Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents 相似文献   

In Intact Cone Photoreceptors,a Ca2+-dependent,Diffusible Factor Modulates the cGMP-gated Ion Channels Differently than in Rods

We investigated the modulation of cGMP-gated ion channels in single cone photoreceptors isolated from a fish retina. A new method allowed us to record currents from an intact outer segment while controlling its cytoplasmic composition by superfusion of the electropermeabilized inner segment. The sensitivity of the channels to agonists in the intact outer segment differs from that measured in membrane patches detached from the same cell. This sensitivity, measured as the ligand concentration necessary to activate half-maximal currents, K _1/2, also increases as Ca²⁺ concentration decreases. In electropermeabilized cones, K _1/2 for cGMP is 335.5 ± 64.4 μM in the presence of 20 μM Ca²⁺, and 84.3 ± 12.6 μM in its absence. For 8Br-cGMP, K _1/2 is 72.7 ± 11.3 μM in the presence of 20 μM Ca²⁺ and 15.3 ± 4.5 μM in its absence. The Ca²⁺-dependent change in agonist sensitivity is larger in extent than that measured in rods. In electropermeabilized tiger salamander rods, K _1/2 for 8Br-cGMP is 17.9 ± 3.8 μM in the presence of 20 μM Ca²⁺ and 7.2 ± 1.2 μM in its absence. The Ca²⁺-dependent modulation is reversible in intact cone outer segments, but is progressively lost in the absence of divalent cations, suggesting that it is mediated by a diffusible factor. Comparison of data in intact cells and detached membrane fragments from cones indicates that this factor is not calmodulin. At 40 μM 8Br-cGMP, the Ca²⁺-dependent change in sensitivity in cones is half-maximal at K _Ca = 286 ± 66 nM Ca²⁺. In rods, by contrast, K _Ca is ∼50 nM Ca²⁺. The difference in magnitude and Ca²⁺ dependence of channel modulation between photoreceptor types suggests that this modulation may play a more significant role in the regulation of photocurrent gain in cones than in rods.     

Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors

We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

Permeability and interaction of Ca2+ with cGMP-gated ion channels differ in retinal rod and cone photoreceptors.

We studied the ionic permeability of cGMP-dependent currents in membrane patches detached from the outer segment of retinal cone and rod photoreceptors. Reversal potentials measured in membranes exposed to symmetric Na+ but with varying cytoplasmic Ca2+ concentrations reveal that the permeability ratio, PCa/PNa, is higher in the cGMP-gated channels of cones (7.6 +/- 0.8) than in those of rods (3.1 +/- 1.0). Ca2+ blocks both channels in a voltage-dependent manner. At any Ca2+ concentration, the channel block is maximal near the ionic reversal potential. The maximal block is essentially identical in channels of cones and rods with respect to its extent and voltage and Ca2+ dependence. The Ca2+ block is relieved by voltage, but the features of this relief differ markedly between rods and cones. Whereas the Boltzmann distribution function describes the relief of block by hyperpolarizing voltages, any given voltage is more effective in relieving the Ca2+ block in cones than in rods. Similarly, depolarizing voltages more effectively relieve Ca2+ block in cones than in rods. Our results suggest that channels contain two binding sites for Ca2+, one of which is similar in the two receptor types. The second site either interacts more strongly with Ca2+ than the first one or it is located differently in the membrane, so as to be less sensitive to membrane voltage. The channels in rods and cones differ in the features of this second site. The difference in Ca2+ permeability between the channels is likely to result in light-dependent changes in cytoplasmic Ca2+ concentration that are larger and faster in cones than in rods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent K+ channels from rat olfactory cilia characterized in planar lipid bilayers

Olfactory cilia contain cyclic nucleotide-gated and Ca2+-dependent Cl- conductances that underlie excitatory chemotransduction, and a Ca2+-dependent K+ (KCa) conductance, apparently involved in inhibitory transduction. Previous single-channel patch-clamp studies on olfactory cilia revealed four different KCas, with different conductances and kinetics. Here, we further characterized these channels in planar bilayers, where blockers could be properly tested. All four ciliary KCas were observed: The 16 pS channel, K0.5,Ca=40 microM and apamin-sensitive; the 30 and 50 pS channel, K0.5,Ca=59 microM, clotrimazole-sensitive and charybdotoxin-insensitive; the 60 pS channel, clotrimazole-sensitive and charybdotoxin-insensitive; and the 210 pS channel, K0.5,Ca=63 microM, blocked by charybdotoxin and iberiotoxin. The presence of the 16 and 210 pS channels was confirmed by immunoblotting.     

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction

The correct spatial and temporal control of Ca²⁺ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca²⁺ concentration. In this review we provide an updated view of how caveolae may regulate both Ca²⁺ entry into cells and Ca²⁺-dependent signal transduction     

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.     

Identification of putative voltage-dependent Ca2+-permeable channels involved in cryptogein-induced Ca2+ transients and defense responses in tobacco BY-2 cells

Ca(2+) is the pivotal second messenger for induction of defense responses induced by treatment of pathogen-derived elicitor or microbial infection in plants. However, molecular bases for elicitor-induced generation of Ca(2+) signals (Ca(2+) transients) are largely unknown. We here identified cDNAs for putative voltage-dependent Ca(2+)-permeable channels, NtTPC1A and NtTPC1B, that are homologous to TPC1 (two pore channel) from suspension-cultured tobacco BY-2 cells. NtTPC1s complemented the growth of a Saccharomyces cerevisiae mutant defective in CCH1, a putative Ca(2+) channel, in a low Ca(2+) medium, suggesting that both products permeate Ca(2+) through the plasma membrane. Cosuppression of NtTPC1s in apoaequorin-expressing BY-2 cells resulted in inhibition of rise in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in response to sucrose and a fungal elicitor cryptogein, while it did not affect hypoosmotic shock-induced [Ca(2+)](cyt) increase. Cosuppression of NtTPC1s also caused suppression of cryptogein-induced programmed cell death and defense-related gene expression. These results suggest that NtTPC1s are involved in Ca(2+) mobilization induced by the cryptogein and sucrose, and have crucial roles in cryptogein-induced signal transduction pathway.     

Making sense out of Ca2+ signals: their role in regulating stomatal movements

Plant cells maintain high Ca²⁺ concentration gradients between the cytosol and the extracellular matrix, as well as intracellular compartments. During evolution, the regulatory mechanisms, maintaining low cytosolic free Ca²⁺ concentrations, most likely provided the backbone for the development of Ca²⁺‐dependent signalling pathways. In this review, the current understanding of molecular mechanisms involved in Ca²⁺ homeostasis of plants cells is evaluated. The question is addressed to which extent the mechanisms, controlling the cytosolic Ca²⁺ concentration, are linked to Ca²⁺‐based signalling. A large number of environmental stimuli can evoke Ca²⁺ signals, but the Ca²⁺‐induced responses are likely to differ depending on the stimulus applied. Two mechanisms are put forward to explain signal specificity of Ca²⁺‐dependent responses. A signal may evoke a specific Ca²⁺ signature that is recognized by downstream signalling components. Alternatively, Ca²⁺ signals are accompanied by Ca²⁺‐independent signalling events that determine the specificity of the response. The existence of such parallel‐acting pathways explains why guard cell responses to abscisic acid (ABA) can occur in the absence, as well as in the presence, of Ca²⁺ signals. Future research may shed new light on the relation between parallel acting Ca²⁺‐dependent and ‐independent events, and may provide insights in their evolutionary origin.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.     

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.     

Cytoplasmic Ca2+ does not inhibit the cardiac muscle sarcoplasmic reticulum ryanodine receptor Ca2+ channel,although Ca2+-induced Ca2+ inactivation of Ca2+ release is observed in native vesicles

Single channel properties of cardiac and fast-twitch skeletal muscle sarcoplasmic reticulum (SR) release channels were compared in a planar bilayer by fusing SR membranes in a Cs⁺-conducting medium. We found that the pharmacology, Cs⁺ conductance and selectivity to monovalent and divalent cations of the two channels were similar. The cardiac SR channel exhibited multiple kinetic states. The open and closed lifetimes were not altered from a range of 10^–7 to 10^–3 M Ca²⁺, but the proportion of closed and open states shifted to shorter closings and openings, respectively.However, while the single channel activity of the skeletal SR channel was activated and inactivated by micromolar and millimolar Ca²⁺, respectively, the cardiac SR channel remained activated in the presence of high [Ca²⁺]. In correlation to these studies, [³H]ryanodine binding by the receptors of the two channel receptors was inhibited by high [Ca²⁺] in skeletal but not in cardiac membranes in the presence of adenine nucleotides. There is, however, a minor inhibition of [³H]ryanodine binding of cardiac SR at millimolar Ca²⁺ in the absence of adenine nucleotides.When Ca²⁺-induced Ca²⁺ release was examined from preloaded native SR vesicles, the release rates followed a normal biphasic curve, with Ca²⁺-induced inactivation at high [Ca²⁺] for both cardiac and skeletal SR. Our data suggest that the molecular basis of regulation of the SR Ca²⁺ release channel in cardiac and skeletal muscle is different, and that the cardiac SR channel isoform lacks a Ca²⁺-inactivated site.This work was supported by research grants from the National Institutes of Health HL13870 and AR38970, and the Texas Affiliate of the American Heart Association, 91A-188. M. Fill was the recipient of an NIH fellowship AR01834.     

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

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

钙-钙调素信号系统参与热激信号转导的研究

根据作者实验室的研究工作结合国内外的研究动态讨论热激信号转导的Ca2 -CaM途径。作者实验室的工作表明,钙一钙调素(Ca^2 -CaM)信号系统参与植物热激信号转导。激光共聚焦扫描显微镜的观察结果表明,37℃热激可引起小麦胞内自由Ca。’浓度迅速提高。在Ca^2 存在条件下,热激也引起小麦CaM基因CaM1-2表达及CaM蛋白含量增加。Ca^2 可促进小麦热激基因hsp26和mp70表达和热激蛋白合成,而Ca^2 螯合剂EGTA、Ca^2 通道阻断剂异搏定和LaCl3、CaM抑制剂W7、TFP和CPZ明显降低热激基因hsp26和mp70表达和热激蛋白合成。EGTA、异搏定、TFP或CPZ也阻止小麦耐热性的获得。小麦CaM基因与热激基因的表达动力学研究表明CaM位于热激信号转导的上游,而Ca^2 是启动热激反应的胞内关键因子。凝胶阻滞分析的结果表明,Ca^2 -CaM在热激信号转导中的作用是通过激活热激转录因子的DNA结合活性来实现的。根据大量实验证据,作者提出在植物细胞内存在一条新的热激信号转导途径——钙一钙调素途径。     

Possible Ca2+-dependent mechanism of apical outer hair cell modulation within the cochlea of the guinea pig

Calcium ions were precipitated with potassium antimonate after injection of the inorganic calcium channel blocker MnCl₂ or the inorganic potassium channel blockers BaCl₂ or CsCl into the perilymph of the scala vestibuli of the guinea pig. The spatial distribution of the formed histochemical reaction products within the organ of Corti was studied by energy-filtering transmission-electron microscopy. Compared with untreated control ears, the number of the formed precipitates drastically increased at the extracellular side of the lamina reticularis after application of the various inorganic channel blockers. The apical side of the outer hair cells and the intervening Deiter cells were covered by a thick layer of calcium precipitates, whereas the number of histochemical reaction products was clearly reduced in the nearby acellular tectorial membrane. The high calcium content within the formed reaction products at the lamina reticularis could be demonstrated by elemental mapping and by electron energy-loss spectroscopy. To ascertain the alterations in the amounts of the calcium precipitates within the tectorial membrane after application of the various inorganic channel blockers, the precipitate densities were determined semiquantitatively by an image processing system and the values obtained compared with those of untreated control specimens. The observed histochemical results are in good agreement with published electrophysiological findings concerning the spatial distribution of ion channels located at the apical outer hair cell membrane. The detected alterations in the spatial distribution of calcium precipitates might correspond to calcium-dependent processes involved in outer hair cell modulation. Received: 4 November 1996 / Accepted: 28 October 1997     

Slo1 tail domains,but not the Ca2+ bowl,are required for the beta 1 subunit to increase the apparent Ca2+ sensitivity of BK channels

Functional large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels can be assembled from four alpha subunits (Slo1) alone, or together with four auxiliary beta1 subunits to greatly increase the apparent Ca(2+) sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the alpha subunit, which includes the RCK2 (regulator of K(+) conductance) domain and Ca(2+) bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca(2+) bowl and high affinity Ca(2+) sensitivity. In the second, the Ca(2+) bowl was disrupted by mutations that greatly reduce the apparent Ca(2+) sensitivity. We found that the beta1 subunit increased the apparent Ca(2+) sensitivity of Slo1 channels, independently of whether the alpha subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca(2+) bowl was mutated. In contrast, beta1 subunits no longer increased Ca(2+) sensitivity when Slo1 tails were replaced by Slo3 tails. The beta1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 beta-estradiol activated these channels in the presence of beta1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca(2+) sensitivity induced by the beta1 subunit does not require either the Ca(2+) bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The beta1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the beta1 subunit-induced increase in apparent Ca(2+) sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the beta1 subunit may be different.     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.     

The role of anion channels and Ca2+ in addition to K+ channels in the physiological volume regulation of murine spermatozoa

Studies in the human, transgenic mice, and cattle indicate that sperm cell volume regulation plays an important role in male fertility as spermatozoa encounter a hypo-osmotic challenge upon ejaculation into the female tract. Physiological regulatory volume decrease (RVD) was examined using flow cytometry in murine sperm released into incubation medium mimicking uterine osmolality and including putative channel inhibitors. The involvement of K+ channels was indicated by the recovery of volume regulation by the K+ ionophore valinomycin in defective sperm from infertile transgenic mice, and from blockage of RVD by quinine in normal sperm. However, in neither case was the recovery complete. The involvement of volume-sensitive osmolyte and anion channels (VSOAC) were investigated using blockers effective in other cell types. NPPB (5-nitro-2(3-phenylpropylamino) benzoic acid) and tamoxifen inhibited RVD but SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid) at 0.4 and 1 mM had no effect whereas DIDS (di-isothiocyanato-stilbene-2,2'-disulphonic acid) at 1 mM enhanced RVD. Verapamil, but not another P-glycoprotein antagonist cyclosporin, caused sperm swelling which persisted in the presence of valinomycin, in Ca2+-free medium and in the presence of thapsigargin, but swelling was abolished by the Ca2+ ionophore A23187. Nifedipine was slightly effective in blocking RVD. Analysis by Western blotting failed to reveal ClC-2 and ClC-3 members of the chloride channel family in murine or rat sperm proteins despite signal bands in positive tissue controls. These findings implicate the involvement of some unidentified VSOAC in sperm volume regulation, which is probably Ca+-dependent.