Ion channels in the plasma membrane ofAmaranthus protoplasts: One cation and one anion channel dominate the conductance

Summary This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons ofAmaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier.The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K⁺) and relative permeability sequence K⁺>Na⁺>Cl^– (10.160.03); whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of sub-conductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (V _m ) is depolarized; at millimolar levels of [Ca²⁺]_cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2mm) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO ₃ ^– >Cl^–>K⁺>Aspartate (2.0410.18 to 0.090.04). The relative permeability for K⁺ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clampV _m at a preferred value in the face of events which would otherwise perturbV _m .     

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K⁺ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K⁺ diffusion potential. The dependence of this ion channel on K⁺ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K⁺ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K⁺ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H⁺ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.Institution paper no.: ECOTRANS publication no. 45.     

Mechanosensitivity of mitochondrial large-conductance calcium-activated potassium channels

Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBK_Ca) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBK_Ca channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBK_Ca channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BK_Ca-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BK_Ca splice variants that variable mechanosensitivity of mitoBK_Ca could be the result of the presence of diverse BK_Ca isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBK_Ca channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.     

Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl^–, K⁺ and Ca²⁺ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK equilibrium potential for potassium transport - E_Cl equilibrium potential for chloride transport - SA stretchactivated Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

哺乳动物及人精子膜离子通道的研究进展

离子的跨膜转动对精子的生理活动起重要的作用。近年,应用膜片钳及人工膜重组等研究通道有关的电生理技术,人们直接观察到哺乳动物及人精子膜上Ｋ、Ｎａ＾＋、Ｃａ＾２＋、Ｃｌ通道的存在。这些结果为揭示精子成熟、获能精卵结合反应等生理过程的某些细节提供了有有的资料,特别是对人精子膜的研究,还为临床应用提供了可能。     

Isolation of protoplasts and ion channel recording of plasma membrane patches and whole-cell recordings of the marine alga, <Emphasis Type="Italic">Ulva pertusa</Emphasis> (Chlorophyta)

Whole-cell patch-clamp techniques were used to study ion channels of a marine alga. High quality protoplasts suitable for electrophysiological studies were isolated from the green marine alga, Ulva pertusa, using enzyme mixtures consisting of cellulase and abalone power and identified by calcofluor fluorescence. The vitality of protoplasts varied depending on the alga growth stage, and those isolated from younger tissue in March maintained a high vitality with high sealing success rate compared with protoplasts isolated from mature or non-growing plants in August or November. In the whole-cell configuration, large inward currents were elicited by negative voltage pulses. The voltage-dependent component was predominantly carried by Cl⁻, as confirmed by the use of the Cl⁻ channel inhibitor DIDS and reversal potential of current-voltage plots. This evidence suggests that hyperpolarization-activated Cl⁻ permeable channels are responsible for the influx of Cl⁻ into U. pertusa cells. Voltage-dependent outward currents were also recorded in several protoplasts, and their properties need further investigation.     

Characterization of whole-cell k<Superscript>+</Superscript> currents across the plasma membrane of pollen grain and tube protoplasts of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>

Outward and inward currents, mainly carried by K⁺, were detected in protoplasts of pollen grains (PG) and pollen tubes (PT) of Lilium longiflorum Thunb. by using the whole-cell configuration of the patch-clamp technique. The outward K⁺ current (I_{K+ out}) was similar in both protoplast types, while the inward K⁺ current (I_{K+ in}) was higher in pollen tube protoplasts. In PT but not in PG protoplasts, inward K⁺ currents were already detectable at negative membrane voltages usually monitored in lily pollen. I_{K+ in} consisted of a slow and a fast current component, as revealed by fitting a sum of two exponential functions to the time-dependent current. The contribution of the fast component to the total inward current was higher in PT than in PG protoplasts, which was even more evident at acidic pH of the external medium. Therefore, based on the measured characteristics, the I_{K+ in} of PT protoplasts may contribute to the endogenous K⁺ currents surrounding a growing pollen tube. Abbreviations: BS, bath solution; BTP, bis-Tris-propane; MES, 2-N-morpholinoethane sulfonic acid; V_act, activation voltage; V_M, membrane voltage; E_rev, reversal potential; I_{K+ in}, inward K⁺ current; I_{K+ out}, outward K⁺ current; PG, pollen grain; PT, pollen tube; PM, pipette medium     

Cytoplasmic calcium affects the gating of potassium channels in the plasma membrane ofChara corallina: a whole-cell study using calcium-channel effectors

The action of a wide range of drugs effective on Ca²⁺ channels in animal tissues has been measured on Ca²⁺ channels open during the action potential of the giant-celled green alga,Chara corallina. Of the organic effectors used, only the 1,4-dihydropyridines were found to inhibit reversibly Ca²⁺ influx, including, unexpectedly, Bay K 8644 and both isomers of 202–791. Methoxyverapamil (D-600), diltiazem, and the diphenylbutylpiperidines, fluspirilene and pimozide were found not to affect the Ca²⁺ influx. Conversely, bepridil greatly and irreversibly stimulated Ca²⁺ influx, and with time, stopped cytoplasmic streaming (which is sensitive to increases in cytoplasmic Ca²⁺). By apparently altering the cytoplasmic Ca²⁺ levels with various drugs, it was found that (with the exception of the inorganic cation, La³⁺) treatments likely to lead to an increase in cytoplasmic Ca²⁺ levels caused an increase in the rate of closure of the K⁺ channels. Similarly, treatments likely to lead to a decrease in cytoplasmic Ca²⁺ decreased the rate of K⁺ channel closure. The main effect of bepridil on the K⁺ channels was to increase the rate of voltage-dependent channel closure. The same effect was obtained upon increasing the external concentration of Ca²⁺, but it is likely that this was due to effects on the external face of the K⁺ channel. Addition of any of the 1,4-dihydropyridines had the opposite effect on the K⁺ channels, slowing the rate of channel closure. They sometimes also reduced K⁺ conductance, but this could well be a direct effect on the K⁺ channel; high concentrations (50 to 100 μM) of bepridil also reduced K⁺ conductance. No effect of photon irradiance or of abscisic acid could be consistently shown on the K⁺ channels. These results indicate a control of the gating of K⁺ channels by cytoplasmic Ca²⁺, with increased free Ca²⁺ levels leading to an increased rate of K⁺-channel closure. As well as inhibiting Ca²⁺ channels, it is suggested that La³⁺ acts on a Ca²⁺-binding site of the K⁺ channel, mimicking the effect of Ca²⁺ and increasing the rate of channel closure.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Regulation by GTP of a Ca2+-activated K+ channel in the apical membrane of rabbit cortical collecting duct cells

Ca²⁺-activated K⁺ channels play an important role in Ca²⁺ signal transduction and may be regulated by mechanisms other than a direct effect of Ca²⁺. Inside-out patches of the apical membrane of confluent transformed rabbit cortical collecting duct cells cultured on collagen were subjected to patch clamp analysis. Two types of K⁺ channel, of medium and high conductance, were observed. The latter channel was characterized by a K⁺/Na⁺ permeability ratio of 10, an inwardly rectified current, a conductance of 80 pS at 0 mV, and an open probability dependent on both voltage and Ca²⁺. Guanosine 5-triphosphate (GTP) but not a guanosine 5-diphosphate (GDP) analogue, adenosine 5-triphosphate (ATP), cytidine 5-triphosphate (CTP), or inosine 5-triphosphate (ITP), inhibited the activity of this Ca²⁺-activated K⁺ channel. The inhibitory effect of GTP was dose dependent, with a 50% inhibitory concentration of 10^–5 m in the absence of Mg²⁺. In the presence of Mg²⁺ (1 mm), which is required for the binding of GTP to G proteins, the 50% inhibitory concentration decreased to 3×10^–12 m. Pertussis toxin or cholera toxin (each at 10 ng/ml) did not prevent the inhibitory effect of GTP. After removal of GTP from the medium bathing an inhibited channel, subsequent application of Ca²⁺ failed to activate the channel. Ca²⁺-activated K⁺ channels of smooth muscle cells and proximal tubule cells did not respond to GTP. Thus, the Ca²⁺-activated K⁺ channel in the apical membrane of collecting duct cells is inhibited by GTP, which appears to exert its effect via a G protein that is insensitive to both cholera and pertussis toxins.     

中华大蟾蜍卵母细胞质膜存在钙依赖性的氯离子通道

本文采用双微电极电压钳方法研究了中华大蟾蜍卵母细胞内源性电压门控型离子通道的成分及其生理特性。卵母细胞去极化至 -30 mV 及更正电压时,有一持续的电压依赖性外向电流出现。钾离子通道拮抗剂四乙基氯化氨(tetraethy-lammonium chloride, TEA, 10 mmol/L)和 4- 氨基吡啶(4-aminopyridine, 4-AP, 10 mmol/L)协同作用时,该电流只能被抑制到最大电流幅度的(23.4±0.72)%。但是,上述浓度的TEA和4-AP 与氯离子通道拮抗剂5- 硝基-2, 3- 苯酚丙胺苯甲酸盐 (5-nitro-2,3-phenypropylamino benzoate, NPPB, 30 μmol/L)、无钙 Ringer 氏液或钙离子通道拮抗剂维拉帕米(40 μmol/L)协同作用时,可分别将此外向电流抑制到最大电流幅度的(2.1±0.08)%、(2.2±0.04)% 和(3.1±0.15)%。结果表明,中华大蟾蜍卵母细胞质膜上除有钾离子电流之外,还存在钙依赖性的氯离子电流。     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

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

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

Ion-conducting channels in a Gram-positive bacterium

The patch-clamp technique was used to obtain information on the existence and properties of ion channels in giant protoplasts obtained from the Gram-positive bacterium Streptococcus faecalis. The membrane proved to contain a pore with numerous conductance states, ranging from 10 pS to several nanosiemens. Application of a slight pressure differential across the membrane resulted in the activation of the channel. The pressure sensitivity points to a relationship between this channel and one recently discovered in E.coli spheroplasts [(1987) Proc. Natl. Acad. Sci. USA 84, 2297–2301] suggesting that pores of this type might be widespread among prokaryotes.     

Ionic channels,ion transport and plant cell membranes: Potential applications of the patch-clamp technique

Conclusion Exciting innovations in the methodologies available for the study of ionic channels (notably in animal cells) have allowed hitherto impossible advances in the comprehension of both structure and function. In using channels like the Na channel and the AChR as examples of these strategies, we have tried to give a concise but up to date account of the current possibilities (in particular, the patch-clamp) for research in membrane physiology. That few of these techniques have been applied to plant cell membranes simply indicates the scope for advancement in the understanding of some problems fundamental to plant physiology. The mechanisms of transport involved in processes known to be important for the life of plant cells (e.g., regulation of cytoplasmic and vacuolar potential differences and pH, maintenance of vacuolar turgor pressure, accumulation of metabolites and their counterions, response to environmental stimuli) are relatively speaking, poorly characterized. In that ion fluxes through plasmalemma and tonoplast membranes are at least in part likely to be via ionic channels for all of these processes, an important step forward would be the application of patch-clamp techniques for the direct demonstration of a channel mechanism and the subsequent elucidation of their role.     

Salt-tolerant ATPase activity in the plasma membrane of the marine angiosperm Zostera marina L

Plasma membrane (PM) H(+)-ATPase and H(+) transport activity were detected in PM fractions prepared from Zostera marina (a seagrass), Vallisneria gigantea (a freshwater grass) and Oryza sativa (rice, a terrestrial plant). The properties of Z. marina PM H(+)-ATPase, specifically, the optimal pH for ATPase activity and the result of trypsin treatment, were similar to those of authentic PM H(+)-ATPases in higher plants. In V. gigantea and O. sativa PM fractions, vanadate-sensitive (P-type) ATPase activities were inhibited by the addition of NaCl. In contrast, activity in the Z. marina PM fraction was not inhibited. The nitrate-sensitive (V-type) and azide-sensitive (F-type) ATPase activities in the Z. marina crude microsomal fraction and the cytoplasmic phosphoenolpyruvate carboxylase activity, however, were inhibited by NaCl, indicating that not all enzyme activities in Z. marina are insensitive to salt. Although the ratio of Na(+) to K(+) (Na(+)/K(+)) in seawater is about 30, Na(+)/K(+) in the Z. marina cells was about 1.0. The salt-tolerant ATPase activity in the plasma membrane must play an important role in maintaining a low Na(+) concentration in the seagrass cells.     

Hodgkin-Huxley analysis of a GCAC1 anion channel in the plasma membrane of guard cells

A quantitative analysis of the time- and voltage-dependent kinetics of the guard cell anion channel (GCAC1) current in guard cell protoplasts from Vicia faba was analyzed using the whole-cell patch clamp technique. The voltage-dependent steady-state activation of GCAC1 current followed a Boltzmann distribution. For the corresponding steady-state value of the activation variable a power of two was derived which yielded suitable fits of the time course of voltage-dependent current activation. The GCAC1 mediated chloride current could successfully be described in terms of the Hodgkin-Huxley equations commonly evoked for the Na channel in nerve. After step depolarizations from a potential in the range of the resting potential to potentials above the equilibrium potential for chloride an activation and also an inactivation could be described. The gating of both processes exhibited an inverse relationship on the polarity of the applied step potentials in the order of milliseconds. Deactivating tail currents decline exponentially. The presented analysis contributes to the understanding of the rising phase of the observed action potentials in guard cells of V. faba. Evidence is presented that the voltage-dependent kinetic properties of the GCAC1 current are different from those properties described for the excitable anion currents in the plasmalemma of Chara corallina (Beilby & Coster, 1979a).The authors gratefully acknowledge the encouragement of Dr. David Colquhoun to apply the Hodgkin-Huxley model to the GCAC1 channel. The work was in part supported by a grant of the Deutsche Forschungsgemeinschaft to R.H. and a grant of the Herman and Lilly Schilling Stiftung to H.-A.K.     

Outward K+ channels in Brassica chinensis pollen protoplasts are regulated by external and internal pH

Summary.  Patch-clamp whole-cell and single-channel recording techniques were used to investigate the regulation of outward K⁺ channels by external and internal protons in Brassica chinensis pollen protoplasts. Outward K⁺ currents and conductance were insensitive to external pH (pH_o) except at pH 4.5. Maximal conductance (G _max) for the outward K⁺ currents was inhibited at acidic external pH. Half-activation voltage (E _1/2) for the outward K⁺ currents shifted to more positive voltages along with the decrease in pH_o. E _1/2 can be described by a modified Henderson–Hasselbalch equation expected from a single titratable binding site. The activation kinetics of the outward K⁺ channels was largely insensitive to pH_o. An internal pH (pH_i) of 4.5 significantly increased outward K⁺ currents and conductance. G _max for the outward K⁺ currents decreased with elevations in pH_i. In contrast to the effect of pH_o, E _1/2 was shifted to more positive voltages with elevations in pH_i. The outward K⁺ currents, G _max and E _1/2 can be described by the modified Henderson–Hasselbalch equation. Furthermore, acidifying pH_i accelerated the activation of the outward K⁺ currents significantly. The differences in electro-physiological properties among previously reported and currently described plant outward K⁺ channels may reflect differences in the structure of these channels. Received May 7, 2002; accepted July 9, 2002; published online November 29, 2002     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.