Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

Tedisamil blocks single large-conductance Ca2+-activated K+ channels in membrane patches from smooth muscle cells of the guinea-pig portal vein

Enzymatically dispersed smooth muscle cells of the guinea-pig portal vein were studied by the patch-clamp technique. They were found to have Ca²⁺-dependent K⁺ channels with the typical properties of the BK channel, i.e. a reversal potential at the calculated equilibrium potential for K⁺ ions, a striking voltage dependence, and a conductance of approximately 200 pS ([K⁺]₀ 50 mM, [K⁺]_i 150 mM, positive patch potentials). Tedisamil, a new bradycardic agent with an inhibitory action on K⁺ currents in heart muscle, reduced the open probability of the BK channels concentration-dependently (1–100 M) when applied at the cytosolic side of membrane inside-out patches. At 100 M [Ca²⁺]_i, the IC₅₀ of tedisamil was 13.8 M (¯x, n=5). Tedisamil increased the frequency of channel closures, and reduced the mean duration of openings from 8 ms to < 1 ms, while the mean duration of closures within bursts (1–2 ms) was not altered. Tedisamil did not affect long closures (> 160 ms) between bursts, either. The mean time of residence of tedisamil at the BK channel was estimated to be 1–2ms. Hence, tedisamil, in comparison to the slow blocker Ba²⁺ and the fast blocker tetraethylammonium, holds the position of an intermediate K⁺ channel blocker.Supported by the Deutsche Forschungsgemeinschaft     

Voltage-dependent K+ channels in the sarcolemma of mouse skeletal muscle

The voltage-dependent K⁺ channels of the mammalian sarcolemma were studied with the patch-clamp technique in intact, enzymatically dissociated fibres from the toe muscle of the mouse. With a physiological solution (containing 2.5 mM K⁺) in the pipette, depolarizing pulses imposed on a cell-attached membrane patch activated K⁺ channels with a conductance of about 17 pS. No channel activity was observed when the pipette solution contained 2mM tetraethylammonium (TEA), or 2 mM 4-aminopyridine (4-AP). Whole cell recordings from these very small muscle fibres showed the well-known delayed rectifier K⁺ outward current with a threshold of about -40mV. The whole-cell current was completely blocked by 2 mM TEA in the bath, suggesting that the TEA-sensitive channels in the patch were also delayed rectifier channels. The inactivation properties of the channels were studied in the cell-attached mode. Averaged single-channel traces showed at least two types of channels discernible by their inactivation time course at a test potential of 60 mV. The fast type inactivated with a time constant of about 150ms, the slow type with a time constant of about 400 ms. A little channel activity always remained during pulses lasting several minutes, indicating either the presence of a very slowly inactivating third type of K⁺ channel, or the tendency of the fast inactivating channels to re-open at constant voltage. No difference was seen in the single-channel amplitudes of the different types of K⁺ channels. The well characterized adenosine-5-triphosphate-(ATP)-sensitive and Ca²⁺-dependent K⁺ channels, although present, were not active under the conditions used. The results suggest that in mouse skeletal muscle the delayed rectifier channels to not only carry the outward current during excitation but are also responsible for the resting K⁺ conductance.     

Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat

A novel class of Ca²⁺-activated K⁺ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these K_Ca,ATP channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of K_Ca,ATP channels to voltage, intracellular Ca²⁺ ([Ca²⁺]_i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K⁺ and are approximately 20 times more selective for K⁺ ions than Na⁺ ions, with a K⁺ permeability (P _K) of 4.6×10^–13cm s^–1. Ca²⁺ ions applied to the intracellular membrane surface of K_Ca,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca²⁺ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of K_Ca,ATP channels by Mg-ATP has an EC₅₀ of approximately 50 M. Although the EC₅₀ is unaffected by [Ca²⁺]_i, channel activation by Mg-ATP is enhanced by increasing [Ca²⁺]_i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of K_Ca,ATP channels to Ca²⁺. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of K_Ca,ATP channels to Ca²⁺ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca²⁺ through voltage-activated Ca²⁺ channels, ultimately leading to increased vascular tone.     

H2O2对大鼠肺动脉平滑肌细胞Kv通道的作用

 目的:观察H₂O₂在常氧时对大鼠肺动脉平滑肌细胞（PASMCs）的Kv的影响, 探讨H₂O₂对肺动脉平滑肌细胞的Kv通道的作用。 方法:用酶解法急性分离单个PASMCs，以全细胞膜片钳技术记录PASMCs膜上的电压依赖性钾通道 (voltage-gated potassium channel, Kv) 电流。 结果:常氧下 H₂O₂可显著增加Kv电流，电流-电压关系曲线左上移；而且Kv电流呈浓度依赖性增加。 结论:常氧下H₂O₂ 可使Kv通道开放。     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly.The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Effects of extracellular K+ and Ca2+ on membrane potential,contraction and86Rb+ efflux in guinea-pig mesotubarium

The effects of varying extracellular concentrations of K⁺ and Ca²⁺ [K⁺]_o and [Ca²⁺]_o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K⁺]_o up to 40 mM, spontaneous action potentials were present, while higher [K⁺]_o gave sustained contractures at a stable membrane potential (−24 to −12 mV for [K⁺]_o from 60 to 120 mM). Tension decreased successively with increasing [K⁺]_o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca²⁺ antagonist, felodipine, increased as the membrane was depolarized with increasing [K⁺]_o and action potentials ceased. These results are compatible with the existence of Ca²⁺ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca²⁺]_o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions.⁸⁶Rb⁺ efflux measurements in 60 mM K⁺ in the absence and presence of felodipine revealed a Ca²⁺-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K⁺), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K⁺ channels by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.     

Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells

Using the patch clamp technique we show that exposure of opossum kidney cells to hypotonic shock evokes an outward rectifying potassium current. The corresponding single channel slope conductance approaches 15 pS at depolarizing voltages. The K current also becomes activated after addition of the ionophore A23187 to an isotonic bath medium containing Ca²⁺. We therefore conclude that the K selective channels are modulated by an elevation of cytoplasmic Ca²⁺. Evidence is presented that release of Ca²⁺ from internal stores is involved.     

Hyperpolarization-activated cationic channels in smooth muscle cells are stretch sensitive

The properties of hyperpolarization-activated channels were studied in single smooth muscle cells from the stomach of the toad, Bufo marinus, using the patchclamp technique. In cell-attached patches, inward channel currents were activated by hyperpolarizing pulses from a holding potential of –20 mV to potentials more negative than –60 mV. The activity of the channels increased and their latency of activation decreased as the hyperpolarization was increased. The slope conductance of the channels with standard high sodium concentration pipette solution was 64.2±9.1 pS (SD, n=17). Stretching the patch, by suction applied to the back of the patch pipette, also increased the activity and shortened the latency of activation. We designate these channels as HA-SACs (hyperpolarization- and stretch-activated channels). HA-SACs were observed in 83% (175/210) of the patches studied. HA-SAC currents were carried by sodium and potassium ions, but their amplitude was increased by replacing extracellular sodium with potassium. Extracellular magnesium and calcium ions significantly reduced the single-channel conductance of HA-SACs. These permeation characteristics and the single-channel conductance of HA-SACs were indistinguishable from those of stretch-activated channels (SACs) previously described in these cells. The following observations are consistent with HA-SACs being a subset of SACs. First, SACs were at times found in cell-attached patches which lacked HA-SACs. Second, the number of channels in a cell-attached patch simultaneously activated by stretch (usually 5–10 and often more) exceeded by far the number simultaneously activated by hyperpolarization (usually one or two). Third, in excised insideout patches, the ability of hyperpolarization to activat HA-SACs was lost within 3–5 min, whereas stretch continued to activate a large number of channels. The last observation also suggests that activation by hyperpolarization requires an intracellular agent, whereas stretch activation does not.     

cAMP-mediated long-term modulation of voltage-dependent K+ channels in cultured colliculi neurons

 Previously, we have described prolonged cAMP-induced inhibition of a K⁺ current in cultured colliculi neurons. The aim of the present study was to characterize the channel responsible for this cAMP-dependent effect. We detected the presence of a non-inactivating voltage-dependent 16-pS K⁺ channel that displayed long-lasting inhibition upon a brief application of cAMP and greater sensitivity to tetraethylammonium than to 4-aminopyridine. In addition to this channel, colliculi neurons express two other voltage-sensitive, non-inactivating K⁺ channels (8 and 49 pS) whose activity is facilitated by a brief application of cAMP, the effect of which is also long-lasting. These results suggest the presence of common sustained cAMP-dependent processes responsible for both up- and down-regulation of these channels in the neurons studied. They indicate that the 16-pS, but not the 8-pS or the 49-pS channels, participates in the cAMP-inhibited macroscopic K⁺ current. Received: 21 April 1998 / Received after revision: 10 July 1998 / Accepted: 5 August 1998     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

Variations of membrane cholesterol alter the kinetics of Ca2+-dependent K+ channels and membrane fluidity in vascular smooth muscle cells

The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca²⁺-dependent K⁺ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (P _o). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in P _o. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca²⁺-dependent K⁺ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca²⁺-dependent K⁺ channels kinetics in different preparations.     

Single-channel and Fura-2 analysis of internal Ca2+ oscillations in HeLa cells: contribution of the receptor-evoked Ca2+ influx and effect of internal pH

Patch-clamp and Fura-2 experiments were performed in order to investigate the calcium oscillations due to H₁ receptor stimulation in HeLa cells. The cytosolic calcium fluctuations occurring directly at the plasma membrane inner face were detected by measuring the activity of calcium-dependent potassium channels. This method also allowed measurement of changes in intracellular potential using as indicator the amplitude of the channel current jump. The average internal calcium concentration was obtained from Fura-2 experiments carried out at either the single-cell level or from a small population of cells in monolayer. The results indicate that the internal calcium oscillations in HeLa cells arise from a biphasic process with an initial phase independent of the presence of external calcium. External calcium was found, however, to become essential once the regular oscillatory process has been established. Removing external calcium after this initial phase produced a rapid decay in the burst frequency and eventually a complete abolition of the oscillations. In addition, the calcium oscillations occurring during the external-calcium-dependent phase could be blocked by calcium entry blockers such as Co²⁺ or La³⁺, or abolished by perfusing the external medium with a high-K⁺ solution. Experiments were also performed in which the cell internal pH (pH_i) was changed by removing the external bicarbonate or by adding NH₄Cl to the bathing solution. The results obtained under these conditions indicate that an increase in internal pH abolishes selectively the appearance of calcium spikes without increasing the basal calcium level, while a cellular acidification maintains or stimulates the calcium oscillatory process. It was also observed that the inhibitory effect of alkaline pH was independent of external calcium, and that calcium oscillations could always be seen at alkaline pH during the initial phase of histamine stimulation. On the basis of these results, it is proposed that the internal calcium oscillations in HeLa cells depend on the release of calcium from internal pools, which are reloaded via a pH-dependent mechanism. Part of the calcium sequestration occurring during the oscillatory process would be carried out, however, by pH-insensitive calcium compartments.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

The effect of high K+ depolarization and verapamil on45Ca2+ fluxes in the canine myocardium

The effects of high K⁺ depolarization and verapamil on Ca²⁺ uptake and the total intracellular Ca²⁺ content of canine ventricular muscle strips (0.5 mm thick) were investigated. High K⁺ (96 mM) increased Ca²⁺ uptake above control and maintained this enhanced uptake throughout a 90 second measuring period. Verapamil (5×10^–6M) significantly (p<0.05) inhibited this high K⁺ stimulated uptake. However, verapamil (5×10^–6M) also had a direct effect on Ca²⁺ fluxes, causing a significant increase in both Ca²⁺ uptake (p<0.001) and total intracellular Ca²⁺ content (p<0.001) in the resting tissue. Therefore, verapamil's apparent inhibition of high K⁺ stimulated Ca²⁺ uptake may have resulted from some mechanism other than Ca²⁺ channel blockade.     

Differential distribution of two Ca2+-dependent and -independent K+ channels throughout receptive and basolateral membranes of bullfrog taste cells

We could identify two types of K⁺ channels, of 80 and 40 pS conductance, respectively, in the bullfrog taste cell membrane using excised and cell-attached configurations of the patch-clamp technique. The taste cell membrane could be divided into four membrane parts — receptive area, apical process, cell body and proximal process. The 80-pS K⁺ channels were dependent on voltage and Ca²⁺ and were located exclusively on the receptive membrane and the apical process membrane. The 40-pS K⁺ channels were independent of voltage and Ca²⁺. The open probability of 40-pS K⁺ channels was decreased by the simultaneous presence of cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), and the suppressive effect was antagonized by protein kinase inhibitor (PKI). Although 40-pS K⁺ channels were found in a high density on the receptive and apical process membranes, the channels also were present in the other two parts of the taste cell membrane. These results suggest that the two different types of K⁺ channel in the bullfrog taste cells may play different roles in gustatory transduction.     

Multiple types of voltage-dependent Ca channels in mammalian intestinal smooth muscle cells

(1) Whole-cell and single channel recording techniques have been applied to smooth muscle cells isolated from guinea-pig taenia coli to examine whether multiple types of Ca channels exist. (2) Whole-cell recordings under physiological Ca concentration (1.8 mM) revealed two current components with fast and slow inactivating kinetics. The fast inactivating component was present when cells were held at very negative potentials (–80 mV). It was insensitive to the dihydropyridine (DHP) derivative, nifedipine. In contrast, the slow inactivating component was present at less negative holding potentials. It was blocked by nifedipine. (3) The two current components were found to have closely similar voltage dependencies for activation. (4) These results suggest that the fast inactivating decay of the Ca current was mediated not only by the entry of Ca into the cell but also by a voltage-dependent process via a different type of Ca channel with fast inactivating kinetics. (5) Recordings from cell-attached membrane patches with 100 mM external Ba clearly showed the existence of multiple types of Ca channels with different conductances. (6) The large conductance channels (30 pS) activated at more positive potentials (0 mV) and their averaged current decayed much more slowly. The DHP Ca antagonist, nifedipine, inhibited the large conductance channels increasing the proportion of blank sweeps and reducing the averaged current. On the other had, the DHP Ca-agonist, BayK 8644, increased the averaged current by increasing the mean open-times of the large conductance channels. The presence of micromolar Cd in the patch pipettes produced a flickering block of the large conductance channels. (7) The small conductance channels (7 pS) activated at more negative potentials (–40 mV 30 mV) and the averaged current decayed rapidly within 100 ms. They showed no sensitivity to nifedipine and Cd ions. (8) In summary, we have identified at least two distinct types of Ca channels with different conductances, different pharmacological sensitivities, and different voltage- and time-dependent kinetics in smooth muscle cells isolated from guinea-pig taenia coli. The large and small conductance channels could be classified asl- andt-type Ca channels, respectively, which have been described in neuronal and heart cells. The contribution of those channels to the macroscopic component of whole-cell Ca current is also discussed.     

Role of external Ca2+ and K+ in gating of cardiac delayed rectifier K+ currents

We sought to determine whether extracellular Ca²⁺ (Ca _e ²⁺ ) and K⁺ (K _e ⁺ ) play essential roles in the normal functioning of cardiac K⁺ channels. Reports by others have shown that removal of Ca _e ²⁺ and K _e ⁺ alters the gating properties of neural delayed rectifier (I _K) and A-type K⁺ currents, resulting in a loss of normal cation selectivity and voltage-dependent gating. We found that removal of Ca _e ²⁺ and K _e ⁺ from the solution bathing guinea pig ventricular myocytes often induced a leak conductance, but did not affect the ionic selectivity or time-dependent activation and deactivation properties of I _K. The effect of [K⁺]_e on the magnitude of the two components of cardiac I _K was also examined. I _K in guinea pig myocytes is comprised of two distinct types of currents: I _Kr (rapidly activating, rectifying) and I _Ks (slowly activating). The differential effect of Ca _e ²⁺ on the two components of I _K (previously shown to shift the voltage dependence of activation of the two currents in opposite directions) was exploited to determine the role of K _e ⁺ on the magnitude of I _Ks and I _Kr. Lowering [K⁺]_e from 4 to 0 mM increased I _Ks, as expected from the change in driving force for K⁺, but decreased I _Kr. The differential effect of [K⁺]_e on the two components of cardiac I _K may explain the reported discrepancies regarding modulation of cardiac I _K conductance by this cation.