Potassium channels of adult locust (Schistocerca gregaria) muscle

Two types of K⁺ channels have been identified in patches of plasma membrane of metathoracic extensor tibiae muscle fibres of adult locust, Schistocerca gregaria. One channel had a maximum conductance of 170 pS, fast open-closed kinetics, and a linear current/ voltage relationship. In inside-out patches it was activated by ‘‘internally applied’’ Ca²⁺, but at unexpectedly low levels (between 10⁻¹⁰ and 10⁻⁹M). The other channel had a maximum conductance of 35 pS, slower open-closed kinetics, and was not activated by Ca²⁺. In cell-attached patches, its channel conductance measured in symmetrical salines was about three times greater for hyperpolarisations than for depolarisations. This inward rectification was proved to be due to block by intracellular Mg²⁺. For both channels, open probability (P _o) and mean open time increased during depolarisations and decreased during hyperpolarisations, resulting in outward rectifications in terms of net current (I _n , product of the single-channel current and P _o). For both channels, the K⁺ conductance was 10 times greater than that for Na⁺. Internally applied tetraethylammonium or tetramethylammonium ions blocked both channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

cAMP Stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance

 Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl^–channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl^–and K⁺ conductances is necessary in many Cl^–-secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K⁺ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl^–conductance, also upregulates endogenous K⁺ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl^–conductance. Of the cAMP-induced whole-cell current increase, 15–25% was due to a 293B-, Ba²⁺and TEA⁺-inhibitable K⁺ conductance. The cRNA of the mutated CFTR (ΔF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K⁺ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K⁺ channels, might explain the coordinated cAMP-mediated increase in Cl^–and K⁺ conductances. Received: 12 March 1996 / Accepted: 10 April 1996     

Calcium uptake and release modulated by counter-ion conductances in the sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum (SR) plays the central role in regulating the free myoplasmic Ca²⁺ level for the contractile activation of skeletal muscle. The initial stages of the voltage-controlled Ca²⁺ release mechanism are known in molecular detail. However, there is still very little known about the later stages of Ca²⁺ uptake and total Ca²⁺ turnover in the contraction–relaxation cycle under normal physiological conditions or under conditions influenced by fatigue or disease. Ca²⁺ uptake and release are both accompanied by ‘counter-ion’ movements across the SR membrane which prevent or reduce the generation of SR membrane potentials and balance for electroneutrality in the SR lumen. The SR membrane is permeable for the cations K⁺, Na⁺, H⁺ and Mg²⁺ and the anion Cl^-. Using electron-probe X-ray microanalysis, it has been shown that during tetanic stimulation the Ca²⁺ release was mainly balanced by uptake of K⁺ and Mg²⁺, leaving a charge deficit that was assumed to be neutralized via H⁺ ion or organic counter-ion movement. The low time resolution of electron-probe X-ray microanalysis leaves the possibility of other transient concentration changes in the SR, e.g. for Cl^- ions. Possible physiological roles of the SR counter-ion conductances can be tested using skinned muscle fibre preparations with intact sarcoplasmic reticulum and removed or chemically permeabilized outer sarcolemma. In skinned fibres, the SR K⁺ conductance can be effectively reduced with SR K⁺ channel blockers such as 4-aminopyridine, tetraethylammonium and decamethonium. Interestingly, these blockers increase Ca²⁺ loading as well as Ca²⁺ release, whereas other less specific blockers, such as 1.10-bis-quanidino-n-decane, seem to reduce Ca²⁺ release, possibly also via blocking Ca²⁺ release channels. Thus, it seems very important also to test the effects of counter-currents carried by K⁺, Mg²⁺, H⁺ or Cl^- ions on intact and voltage-clamped single-fibre preparations.     

Inhibition of SERCA2 Ca2+-ATPases by Cs+

Replacement of K⁺ with Cs⁺ on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane reduces the maximum velocity (V_max) of Ca²⁺ uptake into the SR of saponin-permeabilized rat ventricular myocytes. To compare the sensitivity of the cardiac and smooth muscle/non-muscle forms of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a and -2b respectively) to replacement of K⁺ with Cs⁺, SERCA2a and SERCA2b were expressed in HEK-293 cells. Ca²⁺ uptake into HEK cell microsomes was inhibited by replacement of extravesicular K⁺ with Cs⁺ (V_max of SERCA2a-mediated Ca²⁺ uptake in CsCl was 80% of that in KCl; V_max of SERCA2b-mediated uptake was 70% of that in KCl). The Ca²⁺ sensitivity of uptake was decreased for both SERCA2a- and SERCA2b-mediated uptake and the Hill coefficients were increased in the presence of CsCl. The effects of Cs⁺ on uptake were associated with direct inhibition of the ATPase activity of SERCA2a and SERCA2b. Our results indicate that cation binding sites are present in both SERCA2 isoforms, although the extent to which SERCA2b is inhibited by K⁺ replacement is greater than that of SERCA2a or SERCA1. Consideration of these results and the recent molecular modeling work of others suggests that monovalent cations could interact with the Ca²⁺ binding region of SERCA.     

Segmental heterogeneity of swelling-induced Cl transport in rat small intestine

The effect of cell swelling induced by hypotonic media was studied in segments of rat small intestine. In the Ussing chamber, exposure to a hypotonic medium caused a decrease in short-circuit current (I _sc) and potential difference (V ^ms) in the jejunum, whereas the ileum responded with an increase in I _sc and V ^ms. The transition from one pattern to the other was located about in the middle of the small intestine. Tissue conductance decreased in both segments, probably due to a reduction of paracellular shunt conductance induced by the cell swelling. Voltage scanning experiments revealed that the observed decrease in total tissue conductance in the ileum was caused solely by a decrease in local conductance in the villus region while the crypt conductance did not change, suggesting that the decrease in paracellular conductance of the crypts is compensated by an increase in cellular conductance. The response in both segments was dependent on the presence of Cl⁻ and was blocked by the Cl⁻ channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). It was not affected by the neurotoxin tetrodotoxin. In the jejunum the swelling-induced decrease in I _sc was reduced in the presence of the cyclooxygenase inhibitor, indomethacin, or the lipoxygenase inhibitor, nordihydroguaiaretic acid. In the ileum the Cl⁻ secretion induced by hypotonicity was blocked by the K⁺ channel blocker quinine and was reversed into a decrease in I _sc when serosal Ca²⁺ was zero. We conclude that the observed volume regulatory changes are initiated in the jejunum by an eicosanoid-mediated opening of basolateral Cl⁻ channels and in the ileum by a Ca²⁺-mediated opening of K⁺ channels which enhances apical Cl⁻ efflux. Received: 27 June 1995/Received after revision: 8 December 1995/Accepted: 28 December 1995     

Role of cholinergic-activated KCa1.1 (BK), KCa3.1 (SK4) and KV7.1 (KCNQ1) channels in mouse colonic Cl- secretion

Aim: Colonic crypts are the site of Cl^? secretion. Basolateral K⁺ channels provide the driving force for luminal cystic fibrosis transmembrane regulator‐mediated Cl^? exit. Relevant colonic epithelial K⁺ channels are the intermediate conductance Ca²⁺‐activated K_Ca3.1 (SK4) channel and the cAMP‐activated K_V7.1 (KCNQ1) channel. In addition, big conductance Ca²⁺‐activated K_Ca1.1 (BK) channels may play a role in Ca²⁺‐activated Cl^? secretion. Here we use K_Ca1.1 and K_Ca3.1 knock‐out mice, and the K_V7.1 channel inhibitor 293B (10 μm ) to investigate the role of K_Ca1.1, K_Ca3.1 and K_V7.1 channels in cholinergic‐stimulated Cl^? secretion. Methods: A Ussing chamber was used to quantify agonist‐stimulated increases in short circuit current (I_sc) in distal colon. Chloride secretion was activated by bl. forskolin (FSK, 2 μm ) followed by bl. carbachol (CCH, 100 μm ). Luminal Ba²⁺ (5 mm ) was used to inhibit K_Ca1.1 channels. Results: K_Ca1.1 WT and KO mice displayed identical FSK and CCH‐stimulated I_sc changes, indicating that K_Ca1.1 channels are not involved in FSK‐ and cholinergic‐stimulated Cl^? secretion. CCH‐stimulated ΔI_sc was significantly reduced in K_Ca3.1 KO mice, underscoring the known relevance of this channel in the activation of Cl^? secretion by an intracellular Ca²⁺ increasing agonist. The residual CCH effect observed in K_Ca3.1 KO mice suggests that yet another K⁺ channel is driving the CCH‐stimulated Cl^? secretion. In the presence of the specific K_V7.1 channel blocker 293B, the residual CCH effect was abolished. Conclusions: This demonstrates that both K_Ca3.1 and K_V7.1 channels are activated by cholinergic agonists and drive Cl^? secretion. In contrast, K_Ca1.1 channels are not involved in stimulated electrogenic Cl^? secretion.     

Block of cloned BKCa channels (rSlo)expressed in HEK 293 cells by N-methyl d -glucamine

We have investigated the conductance properties of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels formed by stable expression of the rSlo gene in HEK 293 cells. Single-channel recordings were obtained from inside-out patches excised into solution containing 100 μM Ca²⁺ to ensure a relatively high open probability over the range of membrane potentials studied (–120 to +100 mV). The unitary conductance of these channels at +80 mV was 221.6±5.4 pS in symmetrical 140 mM K⁺. Decreasing the K⁺ concentration on either side of the membrane, while maintaining ionic strength by adding N-methyl d-glucamine (NMDG⁺), reduced the unitary conductance. The reduction in conductance was greater when internal K⁺ was lowered by replacement with NMDG⁺. However, if sucrose was used as the internal K⁺ substitute instead of NMDG⁺ the reduction in unitary conductance was similar to that seen on reducing external K⁺. A rate-theory model whereby NMDG⁺ produces a very rapid block of the BK_Ca channel from the inside, but not the outside, is able to describe our results. Received:18 May 1998 / Received after revision: 17 June 1998 / Accepted: 2 July 1998     

Regulation of ion channels in rat hepatocytes

 Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K⁺ channel with a mean inward conductance of 55 ± 6.5 pS (n = 20) and a mean outward conductance of 25 ± 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl^– channel with a mean outward conductance of 30 ± 4.5 pS (n = 8) and a mean inward conductance of 10 ± 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca²⁺ activity on the intracellular side. Accordingly, the Ca²⁺ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K⁺ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K⁺ channel is inhibited by progesterone, while the outwardly rectifying Cl^– channel is insensitive to progesterone. Received: 21 May 1997 / Received after revision: 7 August 1997 / Accepted: 19 August 1997     

Patch-clamp studies of K+ and Cl− channel currents in canine pancreatic acinar cells

K⁺ and Cl^– channel currents in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp single-channel and whole-cell current recording techniques. In excised inside-out patches, we found a Ca⁺-activated (control range 0.01–0.4 M) and voltage-activated K²⁺-selective channel with a unit conductance of approximately 40 pS in symmetrical K⁺-rich solutions. In intact cells, addition of acetylcholine (1 M) or bombesin tetradecapeptide (0.1 nM) to the bath evoked an increase in frequency of K⁺ channel opening. In whole-cell recordings on cells dialyzed with K⁺-rich and Ca²⁺-free solution containing 0.5 mM EGTA, the resting potential was about –40mV. Depolarizing voltage pulses activated outward K⁺ currents, which were blocked by 10 mM tetraethylammonium, whereas hyperpolarizing pulses evoked smaller inward currents. Acetylcholine or bombesin activated the K⁺ current and enhanced the inward current, which was reduced by a low Cl^– (10 mM) intracellular solution at –90 mV holding potential. These results suggest that both Ca²⁺- and voltage-activated K⁺ channels and Ca²⁺-activated Cl^– channels exist in the plasma membrane of canine pancreatic acinar cells.     

Ion channels in isolated mouse jejunal crypts

 The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K⁺ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K⁺ [P _K ⁺ (1) > P _Rb ⁺ (0.6) >> P _Cs ⁺ (0.09) ≈ P _Na ⁺ (0.07) > P _Li ⁺ (0.04)], had a low, voltage-independent open probability (P _o) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 ± 3 pS), P _o was voltage dependent and it was blocked by cytoplasmic Ba²⁺ (5 mmol/l). An intermediate K⁺ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl^– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 ± 2 pS), its P _o was independent of voltage and unaffected by variations in cytoplasmic Ca²⁺ (100 nmol/l to 1 mmol/l) or ATP (0–1 mmol/l). The NSCC had a linear conductance (20 ± 1 pS), its P _o increased with depolarisation and elevation of cytoplasmic [Ca²⁺] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl^– conductance was activated. This cAMP-dependent Cl^– conductance was distinct from the basolateral Cl^– channel and thus is most likely located in the apical membrane. Received: 25 June 1997 / Accepted: 14 October 1997     

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres

 We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)₂ the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of –0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca²⁺)/P(K⁺)=2.4, P(Ca²⁺)/ P(Na⁺)=2.7, P(Ca²⁺)/P(Tris⁺)=3.1, P(Ca²⁺)/P(Mg²⁺)=1.0 and P(Ca²⁺)/P(Ba²⁺)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)₂ solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)₂ solution. The channel activity in symmetrical 50 mM Ca(gluconate)₂ solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 μM). The results are in agreement with the conclusion that the sarcoball Ca²⁺ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca²⁺-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel. Received: 13 February 1998 / Received after revision: 6 April 1998 / Accepted: 7 April 1998     

Anion and cation channels in the basolateral membrane of rabbit parietal cells

Ion channels in the basolateral membrane of rabbit parietal cells in isolated gastric glands were studied by the patch clamp technique. Whole-cell current-clamp recordings showed that the membrane potential (E _m ) changed systematically as a function of the chloride concentrations of the basolateral bathing solution ([Cl^–]₀), and of the pipette (intracellular) solution. The relationship betweenE _m and [Cl^–]₀ was not affected by additions of histamine, dibutyryl-cAMP, 4-acetoamido-4-isothiocyanostilbene-2,2-disulfonic acid and diphenylamine-2-carboxylate. The whole-cell Cl^– conductance was insensitive to voltage. In cell-attached and cell-free patch membranes, however, single Cl^– channel opening events could not be observed. The value ofE _m depended little on the basolateral K⁺ concentration, but inward-rectifier K⁺ currents were observed in the whole-cell configuration, activated by hyperpolarizing pulses and inhibited by extracellular Ba²⁺. In cell-attached and cell-free patches, openings of single inward-rectifier K⁺ channels and non-selective cation channels were infrequently recorded. Neither cAMP nor Ca²⁺ activated these cation channels. The single K⁺ channel conductance was about 230 pS under the symmetrical high K⁺ conditions and was inhibited by intracellular tetraethylammonium ions (TEA). The non-selective cation channel had a voltage-independent single conductance of 22 pS and was not inhibited by TEA.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

Epigallocatechin-3-gallate has dual,independent effects on the cardiac sarcoplasmic reticulum/endoplasmic reticulum Ca<Superscript>2+</Superscript> ATPase

We determined the effects of epigallocatechin-3-gallate (EGCG) and epicatechin (EC), on pump turnover and Ca²⁺ transport by the cardiac form of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Fluorescence spectroscopy was used to directly measure SERCA ATPase activity and to measure Ca²⁺ uptake into cardiac sarcoplasmic reticulum (SR) vesicles and microsomes derived from human embryonic kidney (HEK) cells expressing human cardiac SERCA2a. We found that EGCG reduces the maximum velocity of Ca²⁺ uptake into cardiac SR vesicles and increases the Ca²⁺-sensitivity of uptake in a concentration-dependent manner. EC is less potent than EGCG in increasing the Ca²⁺-sensitivity of uptake and does not affect maximum uptake velocity. The EGCG-dependent reduction in Ca²⁺ uptake velocity is well correlated with direct inhibition of SERCA. The effect of EGCG on the Ca²⁺-sensitivity of Ca²⁺ uptake into cardiac SR vesicles is affected by the phosphorylation status of phospholamban (PLB). When cardiac SERCA2a is expressed in HEK cells without PLB, EGCG reduces the maximum velocity of Ca²⁺ uptake but does not affect the Ca²⁺-sensitivity of uptake into microsomes derived from these cells indicating that the effect of EGCG on Ca²⁺-sensitivity requires the presence of PLB. Our results show that EGCG has dual effects on SERCA function in cardiac SR vesicles: it directly affects SERCA by reducing maximum uptake velocity; it increases the Ca²⁺-sensitivity of Ca²⁺ uptake in a manner that appears to depend on the interaction between SERCA and PLB.     

Identification of a voltage- and calcium-dependent non-selective cation channel in cultured adult and fetal human nasal epithelial cells

The apical membranes of cultured human nasal epithelial cells from adults and fetuses were investigated with the patch-clamp technique. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were found in 4% of the cell-attached, and 18% of the inside-out and outside-out patches (n=412). Multiple functional channels were present in more than 90% of these patches, with a mean of 3.9 channels per patch (n=55). The current-voltage relationship can be described by the Goldman equations and the single channel conductance was 20.1±0.3 pS (n=29) in adult and 20.7±0.4 pS (n=44) in fetal cells in symmetrical 150mM NaCl solutions. The channels were highly selective for cations: P_Na/P_Cl was 30 in adult and 45 in fetal experiments. They were equally permeable for K⁺ and Na⁺, somewhat less for Cs⁺, and impermeable for choline⁺ and tetraethylammonium⁺. The open probability was voltage dependent: it increased approximately 2-fold with 30mV depolarization in the potential range from −60mV to +60mV. The channels were activated by Ca²⁺ concentrations of about 10⁻⁴M at the cytoplasmic side, but were insensitive to extracellular Ca²⁺ and amiloride (10⁻⁴M). The non-selective cation channels found in apical membranes of cultured fetal nasal epithelial cells were not different from the adult ones.     

The secretory K<Subscript>Ca</Subscript>1.1 channel localises to crypts of distal mouse colon: functional and molecular evidence

The colonic epithelium absorbs and secretes electrolytes and water. Ion and water absorption occurs primarily in surface cells, whereas crypt cells perform secretion. Ion transport in distal colon is regulated by aldosterone, which stimulates both Na⁺ absorption and K⁺ secretion. The electrogenic Na⁺ absorption is mediated by epithelial Na⁺ channel (ENaC) in surface cells. Previously, we identified the large conductance Ca²⁺-activated K⁺ channel, K_Ca1.1 or big potassium (BK) channel, as the only relevant K⁺ secretory pathway in mouse distal colon. The exact localisation of K_Ca1.1 channels along the crypt axis is, however, still controversial. The aim of this project was to further define the localisation of the K_Ca1.1 channel in mouse distal colonic epithelium. Through quantification of mRNA extracted from micro-dissected surface and crypt cells, we confirmed that Na⁺/K⁺/2Cl⁻ (NKCC1) is expressed primarily in the crypts and γ-ENaC primarily in the surface cells. The K_Ca1.1 α-subunit mRNA was like NKCC1, mainly expressed in the crypts. The crypt to surface expression pattern of the channels and transporters was not altered when plasma aldosterone was elevated. The mRNA levels for NKCC1, γ-ENaC and K_Ca1.1 α-subunit were, however, under these circumstances substantially augmented (K_Ca1.1 α-subunit, twofold; NKCC1, twofold and ENaC, tenfold). Functionally, we show that ENaC-mediated Na⁺ absorption and BK channel-mediated K⁺ secretion are two independent processes. These findings show that K_Ca1.1-mediated K⁺ secretion mainly occurs in the crypts of the murine distal colon. This is in agreement with the general model of ion secretion being preferentially located to the crypt and not surface enterocytes.     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

Ryanoids change the permeability of potassium channels of locust (Schistocerca gregaria) muscle

The plasma membrane of locust skeletal muscle contains two types of K⁺ channel; a maxi, Ca²⁺-activated 170 pS channel (BK channel) and an inward rectifier of 35 pS conductance (IK channel). The effects of ryanodine, 9, 21-didehydroryanodine and 9, 21-didehydroryanodol on these channels have been investigated. In the concentration domain 10⁻⁹ M to 10⁻⁵ M, ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V _rev) of the channel currents, measured under physiologically normal K⁺ and Na⁺ gradients, i.e. from ≈60 mV in the absence of ryanodine to ≈15 mV for 10⁻⁵ M ryanodine. The alteration of K⁺ channel selectivity was Ca²⁺ independent. 9, 21-Didehydroryanodine and 9, 21-didehydroryanodol reduced V _rev, but only to ≈35 mV during application of 10⁻⁵ M of these compounds. 9, 21-Didehydroryanodine also diminished the conductances of the locust K⁺ channels. The three ryanoids reduced V _rev of a Ca²⁺-activated, high-conductance channel in inside-out patches excised from mouse interosseal muscle, although the changes were in each case less pronounced than those for the locust K⁺ channels. Also, the action of 9, 21-didehydroryanodine on mouse K⁺ (BK) channels was restricted to a shift of V _rev. For all three ryanoids, the IC₅₀ coefficients (i.e. the concentration of toxin that gave a 50% reduction in V _rev) for the shifts in V _rev were similar for the locust and mouse muscle K⁺ channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

Characterization of the chloride conductance in porcine renal brush-border membrane vesicles

 The chloride conductance in brush-border membrane vesicles prepared from pig kidney cortex was investigated using a light-scattering assay, anion-diffusion-potential-dependent Na⁺-D-glucose cotransport and ³⁶Cl^– influx. K⁺-diffusion-potential-driven salt exit from, or entry into, the vesicles was slow in the presence of gluconate, SO₄ ^2– and F^–, intermediate with Cl^– and Br^–, and fast with I^–, NO₃ ^–, and SCN^–. Stimulation of Na⁺-D-glucose uptake followed a similar anion sequence. Conductive Cl^– flux had a low activation energy and was inhibited by suphhydryl reagents, the stilbene disulphonates 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulphonate (SITS) and 4,4’-diisothiocyanato-2,2’-disulphonate (DIDS), and the arylaminobenzoates diphenylamine-2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Intravesicular Ca²⁺ and extravesicular nucleotides were without effect on conductive Cl^– flux. These characteristics tentatively exclude some known Cl^– channels and leave members of the ClC family as possible candidates responsible for the Cl^– conductance in brush-border membranes. Received: 28 July 1997 / Received after revision: 31 August 1997 / Accepted: 15 September 1997     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998