreover, corticosterone inhibits [Ca(2+)](i) elevation induced by 50 mM high K(+) within just 3 s. The inhibitory effects of corticosterone on the voltage-dependent calcium current and high K(+)-induced calcium influx diminish after adding protein kinase C inhibitor or pretreatment with pertussis toxin for 24 h. Our results demonstrate an nongenomic effect of corticosterone on the excitability of dorsal root ganglion neurons and the effect is mediated through a putative pertussis toxin-sensitive G-protein-coupled receptor and activation of protein kinase C.     

TRPM2 contributes to antigen-stimulated Ca2+ influx in mucosal mast cells

Food allergy (FA) is a common allergic disease without any currently available effective drug therapies. Mucosal mast cells (MMCs) play a particularly important role in FA, and the increase in their cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) is considered to be a principal component of the degranulation process. However, the mechanisms governing Ca²⁺ influx remain poorly understood in MMCs. Recent reports have highlighted the functions of the transient receptor potential melastatin 2 (TRPM2) channel in immunocytes, including its role in monocyte chemokine production and macrophage phagocytic activity. Although TRPM2 gene expression has been demonstrated in mast cells, the significance of such expression remains virtually unknown. In this study, we found that antigen-stimulated degranulation was significantly reduced in mucosal-type bone marrow-derived mast cells (mBMMCs) prepared from TRPM2-knockout (TRPM2-KO) mice (TRPM2-KO mBMMCs) and was suppressed following the administration of three TRPM2 inhibitors with different chemical structures, including econazole, flufenamic acid (FFA), and 2-aminoethoxydiphenyl borate. Furthermore, the antigen-stimulated increase in [Ca²⁺]_cyt was significantly decreased in TRPM2-KO mBMMCs and was also suppressed by the TRPM2 inhibitors econazole and FFA. In addition, thapsigargin-induced increase in [Ca²⁺]_cyt was significantly decreased in TRPM2-KO mBMMCs. These results suggest that TRPM2 may participate in antigen-induced extracellular Ca²⁺ influx and subsequent degranulation. In addition, TRPM2 inhibitors were shown to improve food allergic reactions in a mouse model. Together, these results suggest that TRPM2 inhibitors suppress MMC degranulation via regulation of the increase in [Ca²⁺]_cyt. Thus, TRPM2 may play a key role in degranulation by modulating intracellular Ca²⁺ in MMCs.     

Expression of voltage-gated calcium channel subunits in rat dorsal root ganglion neurons

The ability of a series of specific Galpha carboxyl-terminal antisera, (i.e. anti-Gsalpha, anti-Gi1/2alpha, anti-Gi3alpha/Goalpha, anti-Goalpha/Gi3alpha, and anti-Gq/11alpha) to disrupt (+/-)-baclofen-stimulated high-affinity guanosine triphosphatase (GTPase) activity was explored in rat cerebral cortical membranes to identify the Galpha subunit(s) involved in gamma-aminobutyric acid(B) (GABA(B)) receptor-mediated signal transduction. Pretreatment of the membranes with the AS/7 (anti-Gi1/2alpha) antiserum inhibited GABA(B) receptor-mediated response without affecting the basal activity. The RM/1 (anti-Gsalpha) and QL (anti-Gq/11alpha) antisera failed to inhibit GABA(B) receptor-coupled responses. The results of the EC/2 (anti-Gi3alpha/Goalpha) and GO/1 (anti-Goalpha/Gi3alpha) antisera were difficult to interpret since the basal activities were influenced by these antisera. These results, in conjunction with the data in our previous reconstitution study, indicate that Gi2alpha is a main transducer of GABA(B) receptor-mediated signaling in rat cerebral cortex.     

Voltage-dependent K+ currents in rat cardiac dorsal root ganglion neurons

We have assessed the expression and kinetics of voltage-gated K(+) currents in cardiac dorsal root ganglion (DRG) neurons in rats. The neurons were labelled by prior injection of a fluorescent tracer into the pericardial sack. Ninety-nine neurons were labelled: 24% small (diameter<30 microm), 66% medium-sized (diameter 30 microm>.48 microm) and 10% large (>48 microm) neurons. Current recordings were performed in small and medium-sized neurons. The kinetic and pharmacological properties of K(+) currents recorded in these two groups of neurons were identical and the results obtained from these neurons were pooled. Three types of K(+) currents were identified:a) I(As), slowly activating and slowly time-dependently inactivating current, with V(1/2) of activation -18 mV and current density at +30 mV equal to 164 pA/pF, V(1/2) of inactivation at -84 mV. b) I(Af) current, fast activating and fast time-dependently inactivating current, with V(1/2) of activation at two mV and current density at +30 mV equal to 180 pA/pF, V(1/2) of inactivation at -26 mV. At resting membrane potential I(As) was inactivated, whilst I(Af), available for activation. The I(As) currents recovered faster from inactivation than I(Af) current. 4-Aminopiridyne (4-AP) (10 mM) and tetraethylammonium (TEA) (100 mM) produced 98% and 92% reductions of I(Af) current, respectively and 27% and 66% of I(As) current, respectively. c) The I(K) current that did not inactivate over time. Its V(1/2) of activation was -11 mV and its current density equaled 67 pA/pF. This current was inhibited by 95% (100 mM) TEA, whilst 4-AP (10 mM) produced its 23% reduction. All three K(+) current components (I(As), I(Af) and I(K)) were present in every small and medium-sized cardiac DRG neuron.We suggest that at hyperpolarized membrane potentials the fast reactivating I(As) current limits the action potential firing rate of cardiac DRG neurons. At depolarised membrane potentials the I(Af) K(+) current, the reactivation of which is very slow, does not oppose the firing rate of cardiac DRG neurons.     

Expression of the TRPM8-immunoreactivity in dorsal root ganglion neurons innervating the rat urinary bladder

The neurochemical phenotypes of the transient receptor potential melastatin-8 (TRPM8)-immunoreactive afferent neurons innervating the rat urinary bladder were examined by using a highly sensitive tyramide signal amplification method, combined with wheat-germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing. TRPM8-immunoreactivity was detected in a small proportion of the WGA-HRP-labeled bladder afferent neurons in the dorsal root ganglia of the Th13-L1 (1.14%) and the L6-S1 (1.27%), and these neurons were small in size (<600 μm²). The 82.6 ± 3.8% of the TRPM8-immunoreactive bladder afferent neurons and 80.9 ± 1.5% of the total population of the TRPM8-immunoreactive afferent neurons in the observed dorsal root ganglia expressed NF200. On the other hand, the proportions of the co-expression of TRPM8 and nociceptive markers such as calcitonin gene-related peptide (CGRP), transient receptor potential vanilloid-1 (TRPV1), and isolectin B4 (IB4) in the bladder afferent neurons (81.5 ± 5.2% for CGRP, 36.1 ± 4.0% for TRPV1, and 15.8 ± 5.5% for IB4) were higher in comparison to those in the total population of the TRPM8-immunoreactive afferent neurons (21.9 ± 2.4% for CGRP, 16.6 ± 1.7% for TRPV1, and 5.4 ± 0.5% for IB4), although no significant difference existed for IB4. Our results suggest that the TRPM8-expressing bladder afferents should be classified as Aδ-fibers and C-fibers, while some of these afferents may be involved in nociceptive sensations.     

Immunohistochemical analysis of acid-sensing ion channel 2 expression in rat dorsal root ganglion and effects of axotomy

Several studies have suggested that acid-sensing ion channel 2 (ASIC2) plays a role in mechanoperception and acid sensing in the peripheral nervous system. We examined the expression and distribution of ASIC2 in the rat dorsal root ganglion, the co-localization of ASIC2 with tropomyosin-related kinase (trk) receptors, and the effects of axotomy on ASIC2 expression. ASIC2 immunoreactivity was observed in both neurons and satellite cells. ASIC2-positive neurons accounted for 16.5 +/- 2.4% of the total neurons in normal dorsal root ganglion. Most ASIC2-positive neurons were medium-to-large neurons and were labeled with neurofilament 200 kD (NF200). Within these neurons, ASIC2 was not evenly distributed throughout the cytoplasm, but rather was accumulated prominently in the cytoplasm adjacent to the axon hillock and axonal process. We next examined the co-localization of ASIC2 with trk receptors. trkA was expressed in few ASIC2-positive neurons, and trkB and trkC were observed in 85.2% and 53.4% of ASIC2-positive neurons, respectively, while only 6.9% of ASIC2-positive neurons were co-localized with trkC alone. Peripheral axotomy markedly reduced ASIC2 expression in the axotomized dorsal root ganglion neurons. On the other hand, intense ASIC2 staining was observed in satellite cells. These results show that ASIC2 is expressed in the distinct neurochemical population of sensory neurons as well as satellite cells, and that peripheral axotomy induced marked reductions in ASIC2 in neurons.     

GABAB-mediated upregulation of the high-voltage-activated Ca2+ channels in rat dorsal root ganglia

 The mechanism underlying the enhancement of the high-voltage-activated (HVA) Ca²⁺ current (I _Ca) after application of baclofen, a GABA_B agonist, in neurones of the rat dorsal root ganglia was studied by a combined use of the nystatin perforated patch clamp recording and our rapid superfusion system. Baclofen (50 μM) decreased the peak amplitude of HVA I _Ca and slowed the onset of the current, i.e. produced a typical G-protein-mediated inhibition of I _Ca. However, when baclofen was rapidly removed from the medium, the amplitude of the current was rather augmented, exceeding the control value obtained before application of the drug. This enhancement was not due to a shift of the voltage dependence of Ca²⁺ channel activation or a change in ionic permeability to other ions. The enhancement of HVA I _Ca by baclofen was sensitive to pertussis toxin treatment. The enhancement was evident during superfusion of baclofen. Since the inhibitory effect of baclofen on HVA I _Ca was not attenuated, even after a continuous application of baclofen for 10 min, the enhancement was not due to relief from tonic G-protein-mediated inhibition of the current or a desensitization of the GABA_B receptor–effector system. An extremely prolonged time course of the enhancement of HVA I _Ca by baclofen strongly suggests an involvement of some intracellular signal transduction system. Received: 22 May 1996 / Received after revision: 17 October 1996 / Accepted: 7 January 1997     

前列腺癌的分子生物学研究进展

前列腺癌是男性中的发病率最高的癌症,死亡率仅次于肺癌居第2位,严重影响男性的生殖健康。前列腺特异性抗原(PSA)是一种常见的前列腺癌筛查指标。近来研究表明前列腺癌的发生可能与钙离子有关,TRPM8是Ca2 离子通道家族的一员,是一种在前列腺癌和其他癌细胞中的调节蛋白,在前列腺癌细胞中的表达较正常的前列腺上皮细胞高。虽然前列腺癌患者血清中PSA含量明显升高,但处于灰色区域(4～10ng/ml)的患者,TRPM8是一种潜在的早期诊断指标,也将是药物或基因治疗的潜在靶标。     

Distinct inhibition of voltage-activated Ca2+ channels by delta-opioid agonists in dorsal root ganglion neurons devoid of functional T-type Ca2+ currents

Both mu- and delta-opioid agonists selectively inhibit nociception but have little effect on other sensory modalities. Voltage-activated Ca(2+) channels in the primary sensory neurons are important for the regulation of nociceptive transmission. In this study, we determined the effect of delta-opioid agonists on voltage-activated Ca(2+) channel currents (I(Ca)) in small-diameter rat dorsal root ganglion (DRG) neurons that do and do not bind isolectin B(4) (IB(4)). The delta-opioid agonists [d-Pen(2),d-Pen(5)]-enkephalin (DPDPE) and deltorphin II produced a greater inhibition of high voltage-activated I(Ca) in IB(4)-negative than IB(4)-positive neurons. Furthermore, DPDPE produced a greater inhibition of N-, P/Q-, and L-type I(Ca) in IB(4)-negative than IB(4)-positive neurons. However, DPDPE had no significant effect on the R-type I(Ca) in either type of cells. We were surprised to find that DPDPE failed to inhibit either the T-type or high voltage-activated I(Ca) in all the DRG neurons with T-type I(Ca). Double immunofluorescence labeling showed that the majority of the delta-opioid receptor-immunoreactive DRG neurons had IB(4) labeling, while all DRG neurons immunoreactive to delta-opioid receptors exhibited Cav(3.2) immunoreactivity. Additionally, DPDPE significantly inhibited high voltage-activated I(Ca) in Tyrode's or N-methyl-d-glucamine solution but not in tetraethylammonium solution. This study provides new information that delta-opioid agonists have a distinct effect on voltage-activated Ca(2+) channels in different phenotypes of primary sensory neurons. High voltage-activated Ca(2+) channels are more sensitive to inhibition by delta-opioid agonists in IB(4)-negative than IB(4)-positive neurons, and this opioid effect is restricted to DRG neurons devoid of functional T-type Ca(2+) currents.     

Electrophysiological properties of subpopulations of rat dorsal root ganglion neurons in vitro

Intracellular recordings were made from rat dorsal root ganglion neurons in vitro. Action potentials with an inflection on the falling phase occurred in all cells conducting up to 5.2 m/s and in a proportion of faster conducting cells which decreased with increasing conduction velocity, until no cells conducting faster than 31 m/s had an inflection. Overall, all C-cells (less than 1.3 m/s), 61% of A delta-cells (1.3-12 m/s) and 23% of A alpha/beta-cells (greater than 12 m/s) had inflections. A-cells with inflections were found to be electrophysiologically distinct from those without as they differed in the mean and distribution of every action potential and afterhyperpolarization parameter measured. C-cells differed from all A-cell groups, but the means and distributions of most parameters were closer to those of A-cells with inflections than of A-cells without. In addition, all A- and C-cell action potentials with inflections were tetrodotoxin resistant, while all those without were sensitive. The only parameters whose means differed between A alpha/beta- and A delta-neurons were ones which correlated with conduction velocity (action potential duration, overshoot and maximum rate of rise and fall). The response pattern to prolonged current injection did not correlate with conduction velocity, but slightly more A-cells with inflections were single firing. A-cells with long afterhyperpolarizations always fired singly, while those with shorter durations fired singly or multiply. Somatic following frequency was most strongly limited by long afterhyperpolarization duration; it was also slightly lower in A delta- than in A alpha/beta-cells, and lower in A-cells with inflections than in those without. Fibre following frequencies were highest in the fastest conduction neurons.     

Low- and high-voltage-activated Ca2+ conductances in electrically excitable growth cones of chick dorsal root ganglion neurons

Growth cone Ca2+ currents of chick dorsal root ganglion (DRG) neurons were recorded by a patch pipette located on the cell soma. Somatic and neuritic conductances were selectively blocked either with TTX and Cd2+ or by superfusing with isotonic sucrose using a laminar flow perfusion system. DRG growth cones were electrically excitable and growth cone Ca2+ currents were similar to Ca2+ currents described in DRG somata. In particular low-voltage-activated (LVA) Ca2+ conductances were well represented contrary to previous suggestions in other cell types.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

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.