Dihydropyridine-sensitive calcium channels expressed in canine colonic smooth muscle cells

1993 ◽  
Vol 264 (3) ◽  
pp. C745-C754 ◽  
Author(s):  
A. Rich ◽  
J. L. Kenyon ◽  
J. R. Hume ◽  
K. Overturf ◽  
B. Horowitz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Channels ◽  
Smooth Muscle Cells ◽  
Calcium Current ◽  
Single Channel ◽  
Muscle Cells ◽  
Open Probability ◽  
Boltzmann Function ◽  
Single Channel Currents ◽  
Channel Currents

Experiments were performed to identify and characterize the types of calcium channels that regulate inward calcium current in canine colonic smooth muscle. Freshly dispersed smooth muscle cells from the circular layer of the canine proximal colon were used. Single-channel currents were measured with 80 mM Ba2+ as the charge carrier. Small-conductance (10 +/- 2 pS, EBa = 46 +/- 11 mV, n = 9) and large-conductance (21 +/- 1 pS, EBa = 52 +/- 3 mV, n = 19) single-channel currents were observed during depolarizing voltage steps positive to -30 mV. Both types of single-channel currents were inhibited by the addition of 10(-6) M nifedipine to the bath solution. The smaller current was infrequently observed and therefore was not further characterized. Open probability (P(o)) of the larger current amplitude was strongly dependent on voltage. Activation curves were well described by a Boltzmann function with half activation occurring at 4 mV, and a 5-mV increase in membrane potential resulted in an e-fold increase in P(o). BAY K 8644 (1 microM) shifted the activation curve to the left while nifedipine (1 microM) resulted in a right shift. Molecular analysis showed that only the C class of Ca2+ channel alpha 1-subunit is expressed in this tissue. Furthermore, only a single splice variant (rbc-II) was observed. The results suggest that a single class of dihydropyridine-sensitive calcium channels regulates inward calcium current in canine colonic smooth muscle cells.

scholarly journals BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

2016 ◽  
Vol 113 (16) ◽  
pp. E2335-E2344 ◽  
Author(s):  
Monali Wakle-Prabagaran ◽  
Ramón A. Lorca ◽  
Xiaofeng Ma ◽  
Susan J. Stamnes ◽  
Chinwendu Amazu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Calcium Oscillations ◽  
Proximity Ligation Assay ◽  
Human Myometrium ◽  
Bkca Channel ◽  
Open Probability ◽  
Oscillatory Pattern

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCa and both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCa in an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy.

Interaction of Selective Amino Acid Residues of KCa Channels with Carbon Monoxide

2003 ◽  
Vol 228 (5) ◽  
pp. 474-480 ◽  
Author(s):  
Rui Wang ◽  
Lingyun Wu
Keyword(s):  
Smooth Muscle ◽  
Amino Acid ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Muscle Cells ◽  
Amino Acid Residues ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Channel Proteins

The activation of big-conductance KCa channels in vascular smooth muscle cells by carbon monoxide (CO) has been demonstrated previously. One specific target of CO on KCa channel proteins is the histidine residue. The roles of other amino acid residues on the functionality of KCa channels, as well as their reactions to CO, have been unclear. In the present study, the cell-free single channel recording technique was used to investigate the chemical modification of KCa channels by CO and other chemical agents. The modification of negatively charged carboxyl groups and the ε-amino group of lysine did not affect the open probability, but decreased single-channel conductance of KCa channels. When sulfhydryl groups of cysteine were modified with N-ethylmaleimide, the open probability of KCa channels was decreased, but single-channel conductance was not affected. None of the above chemical modifications affected the CO-induced increase in the open probability of KCa channels. However, N-ethylmaleimide treatment reduced the stimulatory effect of nitric oxide (NO) on KCa channels. Finally, pretreatment of smooth muscle cells with NO abolished the effects of subsequently applied CO on KCa channel proteins. Our study demonstrates that CO and NO acted on different amino acid residues of KCa channel proteins. The interaction of CO and NO determines the functional status of KCa channels in vascular smooth muscle cells

scholarly journals Ca2+ currents in cerebral artery smooth muscle cells of rat at physiological Ca2+ concentrations.

1996 ◽  
Vol 107 (4) ◽  
pp. 459-472 ◽  
Author(s):  
M Rubart ◽  
J B Patlak ◽  
M T Nelson
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Single Channel ◽  
Cerebral Arteries ◽  
Muscle Cells ◽  
Open Probability ◽  
Average Cell ◽  
Whole Cell ◽  
Channel Current ◽  
Fold Reduction

Single Ca2+ channel and whole cell currents were measured in smooth muscle cells dissociated from resistance-sized (100-microns diameter) rat cerebral arteries. We sought to quantify the magnitude of Ca2+ channel currents and activity under the putative physiological conditions of these cells: 2 mM [Ca2+]o, steady depolarizations to potentials between -50 and -20 mV, and (where possible) without extrinsic channel agonists. Single Ca2+ channel conductance was measured over a broad range of Ca2+ concentrations (0.5-80 mM). The saturating conductance ranged from 1.5 pS at 0.5 mM to 7.8 pS at 80 mM, with a value of 3.5 pS at 2 mM Ca (unitary currents of 0.18 pA at -40 mV). Both single channel and whole cell Ca2+ currents were measured during pulses and at steady holding potentials. Ca2+ channel open probability and the lower limit for the total number of channels per cell were estimated by dividing the whole-cell Ca2+ currents by the single channel current. We estimate that an average cell has at least 5,000 functional channels with open probabilities of 3.4 x 10(-4) and 2 x 10(-3) at -40 and -20 mV, respectively. An average of 1-10 (-40 mV and -20 mV, respectively) Ca2+ channels are thus open at physiological potentials, carrying approximately 0.5 pA steady Ca2+ current at -30 mV. We also observed a very slow reduction in open probability during steady test potentials when compared with peak pulse responses. This 4-10-fold reduction in activity could not be accounted for by the channel's normal inactivation at our recording potentials between -50 and -20 mV, implying that an additional slow inactivation process may be important in regulating Ca2+ channel activity during steady depolarization.

Contractile agonists activate voltage-dependent calcium channels in airway smooth muscle cells

1992 ◽  
Vol 263 (1) ◽  
pp. C106-C113 ◽  
Author(s):  
M. Tomasic ◽  
J. P. Boyle ◽  
J. F. Worley ◽  
M. I. Kotlikoff
Keyword(s):  
Smooth Muscle ◽  
Calcium Channels ◽  
Smooth Muscle Cells ◽  
Calcium Channel ◽  
Airway Smooth Muscle ◽  
Muscle Cells ◽  
Cell Types ◽  
State Probability ◽  
Airway Smooth Muscle Cells ◽  
Channel Currents

To determine whether agents that cause contraction of airway smooth muscle affect sarcolemmal calcium channel activity, unitary calcium channel currents (using Ba2+ as the charge carrier) were recorded in on-cell configuration from acutely dissociated (dog, pig, and ferret) and cultured (human) airway smooth muscle cells. Addition of the contractile agonists methacholine or bradykinin increased the open-state probability of the large-conductance calcium channel 37.2- and 45-fold, respectively. The increase in open-state probability was not due to cellular depolarization because increases occurred in the absence of depolarization. Channel activation by the agonist was determined to result in the favoring of a long (16.5 +/- 5.0 ms) open lifetime for the channel, which was not observed under control conditions, in the absence of BAY K 8644. We also report the unitary calcium channel currents from a second, smaller conductance calcium channel. This channel was present in all cell types and had a mean conductance of 9.5 +/- 0.8 pS (80 mM Ba2+). Exposure of cells to agonist also resulted in an increase in the open-channel probability of the small-conductance calcium channel (10.4-fold), which did not result from cellular depolarization. These experiments demonstrate that the molecular pathways exist between contractile agonist receptors and sarcolemmal calcium channels in airway smooth muscle cells. Because membrane patches were not directly exposed to agonist, receptor-channel linkage probably occurs via a second messenger-coupling pathway.

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