Functional characterisation of LKT1, a K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly rectifying K+ channel SKT1 after expression in Xenopus oocytes

A cDNA encoding a novel inwardly rectifying potassium (K⁺ _in) channel, LKT1, was cloned from a root-hair-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The LKT1 mRNA was shown to be most strongly expressed in root hairs by Northern blot analysis. The LKT1 channel is a member of the AKT family of K⁺ _in channels previously identified in Arabidopsis thaliana (L.) Heynh. and potato (Solanum tuberosum L.). Moreover, LKT1 is closely related (97% identical amino acids) to potato SKT1. An electrophysiological comparison of the two channels should therefore assist the identification of possible molecular bases for functional differences. For this comparison, both channels were functionally expressed and electrophysiologically characterised within the same expression system, i.e. Xenopus laevis oocytes. Voltage-clamp measurements identified LKT1 as a K⁺-selective inward rectifier which activates with slow kinetics upon hyperpolarising voltage pulses to potentials more negative than −50 mV. The activation potential of LKT1 is shifted towards positive potentials with respect to SKT1 which might be due to single amino acid exchanges in the rim of the channel's pore region or in the S4 domain. Like SKT1, LKT1 reversibly activated upon shifting the external pH from 6.6 to 5.5, which indicates a physiological role for pH-dependent regulation of AKT-type K⁺ _in channels. The pharmacological inhibitor Cs⁺, applied externally, inhibited K⁺ _in currents mediated by LKT1 and SKT1 half-maximally with a concentration (IC₅₀) of 21 μM and 17 μM, respectively. In conclusion, LKT1 may serve as a low-affinity influx pathway for K⁺ into root hair cells. Comparison of homologous K⁺ _in rectifiers from different plant species expressed in the same heterologous system allows conclusions to be drawn in respect to structure-function relationships. Received: 3 August 1999 / Accepted: 2 November 1999     

TOK homologue in Neurospora crassa: first cloning and functional characterization of an ion channel in a filamentous fungus

In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K⁺ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K⁺ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K⁺ efflux. NcTOKA channel gating was sensitive to extracellular K⁺ such that channel activation was dependent on the reversal potential for K⁺. However, expression of NcTOKA was able to overcome the K⁺ auxotrophy of a yeast mutant missing the K⁺ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K⁺ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K⁺ currents at potentials negative of E_K. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca²⁺, verapamil, quinine, and TEA⁺ but was insensitive to Cs⁺, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.     

Properties of an Inwardly Rectifying ATP-sensitive K+ Channel in the Basolateral Membrane of Renal Proximal Tubule

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na⁺,K⁺-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K⁺ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K⁺ channels (γ_{slope, in} = 24.5 ± 0.6 pS, γ_{chord, out} = 3.7 ± 0.4 pS). The rectification is mediated in part by internal Mg²⁺. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding–unbinding model. The channel conducts Tl⁺ and K⁺, but there is no significant conductance for Na⁺, Rb⁺, Cs⁺, Li⁺, NH₄⁺, or Cl⁻. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0.2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K⁺ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K⁺ channel is the major ATP-sensitive basolateral K⁺ conductance in the proximal tubule.     

Pharmacological dissection and distribution of NaN/Nav1.9, T-type Ca2+ currents, and mechanically activated cation currents in different populations of DRG neurons

Low voltage–activated (LVA) T-type Ca²⁺ (I_CaT) and NaN/Nav1.9 currents regulate DRG neurons by setting the threshold for the action potential. Although alterations in these channels have been implicated in a variety of pathological pain states, their roles in processing sensory information remain poorly understood. Here, we carried out a detailed characterization of LVA currents in DRG neurons by using a method for better separation of NaN/Nav1.9 and I_CaT currents. NaN/Nav1.9 was inhibited by inorganic I_Ca blockers as follows (IC₅₀, μM): La³⁺ (46) > Cd²⁺ (233) > Ni²⁺ (892) and by mibefradil, a non-dihydropyridine I_CaT antagonist. Amiloride, however, a preferential Cav3.2 channel blocker, had no effects on NaN/Nav1.9 current. Using these discriminative tools, we showed that NaN/Nav1.9, Cav3.2, and amiloride- and Ni²⁺-resistant I_CaT (AR-I_CaT) contribute differentially to LVA currents in distinct sensory cell populations. NaN/Nav1.9 carried LVA currents into type-I (CI) and type-II (CII) small nociceptors and medium-Aδ–like nociceptive cells but not in low-threshold mechanoreceptors, including putative Down-hair (D-hair) and Aα/β cells. Cav3.2 predominated in CII-nociceptors and in putative D-hair cells. AR-I_CaT was restricted to CII-nociceptors, putative D-hair cells, and Aα/β-like cells. These cell types distinguished by their current-signature displayed different types of mechanosensitive channels. CI- and CII-nociceptors displayed amiloride-sensitive high-threshold mechanical currents with slow or no adaptation, respectively. Putative D-hair and Aα/β-like cells had low-threshold mechanical currents, which were distinguished by their adapting kinetics and sensitivity to amiloride. Thus, subspecialized DRG cells express specific combinations of LVA and mechanosensitive channels, which are likely to play a key role in shaping responses of DRG neurons transmitting different sensory modalities.     

Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δ strain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K⁺ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K⁺ and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K⁺ with Rb⁺, Cs⁺, or Na⁺ only slightly modulated the magnitude of the inward current, whereas replacement with Li⁺ reduced the inward current by approximately 50%, and tetraethylammonium (TEA⁺) and choline were relatively impermeant. The inward current was blocked by extracellular Ca²⁺ and Mg²⁺ with apparent K_is (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K⁺ increased the magnitude of the inward current independently of the electrochemical driving force for K⁺ influx, consistent with regulation of the inward current by cytosolic K⁺. Uptake of ⁸⁶Rb⁺ by intact trk1Δ trk2Δ cells was inhibited by extracellular Ca²⁺ with a K_i within the range observed for the inward current. Furthermore, increasing extracellular Ca²⁺ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.     

Evaluation of functional interaction between K(+) channel alpha- and beta-subunits and putative inactivation gating by Co-expression in Xenopus laevis oocytes

Animal K⁺ channel α- (pore-forming) subunits form native proteins by association with β-subunits, which are thought to affect channel function by modifying electrophysiological parameters of currents (often by inducing fast inactivation) or by stabilizing the protein complex. We evaluated the functional association of KAT1, a plant K⁺ channel α-subunit, and KAB1 (a putative homolog of animal K⁺ channel β-subunits) by co-expression in Xenopus laevis oocytes. Oocytes expressing KAT1 displayed inward-rectifying, non-inactivating K⁺ currents that were similar in magnitude to those reported in prior studies. K⁺ currents recorded from oocytes expressing both KAT1 and KAB1 had similar gating kinetics. However, co-expression resulted in greater total current, consistent with the possibility that KAB1 is a β-subunit that stabilizes and therefore enhances surface expression of K⁺ channel protein complexes formed by α-subunits such as KAT1. K⁺ channel protein complexes formed by α-subunits such as KAT1 that undergo (voltage-dependent) inactivation do so by means of a “ball and chain” mechanism; the ball portion of the protein complex (which can be formed by the N terminus of either an α- or β-subunit) occludes the channel pore. KAT1 was co-expressed in oocytes with an animal K⁺ channel α-subunit (hKv1.4) known to contain the N-terminal ball and chain. Inward currents through heteromeric hKv1.4:KAT1 channels did undergo typical voltage-dependent inactivation. These results suggest that inward currents through K⁺ channel proteins formed at least in part by KAT1 polypeptides are capable of inactivation, but the structural component facilitating inactivation is not present when channel complexes are formed by either KAT1 or KAB1 in the absence of additional subunits.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

The K+ channel SKT1 is co-expressed with KST1 in potato guard cells--both channels can co-assemble via their conserved KT domains

An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation.     

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.     

The Inhibitory Effects of Ca2+ Channel Blocker Nifedipine on Rat Kv2.1 Potassium Channels

It is well documented that nifedipine, a commonly used dihydropyridine Ca²⁺ channel blocker, has also significant interactions with voltage-gated K⁺ (Kv) channels. But to date, little is known whether nifedipine exerted an action on Kv2.1 channels, a member of the Shab subfamily with slow inactivation. In the present study, we explored the effects of nifedipine on rat Kv2.1 channels expressed in HEK293 cells. Data from whole-cell recording showed that nifedipine substantially reduced Kv2.1 currents with the IC₅₀ value of 37.5 ± 5.7 μM and delayed the time course of activation without effects on the activation curve. Moreover, this drug also significantly shortened the duration of inactivation and deactivation of Kv2.1 currents in a voltage-dependent manner. Interestingly, the half-maximum inactivation potential (V _1/2) of Kv2.1 currents was -11.4 ± 0.9 mV in control and became -38.5 ± 0.4 mV after application of 50 μM nifedipine. The large hyperpolarizing shift (27 mV) of the inactivation curve has not been reported previously and may result in more inactivation for outward delayed rectifier K⁺ currents mediated by Kv2.1 channels at repolarization phases. The Y380R mutant significantly increased the binding affinity of nifedipine to Kv2.1 channels, suggesting an interaction of nifedipine with the outer mouth region of this channel. The data present here will be helpful to understand the diverse effects exerted by nifedipine on various Kv channels.     

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a “shutter-like” mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.     

Regulation of K+ Channels in Maize Roots by Water Stress and Abscisic Acid

Root cortical and stelar protoplasts were isolated from maize (Zea mays L.) plants that were either well watered or water stressed, and the patch-clamp technique was used to investigate their plasma membrane K⁺ channel activity. In the root cortex water stress did not significantly affect inward- or outward-rectifying K⁺ conductances relative to those observed in well-watered plants. In contrast, water stress significantly reduced the magnitude of the outward-rectifying K⁺ current in the root stele but had little effect on the inward-rectifying K⁺ current. Pretreating well-watered plants with abscisic acid also significantly affected K⁺ currents in a way that was consistent with abscisic acid mediating, at least in part, the response of roots to water stress. It is proposed that the K⁺ channels underlying the K⁺ currents in the root stelar cells represent pathways that allow K⁺ exchange between the root symplasm and xylem apoplast. It is suggested that the regulation of K⁺ channel activity in the root in response to water stress could be part of an important adaptation of the plant to survive drying soils.     

P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ∼3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Regulation of the Fast Vacuolar Channel by Cytosolic and Vacuolar Potassium

At resting cytosolic Ca²⁺, passive K⁺ conductance of a higher plant tonoplast is likely dominated by fast vacuolar (FV) channels. This patch-clamp study describes K⁺-sensing behavior of FV channels in Beta vulgaris taproot vacuoles. Variation of K⁺ between 10 and 400 mM had little effect on the FV channel conductance, but a pronounced one on the open probability. Shift of the voltage dependence by cytosolic K⁺ could be explained by screening of the negative surface charge with a density σ = 0.25 e⁻/nm². Vacuolar K⁺ had a specific effect on the FV channel gating at negative potentials without significant effect on closed-open transitions at positive ones. Due to K⁺ effects at either membrane side, the potential at which the FV channel has minimal activity was always situated at ~50 mV below the potassium equilibrium potential, E_K⁺. At tonoplast potentials below or equal to E_K⁺, the FV channel open probability was almost independent on the cytosolic K⁺ but varied in a proportion to the vacuolar K⁺. Therefore, the release of K⁺ from the vacuole via FV channels could be controlled by the vacuolar K⁺ in a feedback manner; the more K⁺ is lost the lower will be the transport rate.     

Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

TRPC3 (or Htrp3) is a human member of the trp family of Ca²⁺-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca²⁺ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca²⁺ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca²⁺ over Na⁺. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca²⁺ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca²⁺. Likewise, infusion of Ca²⁺ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca²⁺-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca²⁺ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca²⁺-permeable channel that supports Ca²⁺-induced Ca²⁺-entry but should not be considered store operated.     

Rat Merkel Cells Are Mechanoreceptors and Osmoreceptors

Merkel cells (MCs) associated with nerve terminals constitute MC-neurite complexes, which are involved in slowly-adapting type I mechanoreception. Although MCs are known to express voltage-gated Ca²⁺ channels and hypotonic-induced membrane deformation is known to lead to Ca²⁺ transients, whether MCs initiate mechanotransduction is currently unknown. To answer to this question, rat MCs were transfected with a reporter vector, which enabled their identification. Their properties were investigated through electrophysiological studies. Voltage-gated K⁺, Ca²⁺ and Ca²⁺-activated K⁺ (K_Ca) channels were identified, as previously described. Here, we also report the activation of Ca²⁺ channels by histamine and their inhibition by acetylcholine. As a major finding, we demonstrated that direct mechanical stimulations induced strong inward Ca²⁺ currents in MCs. Depolarizations were dependent on the strength and the length of the stimulation. Moreover, touch-evoked currents were inhibited by the stretch channel antagonist gadolinium. These data confirm the mechanotransduction capabilities of MCs. Furthermore, we found that activation of the osmoreceptor TRPV4 in FM1-43-labeled MCs provoked neurosecretory granule exocytosis. Since FM1-43 blocks mechanosensory channels, this suggests that hypo-osmolarity activates MCs in the absence of mechanotransduction. Thus, mechanotransduction and osmoreception are likely distinct pathways.     

A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K⁺ channel subunit that assembles with different inward-rectifying K⁺ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K⁺ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K⁺ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K⁺ current in Arabidopsis root epidermal protoplasts as well as K⁺ acquisition and growth in seedlings when channel-mediated K⁺ uptake was limiting. That SYP121 should be important for gating of a K⁺ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K⁺ uptake coordinately with membrane expansion for cell growth.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

Background

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell''s diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents.

Methodology/Principal Findings

The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K⁺ and Cl⁻ currents were strictly dependent on the presence of Ca²⁺. The Ca²⁺-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca²⁺ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca²⁺ permeability pathway leading to increased [Ca²⁺]_i, secondary activation of Ca²⁺-sensitive K⁺ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents.

Conclusions/Significance

The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca²⁺-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca²⁺ content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.