Physiological significance of SK4 channels in pancreatic β-cell oscillations

Pancreatic beta-cells show oscillations in membrane potential (V_m), the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) and finally insulin secretion. It is well accepted that the initiation of a burst phase with action potentials is mediated by voltage-dependent Ca²⁺ (and Na⁺) channels. The mechanism triggering the onset of interburst phases is less clear. The exact nature of the K⁺ channels that hyperpolarize V_m to maintain the rhythmic activity is unknown. In 1999 Göpel and co-workers¹ described a current termed K_slow and claimed that this current terminates the burst phases. KATP current is a part of the K_slow current. We could show that the Ca²⁺-dependent K⁺ current through K⁺ channels of intermediate conductance (SK4, KCa3.1 or IK1) also contributes to the K_slow current. We suggest that the K_slow current is composed of two currents through metabolism-regulated K⁺ channels, K_ATP (regulated by ATP) and SK4 (regulated by Ca²⁺). We further propose that the SK4 component of the K_slow current can trigger oscillations in mice without functioning K_ATP channels.     

Role of K+ channels in EDHF-dependent relaxation induced by acetylcholine in canine coronary artery

Summary To identify the K⁺ channels responsible for endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation, we studied the effects of various K⁺ channel blockers on acetylcholine-induced relaxation, which persists even in the presence of both an inhibitor of nitric oxide synthase and that of cyclooxygenase, in canine coronary artery rings. A nonselective K⁺ channel blocker, tetrabutylammonium (TBA), a large and intermediate conductance Ca²⁺-activated K⁺ channel blocker, charybdotoxin (CTX), and a voltage-dependent K⁺ channel blocker, 4-aminopyridine (4-AP), significantly inhibited this residual relaxation. A combined treatment with CTX and 4-AP almost completely blocked the relaxation. Neither a large (iberiotoxin) nor a small (apamin) conductance Ca²⁺-activated K⁺ channel blocker blocked the relaxation. We also investigated effects of K⁺ channel blockers on basal tone to determine whether or not EDHF is involved in regulating basal tone. TBA and CTX substantially raised basal tone to a greater degree in endothelium-intact preparations than in endothelium-denuded preparations. These results indicate that EDHF may exert its relaxing action through intermediate conductance Ca²⁺-activated and voltage-dependent K⁺ channels in canine coronary arteries. In addition, EDHF may play a role in maintaining basal vascular tone. This study was supported in part by a Grant-in-Aid for Scientific Research (B07457167) from the Ministry of Education, Science and Culture of Japan.     

Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells

Summary Sulphonylureas stimulate insulin secretion by binding to a receptor in the pancreatic beta-cell plasma membrane resulting in inhibition of ATP-sensitive K⁺ channels, membrane depolarization and thus influx of Ca²⁺ through voltage-dependent Ca²⁺ channels. Sulphonylureas can also induce hormone release at fixed membrane potentials without Ca²⁺ entry suggesting that these drugs may have other modes of action. We have determined whether different forms of sulphonylurea-binding proteins are present in insulin-secreting cells and their subcellular localization by density gradient centrifugation. Binding studies using [³H]-glibenclamide showed that islet and insulinoma membranes contained a single high affinity sulphonylurea binding site (K_d = 1 nmol/l). Photo-crosslinking of the drug to the membranes resulted in labelling of two proteins with apparent molecular weights of 170 and 140 kDa. The same analyses of insulinoma subcellular fractions showed that the majority (>90%) of binding proteins were localized to intracellular membranes with only minor levels (<10%) on plasma membranes. The 170 kDa sulphonylurea binding protein was present in both plasma and granule membrane fractions whereas the 140 kDa form was not present in the plasma membrane fraction. The differences in the molecular forms and subcellular distribution of the receptor are consistent with sulphonylureas having multiple sites of action in the pancreatic beta cell.Abbreviations NEDH rats New England Deaconess Hospital rats - DMEM Dulbecco's modified Eagle's medium - PMSF phenylmethylsulphonyl fluoride - ER endoplasmic reticulum     

Toward a Molecular Understanding of Voltage-Gated Potassium Channels

Voltage-Gated Potassium Channels . Many different types of potassium (K⁺) channels exist and they play a central role in the fine tuning of excitability properties. Of the distinct subpopulations of K⁺ channels expressed in different cells, voltage-gated K⁺ channels have been studied most thoroughly at a molecular level. Over the last few years, the joint application of recombinant DNA technology together with electrophysiology, such as the voltage clamp and the patch clamp techniques, has produced a wealth of information. We have begun to unravel the genetic basis of ion channel diversity. In particular, the Xenopus oocyte expression system has turned out to be of enormous experimental value. Oocytes microinjected with “cloned” mRNA have been used to gain insight into biophysical and pharmacologic properties of voltage-gated K⁺, Na⁺, and Ca²⁺ channels. Here, we will review our understanding of K⁺ channel diversity based upon the fact that ion channels are encoded as a large multigene family. We have caught a first glimpse at possible molecular mechanisms underlying several biophysical properties characteristic for voltage-gated ion channels: voltage dependence of activation and inactivation, and ion permeation and selectivity. We will discuss molecular mechanisms of K⁺ channel activation and inactivation. We will also describe experiments that led to the identification of the “pore region,” and we will present a model of a potassium selective ion channel pore.     

Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells

Aims/hypothesis  The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. Methods  Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. Results  Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba²⁺-sensitive inwardly rectifying K⁺ current and two types of voltage-gated K⁺ currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na⁺ currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca²⁺ channels blocked by isradipine (L), ω-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to ω-agatoxin. Conclusions/interpretation  Action potential firing in delta cells is modulated by ATP-sensitive K⁺-channel activity. The membrane potential is stabilised by Ba²⁺-sensitive inwardly rectifying K⁺ channels. Voltage-gated L- and T-type Ca²⁺ channels are required for electrical activity, whereas Na⁺ currents and P/Q-type Ca²⁺ channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K⁺ channels. Exocytosis is tightly linked to Ca²⁺-influx via P/Q-type Ca²⁺ channels. Glucose stimulation of somatostatin secretion involves both K_ATP channel-dependent and -independent processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users. M. Braun and R. Ramracheya contributed equally to this study     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

Trimebutine maleate has inhibitory effects on the voltage-dependent Ca2+ inward current and other membrane currents in intestinal smooth muscle cells

We examined effects of trimebutine maleate on the membrane currents of the intestinal smooth muscle cells by using the tight-seal whole cell clamp technique. Trimebutine suppressed the Ba²⁺ inward current through voltage-dependent Ca²⁺ channels in a dose-dependent manner. The inhibitory effect of trimebutine on the Ba²⁺ inward current was not use-dependent. It shifted the steady-state inactivation curve to the left along the voltage axis. Trimebutine also had inhibitory effects on the other membrane currents of the cells, such as the voltage-dependent K⁺ current, the Ca²⁺-activated oscillating K⁺ current and the acetylcholine-induced inward current. These relatively non-specific inhibitory effects of trimebutine on the membrane currents may explain, at least in part, the dual actions of the drug on the intestinal smooth muscle contractility, i.e. inhibitory as well as excitatory.     

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs

Two Ca²⁺-mobilizing receptors expressed in lactotrophs, endothelin-A (ET_A) and thyrotropin-releasing hormone (TRH), induce a rapid Ca²⁺ release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX)-treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca²⁺ signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs⁺, a relatively specific blocker of inward rectifier K⁺ channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca²⁺ signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs⁺ was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca²⁺-activated SK-and BK-type K⁺ channels, respectively; E-4031, a blocker of ether a-go-go K⁺ channel; and linopirdine, a blocker of M-type K⁺ channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs⁺-sensitive channels, presumably the G_i/o-controlled inward rectifier K⁺ channels, and that this agonist also inhibits PRL release, but downstream of Ca²⁺ influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.     

Association of beta-catenin with the alpha-subunit of neuronal large-conductance Ca2+-activated K+ channels

The association of Ca²⁺-activated K⁺ channels with voltage-gated Ca²⁺ channels at the presynaptic active zones of hair cells, photoreceptors, and neurons contributes to rapid repolarization of the membrane after excitation. Ca²⁺ channels have been shown to bind to a large set of synaptic proteins, but the proteins interacting with Ca²⁺-activated K⁺ channels remain unknown. Here, we report that the large-conductance Ca²⁺-activated K⁺ channel of the chicken's cochlear hair cell interacts with β-catenin. Yeast two-hybrid assays identified the S10 region of the K⁺ channel's α-subunit and the ninth armadillo repeat and carboxyl terminus of β-catenin as necessary for the interaction. An antiserum directed against the α-subunit specifically coprecipitated β-catenin from brain synaptic proteins. β-Catenin is known to associate with the synaptic protein Lin7/Velis/MALS, whose interaction partner Lin2/CASK also binds voltage-gated Ca²⁺ channels. β-Catenin may therefore provide a physical link between the two types of channels at the presynaptic active zone.     

Introduction: Mechanisms of positive inotropic effects

Summary This review deals with the principal mechanisms which are known to play a role in positive inotropism: 1) The myoplasmic Ca²⁺ concentration may be increased by increases in cyclic AMP. Beside receptor-mediated stimulation (isoprenaline) or direct stimulation (forskolin) of the adenylate cyclase, the cyclic AMP may be increased by phosphodiesterase inhibition; 2) Cyclic AMP-independent activation of Ca²⁺ channels can be brought about by alpha-adrenergic agents (phenylephrine) or so-called calcium agonists; 3) Only a small increase in myoplasmic Na⁺ concentration can greatly enhance the force of contraction by an increase in the intracellular Ca²⁺ concentration. This is possible by inhibition of the Na⁺/K⁺-ATPase (glycosides) or by prolongation of the open state of Na⁺ channels (DPI 201-106); 4) A direct inhibition of the Na⁺/Ca²⁺ exchange has been discussed for amiloride; 5) A prolongation of the action potential induced by K⁺ channel-inhibiting agents such as 4-amino-pyridine may increase the myoplasmic Ca²⁺ concentration by a prolongation of the slow Ca²⁺ inward current; 6) An increased Ca²⁺ sensitivity of the contractile proteins has been demonstrated for a number of compounds in vitro; the contribution of such an effect to the overall positive inotropism is unknown because a calcium sensitizer without any effects on calcium or sodium movements is not yet available.     

Contribution of electromechanical coupling between KV and CaV1.2 channels to coronary dysfunction in obesity

Previous investigations indicate that diminished functional expression of voltage-dependent K⁺ (K_V) channels impairs control of coronary blood flow in obesity/metabolic syndrome. The goal of this investigation was to test the hypothesis that K_V channels are electromechanically coupled to Ca_V1.2 channels and that coronary microvascular dysfunction in obesity is related to subsequent increases in Ca_V1.2 channel activity. Initial studies revealed that inhibition of K_V channels with 4-aminopyridine (4AP, 0.3 mM) increased intracellular [Ca²⁺], contracted isolated coronary arterioles and decreased coronary reactive hyperemia. These effects were reversed by blockade of Ca_V1.2 channels. Further studies in chronically instrumented Ossabaw swine showed that inhibition of Ca_V1.2 channels with nifedipine (10 μg/kg, iv) had no effect on coronary blood flow at rest or during exercise in lean swine. However, inhibition of Ca_V1.2 channels significantly increased coronary blood flow, conductance, and the balance between coronary flow and metabolism in obese swine (P < 0.05). These changes were associated with a ~50 % increase in inward Ca_V1.2 current and elevations in expression of the pore-forming subunit (α₁c) of Ca_V1.2 channels in coronary smooth muscle cells from obese swine. Taken together, these findings indicate that electromechanical coupling between K_V and Ca_V1.2 channels is involved in the regulation of coronary vasomotor tone and that increases in Ca_V1.2 channel activity contribute to coronary microvascular dysfunction in the setting of obesity.     

Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel

The activation of eukaryotic SLO K⁺ channels by intracellular cues, mediated by a cytoplasmic structure called the gating ring, is central to their physiological roles. SLO3 channels are exclusively expressed in mammalian sperm, where variations of intracellular pH are critical to cellular function. Previous studies primarily focused on the mouse SLO3 orthologue and revealed that, in murine sperm, SLO3 mediates a voltage- and alkalization-activated K⁺ current essential to male fertility. Here we investigate the activation of the human SLO3 channel by intracellular pH at the functional and structural level. By using electrophysiology in a heterologous system, we show that human SLO3 opens upon intracellular pH increase and that its expression and functional properties are modulated by LRRC52, a testis-specific accessory subunit. We next present the crystal structure of the human SLO3 gating ring. Comparison with the known structures of the corresponding domain from SLO1, a Ca²⁺-activated homologue, suggests that the SLO3 gating ring structure may represent an open state. Together, these results present insights into the function of a protein expected to be critical for human reproduction and provide a framework to study the mechanism of pH gating in SLO3 channels.     

Effects of nitric oxide donors on vascular smooth muscles depend on a type of vascular smooth-muscle preactivation

The abilities of such therapeutic nitrovasodilators as sodium nitroprusside (SNP) and glyceryl trinitrate (GTN) to dilate vascular smooth muscles (VSM) and affect intracellular calcium concentration level ([Ca²⁺]_i) in a rat tail artery were tested under different types of preactivation. To shed light on mechanisms underlying possible differences in the action of these two nitric oxide (NO) donors, simultaneous measurements of [Ca²⁺]_i and contractile force were done. All vascular rings were precontracted either using a high-K⁺-Krebs solution or phenylephrine (PE). It was shown that the effect of both NO donors strongly depended on a type of VSM preactivation. The EC₅₀ for GTN under K⁺ stimulation of VSM comprised (2.48±1.6)×10⁻⁵ M, whereas the mean EC₅₀ under PE stimulation was (3.05±2.3)×10⁻⁴ M (p<0.05, n=9). The EC₅₀ for SNP under K⁺ stimulation of VSM comprised (1.09±0.47)×10⁻⁷ M, whereas the EC₅₀ under PE stimulation was (8.01±2.4)×10⁻⁶ M (p<0.05, n=9). GTN demonstrated a significant discrepancy in the magnitude of changes in [Ca²⁺]_i and related VSM relaxant responses, indicating the ability of GTN to relax VSM in the absence of a proportional decrease in [Ca²⁺]_i. The main peculiarity of SNP action under K⁺ stimulation as compared to PE stimulation was the transient decrease in [Ca²⁺]_i while relaxation was sustained. Therefore, both NO donors demonstrated their ability to produce vasorelaxation as a result of an alteration in myofilament calcium sensitivity. These data clearly indicate that the sensitivity of VSM to NO donors is higher under K⁺ depolarization than that seen under PE stimulation, indicating that Ca²⁺ entry through voltage-operated calcium channels is more sensitive to NO as compared to calcium mobilization by means of Ca²⁺ entry through receptor-operated calcium channels or intracellular Ca²⁺ release, or both.     

Internal pH,Na+, and Ca2+ regulation by trimetazidine during cardiac cell acidosis

Summary The effects of trimetazidine were studied on plasma membrane structures of cardiac cells which control excitability, as well as on cardiac cells that were cultured in normal physiologic conditions and after intracellular acidification.When cardiac cells were kept in normal physiologic conditions, trimetazidine at concentrations ranging from 10^–8 to 3.10^–4 M interacted neither directly nor indirectly with the major ionic transporter systems of cardiac cells, such as ionic channels (Na⁺, K⁺), ATPase, Na⁺/H⁺, and Na⁺/Ca²⁺ exchange systems.Under acid-load conditions trimetazide acts in a dose- and time-dependent manner, in limiting the accumulation of Na⁺ and Ca²⁺ inside cardiac cells and depressing intracellular cell acidosis.It is proposed that trimetazidine plays a key role in limiting the intracellular accumulation of protons that is responsible for cell acidosis during ischemia.Trimetazidine, in protecting cardiac cells against accumulation of protons, limits accumulation of Na⁺ and Ca²⁺.     

Na+/K+ pump inhibition induces cell shrinkage in cultured chick cardiac myocytes

Summary Myocardial cell swelling occurs in ischemia and in reperfusion injury before the onset of irreversible injury. Swelling has been attributed to failure of the Na⁺/K⁺ pump and the accumulation of intracellular Na⁺. To evaluate the role of the pump-leak model of cell volume maintenance, short term changes in cell volume in response to Na⁺/K⁺ pump inhibition were studied in aggregates of cultured embryonic chick cardiac myocytes using optical and biochemical methods. Exposure to 100 M ouabain over 20 min induced cell shrinkage of approximately 10%. Cell water was also decreased by Na⁺/K⁺ pump inhibition; incubation for 1 hr either in the presence of 100 M ouaain or in K⁺-free solution reduced cell water by 18.4% and 28.4% respectively. When exposed to ouabain in the absence of extracellular Ca²⁺, the aggregates swelled by approximately 15%, indicating that extracellular Ca²⁺ was required for the ouabain-induced shrinkage to occur. Ouabain still caused shrinkage, however, in the presence of the Ca²⁺ channel blockers verapamil (10 M) and nifedipine (10 M), suggesting that Na⁺/Ca²⁺ exchange, rather than Ca²⁺ channels, is the route for Ca²⁺ influx during Na⁺/K⁺ pump inhibition. Efflux of amino acids (taurine, aspartate, glutamate, glycine and alanine) from confluent monolayers of chick heart cells exposed to ouabain for 20 min was nearly double that observed in control solution. These results suggest that, during Na⁺/K⁺ pump inhibition, chick heart cells can limit accumulation of intracellular sodium by means of Na⁺/Ca²⁺ exchange, and that a rise in intracellular [Ca²⁺], also mediated by Na⁺/Ca²⁺ exchange, promotes the loss of amino acids and ions to cause cell shrinkage. Therefore, swelling during ischemic injury may not result from Na⁺/K⁺ pump failure alone, but may reflect the exhaustion of alternative volume regulatory transport mechanisms.     

Voltage dependence of K channels in guard-cell protoplasts

Stomatal pores in leaves enable plants to regulate the exchange of gases with their environment. Variations of the pore aperture are mediated by controlled changes of potassium salt concentrations in the surrounding guard cells. The voltage-dependent gating of K⁺-selective channels in the plasma membrane (plasmalemma) of cell-wall-free guard cells (protoplasts) was studied at the molecular level in order to investigate the regulation of K⁺ fluxes during stomatal movements. Inward and outward K⁺ currents across the plasmalemma of guard cells were identified by using the whole-cell configuration of the patch-clamp technique. Depolarizations of the membrane potential from a holding potential of -60 mV to values more positive than -40 mV produced outward currents that were shown to be carried by K⁺. Hyperpolarizations elicited inward K⁺ currents. Inward and outward currents were selective for K⁺ over Na⁺ and could be partially blocked by exposure to extracellular Ba²⁺. In cell-attached and excised membrane patches, previously identified K⁺-selective single channels in guard cells were studied. Averaging of single-channel currents during voltage pulses resulted in activation and deactivation kinetics that were similar to corresponding kinetics of inward and outward currents in whole cells, showing that K⁺-selective channels were the molecular pathways for the K⁺ currents recorded across the plasmalemma of single guard-cell protoplasts. Estimates demonstrate that K⁺ currents through the voltage-gated K⁺ channels recorded in whole guard cells can account for physiological K⁺ fluxes reported to occur during stomatal movements in leaves.     

Activation of the SK potassium channel-calmodulin complex by nanomolar concentrations of terbium

Small conductance Ca²⁺-activated K⁺ (SK) channels sense intracellular Ca²⁺ concentrations via the associated Ca²⁺-binding protein calmodulin. Structural and functional studies have revealed essential properties of the interaction between calmodulin and SK channels. However, it is not fully understood how the binding of Ca²⁺ to calmodulin leads to channel opening. Drawing on previous biochemical studies of free calmodulin using lanthanide ions as Ca²⁺ substitutes, we have used the lanthanide ion, Tb³⁺, as an alternative ligand to study the activation properties of SK channels. We found that SK channels can be fully activated by nanomolar concentrations of Tb³⁺, indicating an apparent affinity >100-fold higher than Ca²⁺. Competition experiments show that Tb³⁺ binds to the same sites as Ca²⁺ to activate the channels. Additionally, SK channels activated by Tb³⁺ demonstrate a remarkably slow deactivation process. Comparison of our results with previous biochemical studies suggests that in the intact SK channel complex, the N-lobe of calmodulin provides ligand-binding sites for channel gating, and that its ligand-binding properties are comparable to those of the N-lobe in isolated calmodulin.     

Comparison of K+ channel properties in freshly isolated myocytes from thoracic aorta of WKY and SHR

Altered function of smooth muscle cell K⁺ channels have been reported in hypertension, but the contribution of various K⁺ channel types to these changes has not been completely determined. The purpose of this study was to compare the contribution of K⁺ channel types to whole cell K⁺ currents recorded from isolated thoracic aorta myocytes of 13 to 15 week old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Cells were isolated by collagenase and elastase digestion, and K⁺ currents recorded using whole cell voltage clamp methods at room temperature. Cells were superfused with a solution containing (in mmol/L) 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose. Pipettes were filled with a solution containing (in mmol/L) 120 KCl, 5 NaCl, 5 MgATP, 20 HEPES, and 10 BAPTA. The K⁺ currents (I_K) recorded from a holding potential (HP) of −80 mV were smaller in the SHR compared to those in WKY (for example, at 20 mV: WKY = 6.1 ± 0.6 pA/pF and SHR = 3.7 ± 0.2 pA/pF). Values of cell capacitance were not different between the two groups (WKY = 25.2 ± 3.2 pF and SHR = 26.6 ± 1.9 pF). A component of I_K inhibited by voltage (K_v) over the range from −80 to −20 mV was smaller in SHR. The voltage dependence of K_v availability and activation were not significantly different between the two groups. I_K recorded from a HP = −20 mV (K_Ca) was not different between the two groups. Difference currents calculated from I_K measured at HP of −80 and −20 mV (that is, K_v) were smaller in SHR as was the fraction of I_K inhibited by 4-aminopyridine. These results suggest that under conditions of low intracellular [Ca²⁺] there are no differences in K_Ca currents, but the K_v currents are smaller in SHR. Inhibition of K_v by 4-aminopyridine (0.1 to 10 mmol/L) caused larger increases in basal tone in WKY aorta. These results suggest that K_v channels contribute to resting K⁺ conductance in both WKY and SHR aorta, but with a relatively larger contribution in the WKY.     

Glucose-dependent and -independent electrical activity in islets of Langerhans of Psammomys obesus, an animal model of nutritionally induced obesity and diabetes

Glucose-induced insulin secretion from pancreatic β-cells involves metabolism-induced membrane depolarization and voltage-dependent Ca²⁺ influx. The electrical events in β-cell glucose sensing have been studied intensely using mouse islets of Langerhans, but data from other species, including models of type 2 diabetes mellitus (T2DM), are lacking. In this work, we made intracellular recordings of electrical activity from cells within islets of the gerbil Psammomys obesus (fat sand rat), a model of dietary-induced T2DM. Most islet cells from lean, non-diabetic sand rats displayed glucose-induced, K_ATP channel-dependent, oscillatory electrical activity that was similar to the classic “bursting” pattern of mouse β-cells. However, the oscillations were slower in sand rat islets, and the dose-response curve of electrical activity versus glucose concentration was left-shifted. Of the non-bursting cells, some produced action potentials continuously, while others displayed electrical activity that was largely independent of glucose. The latter activity consisted of continuous or intermittent action potential firing, and persisted for long periods in the absence of glucose. The glucose-insensitive activity was suppressed by diazoxide, indicating that the cells expressed K_ATP channels. Sand rat islets produced intracellular Ca²⁺ oscillations reminiscent of the oscillatory electrical pattern observed in most cells, albeit with a longer period. Finally, we found that the glucose dependence of insulin secretion from sand rat islets closely paralleled that of the bursting electrical activity. We conclude that while subpopulations of K_ATP-expressing cells in sand rat islets display heterogeneous electrical responses to glucose, insulin secretion most closely follows the oscillatory activity. The ease of recording membrane potential from sand rat islets makes this a useful model for studies of β-cell electrical signaling during the development of T2DM.