Pharmacological studies of Cav3.1 T-type calcium channels using automated patch-clamp techniques

T-type calcium channels are involved in a variety of physiological and pathophysiological processes, and thus could be therapeutic targets. However, there is no T-type channel selective blocker for use in clinical practice, demanding a need for the development of novel drugs where a higher-throughput screening system is required. Here we present pharmacological studies on Ca(v)3.1 T-type channels using automated patch-clamp. The IC(50) values obtained from automated patch-clamp and conventional one showed a good correlation (correlation coefficient of 0.82), suggesting that the automated patch-clamp is an efficient and reliable method for ranking the drug potencies for T-type channels.     

Discovery and evaluation of Cav3.1-selective T-type calcium channel blockers

We identified and characterized a series of pyrazole amides as potent, selective Ca_v3.1-blockers. This series culminated with the identification of pyrazole amides 5a and 12d, with excellent potencies and/or selectivities toward the Ca_v3.2- and Ca_v3.3-channels. This compound displays poor DMPK properties, making its use difficult for in vivo applications. Nevertheless, this compound as well as analogous ones are well-suited for in vitro studies.     

Low voltage-activated calcium channels in vascular smooth muscle: T-type channels and AVP-stimulated calcium spiking

An important path of extracellular calcium influx in vascular smooth muscle (VSM) cells is through voltage-activated Ca2+ channels of the plasma membrane. Both high (HVA)- and low (LVA)-voltage-activated Ca2+ currents are present in VSM cells, yet little is known about the relevance of the LVA T-type channels. In this report, we provide molecular evidence for T-type Ca2+ channels in rat arterial VSM and characterize endogenous LVA Ca2+ currents in the aortic smooth muscle-derived cell line A7r5. AVP is a vasoconstrictor hormone that, at physiological concentrations, stimulates Ca2+ oscillations (spiking) in monolayer cultures of A7r5 cells. The present study investigated the role of T-type Ca2+ channels in this response with a combination of pharmacological and molecular approaches. We demonstrate that AVP-stimulated Ca2+ spiking can be abolished by mibefradil at low concentrations (<1 microM) that should not inhibit L-type currents. Infection of A7r5 cells with an adenovirus containing the Cav3.2 T-type channel resulted in robust LVA Ca2+ currents but did not alter the AVP-stimulated Ca2+ spiking response. Together these data suggest that T-type Ca2+ channels are necessary for the onset of AVP-stimulated calcium oscillations; however, LVA Ca2+ entry through these channels is not limiting for repetitive Ca2+ spiking observed in A7r5 cells.     

Protein kinases modulate store-operated channels in pulmonary artery smooth muscle cells

Background  

This study investigates whether protein kinase G (PKG), protein kinase A (PKA) and protein kinase C (PKC) are involved in the regulatory mechanisms of store-operated channel (SOC) in pulmonary arteries.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Ca(v)3 channels: Ca(v)3.1, Ca(v)3.2, and Ca(v)3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 microM) of nickel were found to selectively block the Ca(v)3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Ca(v)3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Ca(v)3.2 and the nickel-insensitive Ca(v)3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Ca(v)3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3-S4 loop of domain I significantly attenuated the nickel block of Ca(v)3.2, mimicking the nickel-insensitive blocking potency of Ca(v)3.1. These findings indicate that His-191 in the S3-S4 loop is a critical residue conferring nickel block to Ca(v)3.2 and reveal a novel role for the S3-S4 loop to control ion permeation through T-type Ca2+ channels.     

Monovalent currents through the T-type Cav3.1 channels and their block by Mg2+

We have investigated the permeability of the Cav3.1 channel for Ca2+ and different monovalent cations and the block of the currents by Mg2+ ions. In the absence of extracellular divalent cations, the Cav3.1 channel was more permeable for Na+ than for Cs+ and impermeable for NMDG+. Monovalent currents were inhibited by Mg2+ of near physiological concentration by three orders of magnitude more effectively than the Ca2+ current. Inhibition of outward, but not inward current by Mg2+ was voltage-dependent. Furthermore, magnesium slowed down channel deactivation presumably by interacting with an open channel state.     

Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels

The human umbilical cord is a biological sample that can be easily obtained just after birth. A methodology was developed to perform cultures of human umbilical artery smooth muscle cells (HUASMC) expressing contractile proteins and functional ionic channels. To avoid fibroblast and endothelial cell contamination, we mechanically separated the tunica media, which only contains HUASMC and matrix proteins. To isolate the cells, collagenase V and elastase were used as hydrolyzing enzymes. The isolated cells were plated in collagen-coated dishes to obtain cultures of HUASMC. The cells obtained after different passages (1 to 6) exhibit the characteristic vascular smooth cell morphology and express smooth muscle alpha-2 actin, myosin heavy chain SM1, and alpha subunits of L- and T-type calcium channels (Cav 1.2, Cav 1.2, and Cav 3.2). Electrophysiology recordings for L- and T-type calcium channels were made, indicating that these channels are functional in the cultured cells. In conclusion, the procedure developed allows obtaining cultures of HUASMC expressing contractile proteins and also functional ionic channels. These cells could be used to study cellular and molecular aspects about the regulation of the vascular function.     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

Functional roles of Cav1.3, Cav3.1 and HCN channels in automaticity of mouse atrioventricular cells

The atrioventricular node controls cardiac impulse conduction and generates pacemaker activity in case of failure of the sino-atrial node. Understanding the mechanisms of atrioventricular automaticity is important for managing human pathologies of heart rate and conduction. However, the physiology of atrioventricular automaticity is still poorly understood. We have investigated the role of three key ion channel-mediated pacemaker mechanisms namely, Ca_v1.3, Ca_v3.1 and HCN channels in automaticity of atrioventricular node cells (AVNCs). We studied atrioventricular conduction and pacemaking of AVNCs in wild-type mice and mice lacking Ca_v3.1 (Ca_v3.1^-/-), Ca_v1.3 (Ca_v1.3^-/-), channels or both (Ca_v1.3^-/-/Ca_v3.1^-/-). The role of HCN channels in the modulation of atrioventricular cells pacemaking was studied by conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity. Inactivation of Ca_v3.1 channels impaired AVNCs pacemaker activity by favoring sporadic block of automaticity leading to cellular arrhythmia. Furthermore, Ca_v3.1 channels were critical for AVNCs to reach high pacemaking rates under isoproterenol. Unexpectedly, Ca_v1.3 channels were required for spontaneous automaticity, because Ca_v1.3^-/- and Ca_v1.3^-/-/Ca_v3.1^-/- AVNCs were completely silent under physiological conditions. Abolition of the cAMP sensitivity of HCN channels reduced automaticity under basal conditions, but maximal rates of AVNCs could be restored to that of control mice by isoproterenol. In conclusion, while Ca_v1.3 channels are required for automaticity, Ca_v3.1 channels are important for maximal pacing rates of mouse AVNCs. HCN channels are important for basal AVNCs automaticity but do not appear to be determinant for β-adrenergic regulation.     

大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理检测

目的：研究大鼠肺动脉平滑肌细胞钙激活氯通道电流的电生理特性。方法：膜片钳全细胞和膜内向外记录模式检测大鼠肺动脉平滑肌细胞上钙激活氯通道全细胞电流和单通道电流。结果：大鼠肺动脉平滑肌细胞记录到稳定的钙激活氯通道电流（ICl（Ca））;ICl（Ca）表现出典型的外向整流特性和电压时间依赖性激活。结论：大鼠肺动脉平滑肌细胞膜上存在电压、时间依赖性氯通道电流,钙激活氯通道通过促进肺动脉平滑肌细胞去极化而成为调节肺动脉特性的关键调节因子。     

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 microM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 microM) or Ro-31-8425 (0.1 microM), inhibitors of both conventional and novel PKC isozymes, significantly activated I(Cl.vol) and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 microM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with epsilon V1-2 (10 microM), a translocation inhibitory peptide derived from the V1 region of epsilon PKC, activated I(Cl.vol) under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with beta C2-2 (10 microM), a translocation inhibitory peptide derived from the C2 region of beta PKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of epsilon PKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound epsilon PKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.     

Regulation of T-type calcium channels in the peripheral pain pathway

Recent evidence strongly suggests that both central and peripheral T-type Ca(2+) channels enhance somatic and visceral nociceptive inputs, as well as that regulation of T-type Ca(2+) channel function can result in significant changes of pain threshold in a variety of animal models. Therefore, T-type Ca(2+) channels in peripheral and central pain pathways, although previously unrecognized, may have great importance as targets for developing new therapies against pain. This is particularly critical in cases in which currently available treatments are limited due to serious side effects or are not consistently effective (e.g., chronic neuropathic pain). In this review, we summarize recent studies of the regulation of T-type channels in peripheral sensory neurons by means of redox agents and neuroactive steroids, as well as studies of the function of these channels in the pathophysiology of neuropathic pain.     

Perinatal hypoxia triggers alterations in K+ channels of adult pulmonary artery smooth muscle cells

Adverse events during the perinatal period, like hypoxia, have been associated with adult diseases. In pulmonary vessels, K(+) channels play an important role in the regulation of vascular tone. In the fetus, Ca(2+)-activated K(+) channels (K(Ca)) are predominant, whereas from birth voltage-gated K(+) channels (K(V)) prevail in the adult. We postulated that perinatal hypoxia could alter this maturational shift and influence regulation of pulmonary vascular tone in relation to K(+) channels in adulthood. We evaluated the effects of perinatal hypoxia on K(V) and K(Ca) channels in the adult main pulmonary artery (PA) using a murine model. Electrophysiological measurements showed a greater outward current in PA smooth muscle cells of mice born in hypoxia than in controls. In controls, only K(V) channels contributed to this current, whereas in mice born in hypoxia both K(V) and K(Ca) channels were implicated. K(V) channel activity was even higher in mice born in hypoxia than in controls. Therefore, perinatal hypoxia results in increased K(Ca) and K(V) channel activity in adult PA. Moreover, PA of adults born in hypoxia displayed higher large-conductance K(Ca) alpha-subunit and K(V)1.5 alpha-subunit protein expression than controls. Interestingly, relaxation induced by nitric oxide (NO) donors [S-nitroso-N-acetyl-D,l-penicillamine, 2-(N,N-diethylamino)-diazenolate-2-oxide] in isolated PA of control mice was not mediated by K(Ca) channels and only slightly by K(V) channels, whereas following perinatal hypoxia both K(Ca) and K(V) channels contributed to this relaxation. Thus perinatal hypoxia results in altered expression and activity of different K(+) channels in the adult main PA, which could contribute to modifications of pulmonary vasoreactivity.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

External cadmium and internal calcium block of single calcium channels in smooth muscle cells from rabbit mesenteric artery.

The patch clamp technique was used to record unitary currents through single calcium channels from smooth muscle cells of rabbit mesenteric arteries. The effects of external cadmium and cobalt and internal calcium, barium, cadmium, and magnesium on single channel currents were investigated with 80 mM barium as the charge carrier and Bay K 8644 to prolong openings. External cadmium shortened the mean open time of single Ca channels. Cadmium blocking and unblocking rate constants of 16.5 mM-1 ms-1 and 0.6 ms-1, respectively, were determined, corresponding to dissociation constant Kd of 36 microM at -20 mV. These results are very similar to those reported for cardiac muscle Ca channels (Lansman, J. B., P. Hess, and R. W. Tsien. 1986. J. Gen. Physiol. 88:321-347). In contrast, Cd2+ (01-10 mM), when applied to the internal surface of Ca channels in inside-out patches, did not affect the mean open time, mean unitary current, or the variance of the open channel current. Internal calcium induced a flickery block, with a Kd of 5.8 mM. Mean blocking and unblocking rate constants for calcium of 0.56 mM-1 ms-1 and 3.22 ms-1, respectively, were determined. Internal barium (8 mM) reduced the mean unitary current by 36%. We conclude that under our experimental conditions, the Ca channel is not symmetrical with respect to inorganic ion block and that intracellular calcium can modulate Ca channel currents via a low-affinity binding site.     

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca(2+) current (I(Ca)) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca(2+), depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I(Ca) which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V(test) of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 microM), an L-type Ca(2+) channels antagonist, completely inhibited I(Ca), while the L-type Ca(2+) channels agonist Bay-K 8644 (1 microM) significantly increased I(Ca) amplitude. Moreover, the selective blocker of P-/Q-type Ca(2+) channels omega-agatoxin IVA partially blocked I(Ca) (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca(2+) channels, both nifedipine-sensitive, underlie the I(Ca) registered using low extracellular Ca(2+). The presence of the P-/Q-type Ca(2+) channels was confirmed by immunoblot analysis. When I(Ca) was recorded in a high concentration (30 mM) of extracellular Ca(2+) or Ba(2+) as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca(2+) channels blocker mibefradil (10 microM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca(2+) currents in human umbilical artery smooth muscle cells.