Maxi K+ channels from the apical membranes of rabbit oviduct epithelial cells

Large conductance (approximately 210 pS), K⁺-selective channels were identified in excised, insideout patches obtained from the apical membranes of both ciliated and nonciliated epithelial cells grown as monolayers from the primary culture of rabbit oviduct. The open probability of channels showing stable gating was increased at positive membrane potentials and was sensitive to the concentration of free calcium ions at the cytosolic surface of the patch ([Ca²⁺] _i ). In these respects, the channel resembled maxi K⁺ channels found in a number of other cell types. The distributions of dwell-times in the open state were most consistently described by two exponential components. Four exponential components were fitted to the distributions of dwelltimes in the closed state. Depolarizations and [Ca²⁺] _i increases had similar effects on the distribution of open dwell-times, causing increases in the two open time constants ( _o1 and _o2) and the fraction of events accounted for by the longer component of the distribution. In contrast, calcium ions and voltage had distinct effects on the distribution of closed dwelltimes. While the three shorter closed time constants ( _c1, _c2 and _c3) were reduced by depolarizing membrane potentials, increases in [Ca²⁺] _i caused decreases in the longer time constants ( _c3 and _c4). It is concluded that oviduct large conductance Ca²⁺-activated K⁺ channels can enter at least two major open states and four closed states.A.F.J. was supported by a research fellowship from the Japan Society for the Promotion of Science and received a grant for laboratory expenses from the Ministry of Education, Science and Culture, Japan. The authors wish to thank Dr. Shigetoshi Oiki for valuable discussion of the analysis of gating kinetics and Dr. Jeman Kim (Kyoto Pharmaceutical University) for making the transmission electron micrographs.     

Analysis of the T-type calcium channel in embryonic chick ventricular myocytes

Summary T-type calcium channels (I _T channels) were studied in cell-attached patch electrode recordings from the ventricular cell membrane of 14-day embryonic chick heart. All experiments were performed in the absence of Ca²⁺ with Na⁺ (120mm) as the charge carrier.I _T channels were distinguished from L-type calcium channels (I _L) by their more negative activation and inactivation potential ranges; their smaller unitary slope conductance (26 pS), and their insensitivity to isoproterenol or D600. Inactivation kinetics were voltage dependent. The time constant of inactivation was 37 msec when the membrane potential was depolarized 40 mV from rest (R+40 mV), and 20 msec atR+60 mV. The frequency histogram of channel open times ₀ was fit by a single-exponential curve while that of closed times _c was biexponeintial. _o was the same atR+40 mV andR+60 mV whereas _c was shortened atR+60 mV. The open-state probability (P _o) increased with depolarization: 0.35 atR+40 mV, 0.8 atR+60 mV and 0.88 atR+80 mV. This increase inP _o at depolarized potentials could be accounted for by the decrease in _c.     

Involvement of apamin-sensitive SK channels in spike frequency adaptation of neurons in nucleus tractus solitarii of the rat

We delineated the role of Ca²⁺-activated K⁺ channels in the phenomenon of spike frequency adaptation (SFA) exhibited by neurons in the caudal region of nucleus tractus solitarius (cNTS) using intracellular recording coupled with the current-clamp technique in rat brain slices. Intracellular injection of a constant depolarizing current evoked a train of action potentials whose discharge frequency declined rapidly to a lower steady-state level of irregular discharges. This manifested phenomenon of SFA was found to be related to extracellular Ca²⁺. Low Ca²⁺ (0.25 mM) or Cd²⁺ (0.5 mM) in the perfusing medium resulted in a significant increase in the adaptation time constant (_adap) and an appreciable reduction in the percentage adaptation of spike frequency (F_adap). In addition, the evoked discharges were converted from an irregular to a regular pattern, accompanied by a profound increase in mean firing rate. Intriguingly, similar alterations in _adap, F_adap, discharge pattern and discharge rate were elicited by apamin (1 µM), a selective blocker for small-conductance Ca²⁺-activated K⁺ (SK) channels. On the other hand, charybdotoxin (0.1 µM), a selective blocker for large-conductance Ca²⁺-activated K⁺ channels, was ineffective. Our results suggest that SK channels of cNTS neurons may subserve the generation of both SFA and irregular discharge patterns displayed by action potentials evoked with a prolonged depolarizing current.     

Evidence for two K+ currents activated upon hyperpolarization ofParamecium tetraurelia

Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K⁺ solutions elicits a complex of Ca²⁺ and K⁺ currents. The tail current that accompanies a return to holding potential (–40 mV) contains two K⁺ components. The tail current elicited by a step to –110 mV of 50-msec duration contains fast-decaying (3.5 msec) and slow-decaying (20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K⁺], suggesting that they represent pure K⁺ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K⁺ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca²⁺ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K⁺ currents are inhibited by extracellular TEA⁺ in a concentration-dependent, noncooperative manner, whereas the fast K⁺ current alone is inhibited by 0.7mm quinidine.     

Characterization of calcium fluxes across the envelope of intact spinach chloroplasts

Calcium fluxes across the envelope of intact spinach chloroplasts (Spinacia oleracea L.) in the light and in the dark were investigated using the metallochromic indicator arsenazo III. Light induces Ca²⁺ influx into chloroplasts. The action spectrum of light-induced Ca²⁺ influx and the inhibitory effect of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) indicate an involement of photosynthetic electron transport in this process. The driving force for light-induced Ca²⁺ influx is most likely a change in the membrane potential component of the proton motive force. This was demonstrated by the use of agents modifying the membrane potential (lipophilic cations, ionophores, different KCl concentrations). The activation energy of the observed Ca²⁺ influx is about 92 kJ mol^-1. Verapamil and nifedipine, two Ca²⁺-channel blockers, have no inhibitory effect on light-induced Ca²⁺ influx, but enhance ferricyanide-dependent oxygen evolution. Inhibition of Ca²⁺ influx by ruthenium red reduces the light-dependent decrease in stromal NAD⁺ level.Abbreviations and symbols Chl chlorophyll - DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea - FCCP earbonyl cyanide p-trifluoromethoxyphenylhydrazone - PGA 3-phosphoglyceric acid - ABA⁺ tetrabutylammonium chloride - TPP⁺ tetraphenylphosphonium chloride - E membrane potential     

Potassium channels and different states ofChara plasmalemma

Summary The current-voltage (I/V) technique was employed to investigate the different electrophysiological states of theChara plasmalemma and their interaction under a range of conditions. In K⁺ state the membrane became very permeable (conductances >20 S m 2) as [K⁺]₀ increased to 10mm. As the cells were then easily damaged by the voltage-clamp procedures, it was difficult to determine the saturation K⁺ conductance. TEA (tetraethylammonium chloride) reversibly blocked the K⁺ channels, but had no effect on theI/V curve of the pump state, indicating that the K⁺ channels were not participating in this state. Acid pH₀ (4.5) diminished the K⁺ conductance, but did not alter the response of the K⁺ channels to change in [K⁺]₀. Alkaline pH₀ (11.0) madeChara resting PD bistable: the PD either stayed near the estimatedE _K and theI/V curve showed a negative conductance region typical of the K⁺ state, or it hyperpolarized and the near-linearI/V profile of the proton-permeable state was observed.     

Responsiveness of cardiac Na+ channels to antiarrhythmic drugs: The role of inactivation

Summary Elementary Na⁺ currents were recorded at 9°C in inside-out patches from cultured neonatal rat heart myocytes. In characterizing the sensitivity of cooled, slowly inactivating cardiac Na⁺ channels to several antiarrhythmic drugs including propafenone, lidocaine and quinidine, the study aimed to define the role of Na⁺ inactivation for open channel blockade.In concentrations (1–10 mol/liter) effective to depressNP _o significantly, propafenone completely failed to influence the open state of slowly inactivating Na⁺ channels. With 1 mol/liter, _open changed insignificantly to 96±7% of the control. Even a small number of ultralong openings of 6 msec or longer exceeding _open of the whole ensemble several-fold and attaining _open (at –45 mV) in cooled, (-)-DPI-modified, noninactivating Na⁺ channels proved to be drug resistant and could not be flicker-blocked by 10 mol/liter propafenone. The same drug concentration induced in(-)-DPI-modified Na⁺ channels a discrete block with association and dissociation rate constants of 16.1 ± 5.3 × 10⁶ mol^–1 sec^–1 and 675 ± 25 sec^–1, respectively. Quinidine, known to have a considerable affinity for activated Na⁺ channels, in lower concentrations (5 mol/liter) left _open unchanged or reduced, in higher concentrations (10 mol/liter) _open only slightly to 81% of the predrug value whereasNP _o declined to 30%, but repetitive blocking events during the conducting state could never be observed. Basically the same drug resistance of the open state was seen in cardiac Na⁺ channels whose open-state kinetics had been modulated by the cytoplasmic presence of F^– ions. But in this case, propafenone reduced reopening and selectively abolished a long-lasting open state. This drug action is unlikely related to the inhibitory effect onNP _o since hyperpolarization and the accompanying block attenuation did not restore the channel kinetics. It is concluded that cardiac Na⁺ channels cannot be flicker-blocked by antiarrhythmic drugs unless Na⁺ inactivation is removed.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Modulation of single cardiac Na+ channels by cytosolic Mg++ ions

Elementary Na⁺ currents were recorded in inside-out patches excised from cultured neonatal rat heart myocytes in order to study the influence of cytosolic Mg⁺⁺ and other bivalent cations present at the cytoplasmic membrane surface on cardiac Na⁺ channel gating. Exposing the cytoplasmic membrane surface to a Mg⁺⁺-free environment shortened the open state of cardiac Na⁺ channels significantly. _open declined to 62±2% of the value obtained at 5 mmol/l Mg_i ⁺⁺. Other channel properties including the tendency to reopen and the elementary current size either changed insignificantly within a 10% range or remained completely unchanged. An almost identical change of _open can be caused by switching from a Mn⁺⁺ (5 mmol/l) containing internal solution to a Mn⁺⁺-free internal solution. But _open failed to significantly respond to a variation in internal Ni⁺⁺ from 5 mmol/l to 0 mmol/l. The same response to internal Mg⁺⁺ withdrawal was obtained with (–)-DPI-modified, non-inactivating Na⁺ channels, indicating that the exit rate from the open state remains as sensitive to cytosolic Mg⁺⁺ variations as in normal Na⁺ channels with operating inactivation. Offprint requests to: M. Kohlhardt     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Characterization of Na+−Ca2+ exchange activity in plasma membrane vesicles from postmortem human brain

Procedures were developed for measurement of Na⁺/Ca²⁺ exchange in resealed plasma membrane vesicles from postmortem human brain. The vesicle preparation method permits use of stored frozen tissue with minimal processing required prior to freezing. Vesicles prepared in this manner transport Ca²⁺ in the presence of a Na⁺ gradient. The kinetic characteristics of the Na⁺/Ca²⁺ exchange process were determined in membrane vesicles isolated from hippocampus and cortex. The K_act for Ca²⁺ was estimated to be 32 M for hippocampal and 17 M for cortical tissue. The maximal rate of Ca²⁺ uptake (V_max) was 3.5 nmol/mg protein/15 sec and 3.3 nmol/mg protein/15 sec for hippocampal and cortical tissue, respectively. Exchange activity was dependent on the Na⁺ gradient, and was optimal in the high pH range. Therefore, membranes in which Na⁺-dependent o Ca²⁺ transport activity is preserved can be isolated from postmortem human brain and could be used to determine the influence of pathological conditions on this transport system.     

Chemiosmotic systems in bioenergetics: H+-cycles and Na+-cycles

The development of membrane bioenergetic studies during the last 25 years has clearly demonstrated the validity of the Mitchellian chemiosmotic H⁺ cycle concept. The circulation of H⁺ ions was shown to couple respiration-dependent or light-dependent energy-releasing reactions to ATP formation and performance of other types of membrane-linked work in mitochondria, chloroplasts, some bacteria, tonoplasts, secretory granules and plant and fungal outer cell membranes. A concrete version of the direct chemiosmotic mechanism, in which H⁺ potential formation is a simple consequence of the chemistry of the energy-releasing reaction, is already proved for the photosynthetic reaction centre complexes.Recent progress in the studies on chemiosmotic systems has made it possible to extend the coupling-ion principle to an ion other than H⁺. It was found that, in ceertain bacteria, as well as in the outer membrane of the animal cell, Na⁺ effectively substitutes for H⁺ as the coupling ion (the chemiosmotic Na⁺ cycle). A precedent is set when the Na⁺ cycle appears to be the only mechanism of energy production in the bacterial cell. In the more typical case, however, the H⁺ and Na⁺ cycles coexist in one and the same membrane (bacteria) or in two diffeerent membranes of one and the same cell (animals). The sets of and generators as well as and consumers found in different types of biomembranes, are listed and discussed.     

Non-Markovian Gating of Ca2+-Activated K+ Channels in Cultured Kidney Cells Vero. Rescaled Range Analysis

Using the patch-voltage clamp technique and the rescaled range method, activity of single large conductance Ca²⁺-activated K⁺ channels (K_Ca channels) was studied. For the sequences of alternating open and shut time intervals, the dependence R/S vs. N in the double logarithmic coordinates presented a curve with two slopes, H₁ =0.60 ± 0.04, and H₂ = 0.88 ± 0.21, where H₁ and H₂ characterized the Hurst exponents for shot and long time ranges, respectively. Similar results were obtained for reduced data sets consisting of only open or only shut intervals. Randomization of the experimental data resulted in a single slope, H, of 0.52 ± 0.02. Simulations were performed with eight-state Markovian model without memory. The calculated Hurst exponent presented in average 0.54 ± 0.02. The results suggest that the activity of single Ca²⁺-activated K⁺ channel exhibits two regimes, with slight positive correlation at short time ranges (H₁ =0.6), and strong positive correlation at long time ranges (H₂ = 0.88); therefore the channel gating as a whole is not a steady-state Markovian process.     

Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers

Plasma membrane was purified from roots of rye (Secale cereale L. cv. Rheidol) by aqueous-polymer two-phase partitioning and incorporated into planar bilayers of 1-palmitoyl-2-oleoyl phosphatidylethanolamine by stirring with an osmotic gradient. Since plasmamembrane vesicles were predominantly oriented with their cytoplasmic face internal, when fused to the bilayer the cytoplasmic side of channels faced the trans chamber. In asymmetrical (cis:trans) 280100 mM KCl, five distinct K⁺-selective channels were detected with mean chord-conductances (between +30 and -30 mV; volyages cis with respect to trans) of 500 pS, 194 pS, 49 pS, 21 pS and 10 pS. The frequencies of incorporation of these K⁺ channels into the bilayer were 48, 21, 50, 10 and 9%, in the order given (data from 159 bilayers). Only the 49 pS channel was characterized further in this paper, but the remarkable diversity of K⁺ channels found in this preparation is noteworthy and is the subject of further study. In symmetrical KCl solutions, the 49 pS channel exhibited non-ohmic unitary-current/voltage relationships. The chord-conductance (between +30 and-30 mV) of the channel in symmetrical 100 mM KCl was 39 pS. The unitary current was greater at positive voltages than at corresponding negative voltages and showed considerable rectification with increasing positive and negative voltages. This would represent inward rectification in vivo. Gating of the channel was not voltage-dependent and the channel was open for approx. 80% of the time. Presumably this is not the case in vivo, but we are at present uncertain of the in vivo controls of channel gating. The distribution of channel-open times could be approximated by the sum of two negative exponential functions, yielding two open-state time constants (_o, the apparent mean lifetime of the channel-open state) of 1.0 ms and 5.7 s. The distribution of channel-closed times was best approximated by the sum of three negative exponential functions, yielding time constants (_c, the apparent mean lifetime of the channel-closed state) of 1.1 ms, 51 ms and 11 s. This indicates at least a five-state kinetic model for the activity of the channel. The selectivity of the 49 pS channel, determined from both reversal potentials under biionic conditions (100 mM KCl100 mM cation chloride) and from conductance measurements in symmetrical 100 mM cation chloride, was Rb⁺ K⁺ > Cs⁺ > Na⁺ > Li⁺ > tetraethylammonium (TEA⁺). The 49 pS channel was reversibly inhibited by quinine (1 mM) but TEA⁺ (10 mM), Ba²⁺ (3 mM), Ca²⁺ (1 mM), 4-aminopyridine (1 mM) and charybdotoxin (3 M) were without effect when applied to the extracellular (cis) surface.Abbreviations and Symbols GHK Goldman-Hodgkin-Katz - I/V current/voltage - PEG polyethyleneglycol - P_o probability o f the channel being open - TEA⁺ tetraethylammonium - _c apparent mean lifetime of the channel-closed state - _o apparent mean lifetime of the channel-open state P.J.W. was supported by a grant from the Science and Engineering Research Council Membrane Initiative (GR/F 33971) to Professor E.A.C. MacRobbie and M.T. by the Glaxo Junior Research Fellowship at Churchill College, Cambridge. We thank Dr. D.T. Cooke (AFRC, Long Ashton Research Station, University of Bristol, UK) and Ms. J. Marshall (University of York, UK) for their advice and assistance with the aqueous-polymer two-phase partitioning of plasma membrane from rye roots, Mr. J. Banfield and Miss P. Parmar (University of Cambridge, UK) for technical assistance and Professor E.A.C. MacRobbie, Dr. G. Thiel (University of Cambridge, UK), Dr. M.R. Blatt (Wye College, University of London, UK), Dr. D. Sanders and Dr. E. Johannes (University of York, UK) for helpful discussions.     

Genetic studies on the muscle protein turnover rate of coturnix quail

The validation of the urinary excretion of N-methylhistidine (N-MH) by quail as an index of the muscle protein turnover rate was tested using the criterion of the rate of recovery of radioactivity in urine following an intraperitoneal dose of l-[3-¹⁴C]methylhistidine. A genetic study on muscle protein turnover in quail was conducted using three genetically diverse lines (LL, large body size; SS, small body size; RR, random-bred control line) selected for body size. When l-[3-¹⁴C]methylhistidine was administered to 20-week-old male and female coturnix quail by direct intraperitoneal injection, approximately 90% of the l-[3-¹⁴C]methylhistidine was recovered by 96 hr postinjection. Recoveries were low in the egg and muscle. These results show that N-MH released from myofibrillar protein is not reutilized and the excretion of N-MH is a satisfactory index of muscle protein breakdown. In all lines, the amount of urinary N-MH excretion and fractional synthesis (K_s) and degradation (K_d) rates at the high growing period were higher than those at the low growing period. The K_s and K_d are significantly different among selected lines at both 3 and 6 weeks of age. At 3 weeks of age, the fractional rate of synthesis of the LL line (13.2%/day) was higher than that of the RR line (11.5%/day), whereas the SS (8.1%/day) was lower than that of the RR line (11.5%/day). The fractional rates of degradation of both the LL line (4.1%/day) and the SS line (5.6%/day) were lower than that of the RR line (7.0%/day) at 3 weeks of age. From these results, it was recognized that selection for body size gave rise to the changes in the muscle protein turnover rate.     

Chemically modified cardiac Na+ channels and their sensitivity to antiarrhythmics: Is there a hidden drug receptor?

Elementary Na⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na⁺ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na⁺ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na⁺ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O₁–O₂ reaction (_open(1) 1.4±0.3 msec; _open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (_{open 3.0±0.5} msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP _o significantly can interact with both types of noninactivating Na⁺ channels to reduce the dwell time in the conducting configuration. lodate-modified Na⁺ channels became drug sensitive during the O₂ state. At –40 mV, for example, lidocaine reduced _open(2) to 62±5% of the control without detectable changes in _open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na⁺ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce _open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, a_lidocaine was 0.64×10⁶ mol^–1 sec^–1 and a_diprafenone 13.6×10⁶ mol^–1 sec^–1. Trypsin-modified Na⁺ channels also appear capable of discriminating among these antiarrhythmics, the ratio a_diprafenone/a_lidocaine even exceeded the value in iodate-modified Na⁺ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na⁺ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na⁺ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn.     

Parallel control of the inward-rectifier K+ channel by cytosolic free Ca2+ and pH inVicia guard cells

The influence of cytosolic pH (pH_i) in controlling K⁺-channel activity and its interaction with cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K⁺-channel currents were recorded under voltage clamp while pH_i or [Ca²⁺]_i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K⁺ concentration, current through inward-rectifying K⁺ channels (I_K,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na⁺-acetate outside imposed acid loads, lowering pH_i from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pH_i was independent of the weak acid used, and indicated a H⁺-buffering capacity which rose from 90 mM H⁺/pH unit near 7.5 to 160 mM H⁺/pH unit near pH_i 7.0. With acid-going pH_i, (I_K,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pH_i lasting 3–5 min and was accompanied by a reduction in (I_K,in) before recovery of the initial resting pH_i and current amplitude. The pH_i-sensitivity of the current was consistent with a single, titratable site for H⁺ binding with a pK_a near 6.3. Acid pH_i loads also affected current through the outward-rectifying K⁺ channels (I_K,out) in a manner antiparallel to (I_K,in) The effect on I_K, out was also scalar, but showed an apparent pK_a of 7.4 and was best accommodated by a cooperative binding of two H⁺. Parallel measurements showed that Na⁺-butyrate loads were generally without significant effect on [Ca²⁺]_i, except when pH_i was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca²⁺]_i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pH_i changes and K⁺-channel responses. The action on [Ca²⁺]_i coincided with a greater variability in (I_K,in) stimulation evident at pH_i values around 7.0 and below, and with negative displacements in the voltage-dependence of (I_K,in) gating. These results distinguish the actions of pH_i and [Ca²⁺]_i in modulating (I_K,in) they delimit the effect of pH_i to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pH_i as a second messenger capable of acting in parallel with, but independent of [Ca²⁺]_i in controlling the K⁺ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca²⁺]_i cytosolic free Ca²⁺ concentration - g_K ensemble (steady-state) K⁺-channel conductance - I_K,out, I_K,in outward-, inward-rectifying K⁺ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pH_i cytosolic pH - V membrane potential     

Thermodynamically specific gating kinetics of cardiac mammalian K (ATP) + channels in a physiological environment near 37°C

Elementary K⁺ currents through isolated ATP-sensitive K⁺ channels from neonatal rat cardiocytes were recorded to study their temperature dependence between 9°C and 39°C. Elementary current size and, thus, K⁺ permeation through the open pore varied monotonically with temperature with a Q₁₀ of 1.25 corresponding to a low activation energy of 3.9 kcal/mol. Open-state kinetics showed a complicated temperature dependence with Q₁₀ values of up to 2.94. Arrhenius anomalies of _open(1) and _open(2) indicate the occurrence of thermallyinduced perturbations with a dominating influence on channel portions that are involved in gating but are obviously ineffective in altering pore-forming segments. At 39°C, open-state exit reactions were associated with the highest activation energy (O₂ exit reaction: 12.1 kcal/ mol) and the largest amount of entropy. A transition from 19°C to 9°C elucidated a paradoxical kinetic response, shortening of both O-states, irrespective of the absence or presence of cAMP-dependent phosphorylation. Another member of the K⁺ channel family and also a constituent of neonatal rat cardiocyte membranes, 66 pS outwardly-rectifying channels, was found to react predictably since _open increased on cooling. Obviously, cardiac K _(ATP) ⁺ channels do not share this exceptional kinetic responsiveness to a temperature transition from 19°C to 9°C with other K⁺ channels and have a unique sensitivity to thermally-induced perturbations.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.