Membrane stretch and cytoplasmic Ca2+ independently modulate stretch-activated BK channel activity

Large conductance Ca²⁺-activated K⁺ (BK) channels are responsible for changes in chemical and physical signals such as Ca²⁺, Mg²⁺ and membrane potentials. Previously, we reported that a BK channel cloned from chick heart (SAKCaC) is activated by membrane stretch. Molecular cloning and subsequent functional characterization of SAKCaC have shown that both the membrane stretch and intracellular Ca²⁺ signal allosterically regulate the channel activity via the linker of the gating ring complex. Here we investigate how these two gating principles interact with each other. We found that stretch force activated SAKCaC in the absence of cytoplasmic Ca²⁺. Lack of Ca²⁺ bowl (a calcium binding motif) in SAKCaC diminished the Ca²⁺-dependent activation, but the mechanosensitivity of channel was intact. We also found that the abrogation of STREX (a proposed mechanosensing apparatus) in SAKCaC abolished the mechanosensitivity without altering the Ca²⁺ sensitivity of channels. These observations indicate that membrane stretch and intracellular Ca²⁺ could independently modulate SAKCaC activity.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Emerging Role of the Calcium-Activated,Small Conductance,SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4

The Ca²⁺-activated, maxi-K (BK) K⁺ channel, with low Ca²⁺-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K⁺ secretion. In the present study we demonstrate that the Ca²⁺-activated, SK3 (K_Ca2.3) K⁺ channel, with high Ca²⁺-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K⁺ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca²⁺-dependent K⁺ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca²⁺-permeable TRPV4 channel, thereby inducing Ca²⁺ influx and elevating intracellular Ca²⁺ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca²⁺ influx, leading to elevated intracellular Ca²⁺ levels, activates this high Ca²⁺-affinity K⁺ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca²⁺-dependent regulation of membrane potential and K⁺ secretion.     

Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl⁻ and K⁺ channels. Besides regulating cytosolic Cl⁻ activity, osmolyte flux and, thus, cell volume, most Cl⁻ channels allow HCO₃⁻ exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K⁺ exit through K⁺ channels may decrease intracellular K⁺ concentration, which in turn favors apoptotic cell death. K⁺ channel activity further maintains the cell membrane potential, a critical determinant of Ca²⁺ entry through Ca²⁺ channels. Cytosolic Ca²⁺ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca²⁺ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Spontaneous inactivation of the Ca-sensitive K channels of human red cells at high intracellular Ca activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a (dCa²⁺/dt)-sensitive process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about −65 mV was observed. At a membrane potential of about −70 mV and an intracellular concentration of about 2·10⁻⁴M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Single nonselective cation channels and Ca2+-activated K+ channels in aortic endothelial cells

Summary In cultured bovine aortic endothelial cells, elementary K⁺ currents were studied in cell-attached and inside-out patches using the standard patch-clamp technique. Two different cationic channels were found, a large channel with a mean unitary conductance of 150±10 pS and a small channel with a mean unitary conductance of 12.5±1.1 pS. The 150-pS channel proved to be voltag- and Ca²⁺-activatable and seems to be a K⁺ channel. Its open probability increased on membrane depolarization and, at a given membrane potential, was greatly enhanced by elevating the Ca²⁺ concentration at the cytoplasmic side of the membrane from 10^–7 to 10^–4 m. 150-pS channels were not influenced by the patch configuration in that patch excision neither induced rundown nor evoked channel activity in silent cell-attached patches. However, they were only seen in two out of 55 patches. The 12-pS channel was predominant, a nonselective cationic channel with almost the same permeability for K⁺ and Na⁺ whose open probability was minimal near –60 mV but increased on membrane hyperpolarization. An increase in internal Ca²⁺ from 10^–7 to 10^–4 m left the open probability unchanged. Although the K⁺ selectivity of the 150-pS channels remains to be elucidated, it is concluded that they may be involved in controlling Ca²⁺-dependent cellular functions. Under physiological conditions, 12-pS nonselective channels may provide an inward cationic pathway for Na⁺.     

Fluid shear stress induces calcium transients in osteoblasts through depolarization of osteoblastic membrane

Intracellular calcium transient ([Ca²⁺]_i transient) induced by fluid shear stress (FSS) plays an important role in osteoblastic mechanotransduction. Changes of membrane potential usually affect [Ca²⁺]_i level. Here, we sought to determine whether there was a relationship between membrane potential and FSS-induced [Ca²⁺]_i transient in osteoblasts. Fluorescent dyes DiBAC₄(3) and fura-2 AM were respectively used to detect membrane potential and [Ca²⁺]_i. Our results showed that FSS firstly induced depolarization of membrane potential and then a transient rising of [Ca²⁺]_i in osteoblasts. There was a same threshold for FSS to induce depolarization of membrane potential and [Ca²⁺]_i transients. Replacing extracellular Na⁺ with tetraethylammonium or blocking stretch-activated channels (SACs) with gadolinium both effectively inhibited FSS-induced membrane depolarization and [Ca²⁺]_i transients. However, voltage-activated K⁺ channel inhibitor, 4-Aminopyridine, did not affect these responses. Removing extracellular Ca²⁺ or blocking of L-type voltage-sensitive Ca²⁺ channels (L-VSCCs) with nifedipine inhibited FSS-induced [Ca²⁺]_i transients in osteoblasts too. Quantifying membrane potential with patch clamp showed that the resting potential of osteoblasts was −43.3 mV and the depolarization induced by FSS was about 44 mV. Voltage clamp indicated that this depolarization was enough to activated L-VSCCs in osteoblasts. These results suggested a time line of Ca²⁺ mobilization wherein FSS activated SACs to promote Na⁺ entry to depolarize membrane that, in turn, activated L-VSCCs and Ca²⁺ influx though L-VSCCs switched on [Ca²⁺]_i response in osteoblasts.     

Oscillating activity of a calcium-activated K+ channel in normal and cancerous mammary cells in culture

Summary Calcium-activated potassium channels were the channels most frequently observed in primary cultured normal mammary cell and in the established mammary tumor cell, MMT060562. In both cells, single-channel and whole-cell clamp recordings sometimes showed slow oscillations of the Ca²⁺-gated K⁺ current. The characteristics of the Ca²⁺-activated K⁺ channels in normal and cancerous mammary cells were quite similar. The slope conductances changed from 8 to 70 pS depending on the mode of recording and the ionic composition in the patch electrode. The open probability of this channel increased between 0.1 to 1 m of the intracellular Ca²⁺, but it was independent of the membrane potential.Charybdotoxin reduced the activity of the Ca²⁺-activated K⁺ channel and the oscillation of the membrane current, but apamin had no apparent effect. The application of tetraethylammonium (TEA) from outside and BaCl₂ from inside of the cell diminished the activity of the channel. The properties of this channel were different from those of both the large conductance (BK or MAXI K) and small conductance (SK) type Ca²⁺-activated K⁺ channels.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Human macrophages contain a stretch-sensitive potassium channel that is activated by adherence and cytokines

A variety of stimuli, including cytokines and adhesion to surfaces and matrix proteins, can regulate macrophage function, in part through changes in Ca²⁺-dependent second messengers. While fluctuation in in-tracellular Ca²⁺ is an important modulator of cellular activation, little attention has been paid to the roles of other ions whose cytoplasmic concentrations can be rapidly regulated by ion channels. To examine the role of ion channels in macrophage function, we undertook patch clamp studies of human culture-derived macrophages grown under serum-free conditions. The major ionic current in these cells was carried by an outwardly rectifying K⁺ channel, which had a single-channel conductance of 229 pS in symmetrical K⁺-rich solution and macroscopic whole-cell conductance of 9.8 nS. These channels opened infrequently in resting cells but were activated immediately by (i) adhesion of mobile cells onto a substrate, (ii) stretch applied to isolated membrane patches in Ca²⁺-free buffers, (iii) intracellular Ca²⁺ (EC₅₀ of 0.4 m), and (iv) the cytokine IL-2. Furthermore, barium and 4-aminopyridine, blockers of this channel, altered the organization and structure of the cytoskeletal proteins actin, tubulin and vimentin. These cytoskeletal changes were associated with reversible alteration to the morphology of the cells. Thus, we have identified an outwardly rectifying K⁺ channel that appeared to be involved in cytokine and adherence-mediated macrophage activation, and in the maintenance of cytoskeletal integrity and cell shape.We thank Ken Wyse and Sue Bennett for excellent technical assistance. This work was supported by the National Health & Medical Research Council of Australia, the National Heart Foundation of Australia, the Clive & Vera Ramaciotti Foundation of Australia, the St Vincent's Hospital Clinic Foundation and a St Vincent's Hospital Research Grant.     

Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca²⁺-dependent K⁺ channel (maxi-K_Ca), a K⁺ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca²⁺ ions. The resulting Ca²⁺-flux may contribute to the photodynamically induced increase of the intracellular Ca²⁺ concentration observed in various cell lines. Received: 26 May 1998/Revised: 8 September 1998     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

Ca2+-activated K+ channels from cultured renal medullary thick ascending limb cells: Effects of pH

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb cells (MTAL) using the patch-clamp technique. The purpose was to determine the effect of acidic pH on channel properties in excised patches of apical cell membrane. At pH 7.4, increasing Ca²⁺ on the intracellular side or applying positive voltages increases channel open probability. Reducing pH to 5.8 on the intracellular face of the channel decreases channel open probability at each voltage and Ca²⁺ concentration. Channel mean open times display two distributions and mean closed times display three distributions. Increasing Ca²⁺ or applying depolarizing voltages lengthens each of the mean open times and shortens each of the closed times. Lowering pH to 5.8 decreases the mean open times and increases mean closed times at each Ca²⁺ and voltage with the greatest effect on the mean closed times. In contrast, both single-channel conductance and channel kinetics are unaffected when pH is reduced to 5.8 on the extracellular face of the membrane. We conclude that protons interfere with Ca²⁺ binding to the gate of Ca²⁺-activated K⁺ channels reducing the probability of channel opening.     

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca²⁺-dependent K⁺ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K⁺ channel with a mean conductance of 112 pS is reversibly blocked by ATP (K _i = 100 m), TEA (K _i =0.8 mm) and 10 mm Ba²⁺ and irreversibly blocked by 10 nm glibenclamide and 10 m tolbutamide. It is Ca²⁺ and voltage independent. Its open state probability (P _o) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca²⁺-dependent K⁺ channel with a mean conductance of 114 pS shows a bell-shaped Ca²⁺ dependence of P _o with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The P _o is voltage independent at the physiologically relevant V range. Ba²⁺ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba²⁺ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K⁺ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca²⁺-dependent K⁺ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca²⁺ concentration.This study was supported by the Deutsche Forschungsgemeinschaft (W.-R. Schlue) and by a fellowship of the Konrad-Adenauer-Stiftung (G. Frey). We thank Dr. Draeger (Hoechst AG) for the gift of glibenclamide. The data are part of a future Ph.D. thesis of G. Frey.     

Molecular Biology of Background K Channels: Insights from K2P Knockout Mice

K_2P channels are a family of cellular proteins that are essential for electrical signaling throughout the body. There are six K_2P channel subfamilies, consisting of 15 distinct mammalian genes. K_2P channels display a remarkable range of regulation by cellular, physical and pharmacologic agents, including protein kinases, intracellular Ca²⁺, changes in internal and external pH, anesthetic agents, heat, stretch and membrane deformers. The molecular and cellular mechanisms underlying this regulation are complex and cooperate at many different levels. Recent research has provided strong evidence that the spatiotemporal-specific expression of K_2P channels are determinants of physiologic selectivity and specificity. In recent years, knockout mice have been generated with inactivated K_2P channel genes. These animals shed new light on the contribution of K_2P channels to normal and abnormal physiology. In this review, we summarize the published data on these mice to broaden the understanding of the role of K_2P channel activity.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

Effect of the diazonium salt of sulfanilic acid on human erythrocyte ATPase activity

External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg²⁺-dependent ATPase but does markedly inhibit the Ca²⁺-stimulated ATPase activity. Inhibition of the (Na⁺ + K⁺)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na⁺ + K⁺)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg²⁺-dependent and Ca²⁺-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na⁺ + K⁺)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg²⁺-dependent ATPase from inhibition. The Ca²⁺-stimulated ATPase activity is not protected.