Longer Mean Open Times Research Articles

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

Gating by Voltage and Ca2+ in Human Connexin (cx26) Hemichannels

Opening of connexin hemichannels permits the release of small metabolites, such as ATP and glutamate, which play an important autocrine/paracrine signaling in a variety of cell types. The recently solved crystal structure of the Cx26 gap junction channel allows us to explore in greater detail the relationship between the structure and function of both, hemichannels and gap junction channels. Here, we begin by revisiting the activation mechanisms of human Cx26 (hCx26) hemichannels by voltage and Ca2+. Using the two electrode oocyte voltage-clamp technique, we found that depolarization up to +60 mV induced activation of hemichannel currents, and repolarization produced large tail-currents with slow deactivation (τ ∼10 s). Interestingly, the magnitudes of the tail-currents were dependent on the lengths of the depolarizating pulses rather than the magnitudes of the currents activated during the pulses. Strikingly, cell-attached single channel recordings showed that depolarizating pulses (≤40 mV) stimulated only infrequent and brief openings of hCx26 hemichannels. However, the repolarizating pulses induced opening of single hemichannel currents (likely corresponding to the tail currents) with smaller conductance and longer mean open times. In addition, we found that low Ca2+ (500 μM) increased macroscopic hemichannel currents at positive potentials and slowed deactivation of the tail currents. We are currently performing single channel recordings to elucidate how Ca2+ modulates channel activation and deactivation kinetics in response to depolarizing and repolarizing voltages. Our results indicate that while depolarization causes opening of some hCx26 hemichannels, it mostly shifts the hemichannels to a non-conductive state that will likely open after repolarization; and that Ca2+ ions may play a role by regulating the energetics of these transitions. Supported by The Grass Foundation.

Characteristics of a Store-operated Calcium-permeable Channel: SARCOENDOPLASMIC RETICULUM CALCIUM PUMP FUNCTION CONTROLS CHANNEL GATING

We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.

Comparison of spontaneous and noradrenaline-evoked non-selective cation channels in rabbit portal vein myocytes.

The properties of non-selective cation channels were studied in rabbit portal vein smooth muscle cells in K+-free conditions with patch pipette techniques. In about 45 % of isolated outside-out patches spontaneous channel currents with a unitary conductance of 23 pS were observed. The reversal potential was +11 mV which was shifted to more negative values and the unitary conductance was reduced when part of the external Na+ was replaced by Tris. At negative potentials the probability of opening (Po) was low and the open time distributions were described by two exponentials with time constants of about 1 and 7 ms. At positive potentials Po and the longer mean open time were greatly increased. The channel exhibited bursting behaviour and the burst duration distributions were described by two exponentials with time constants of about 3 and 15 ms. At positive potentials the longer burst duration was increased substantially. Application of noradrenaline and the diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to the external membrane of quiescent patches evoked single-channel activity with a unitary conductance of about 23 pS and with similar kinetic behaviour to that of the spontaneous channel currents. In conclusion, noradrenaline and OAG activate non-selective cation channel currents in excised patches of rabbit portal vein myocytes with a unitary conductance and kinetic properties that are similar to those of spontaneous channel currents. These channels have two open states and exhibit bursting behaviour. It is suggested that these channels underlie the whole-cell currents evoked by noradrenaline and OAG.

BK-Type KCaChannels in Two Parasympathetic Cell Types: Differences in Kinetic Properties and Developmental Expression

The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca(2+)-activated K(+) channels (K(Ca)), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic K(Ca) carried by a kinetically homogeneous population of large-conductance (BK-type) K(Ca) channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic K(Ca) produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying K(Ca) channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of K(Ca) channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of K(Ca) channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance K(Ca) channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional K(Ca) channels appears to be regulated differently in the two cell types. Although both cell types acquire functional K(Ca) at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional K(Ca) channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain K(Ca) stimulatory activity. However, K(Ca) stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.

Further Characterization of the Type 3 Ryanodine Receptor (RyR3) Purified from Rabbit Diaphragm

We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (Po), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity with Po approximately 1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity.

Two types of BK channels in immature rat neocortical pyramidal neurons.

1. The properties of large conductance Ca(2+)-activated K+ channels (BK channels) were investigaed in neocortical infragranular pyramidal neurons by the use of inside-out patch recordings. Neurons were acutely isolated from slices of newborn to 28-day-old rats (P0-P28) by using minimal protease exposure followed by trituration with a vibrating glass probe. Two types of BK channels, slow-gating and fast-gating, were observed in immature neurons (P0-P5), whereas only slow-gating BK were found in more mature neurons. Fast-gating BK channels differed in conductance, voltage dependence, and kinetics from the slow-gating ones. 2. The properties of fast-gating channels included a conductance of 145 +/- 12.9 (SE) pS; frequent openings with short mean open times that were relatively voltage-independent, mean closed times that showed a voltage-dependent increase, a voltage-dependent decrease in open probability (Po). The properties of slow-gating channels contrasted with those of the fast-gating ones, in that the former had a conductance of 181 +/- 3.9 pS, longer mean open times that showed a voltage-dependent increase, mean closed times that showed a marked voltage-dependent decrease, a voltage-dependent increase in Po, and slight inward rectification. The significance of these developmental variations in channel properties is discussed.

Selective binding of FKBP12.6 by the cardiac ryanodine receptor.

The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer)4(FKBP12)4 and (RyR2 protomer)4(FKBP12.6)4, respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35S-labeled FKBP12 and 35S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12. 6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

5330911 Surfaces having desirable cell adhesive effects

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992–22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (μM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 μM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

Calcium-activated potassium channels in goldfish hair cells.

1. Characteristics of Ca(2+)-activated K+ channels in the basolateral membrane of hair cells isolated from the caudal part of the goldfish saccular macula were studied mainly with the inside-out mode of the patch clamp method. 2. Several types of Ca(2+)-activated K+ channels differing in unitary conductance were identified. The conductances (n = 156) ranged from 130 to 320 pS (when measured in symmetrical 125 mM KCl) and could be roughly separated into four groups, centred on values of 150, 200, 250 and 300 pS. The pharmacological profile, assessed by, for example, tetraethylammonium blockade, and the relatively large conductance indicated that these channels can be classified as large-conductance Ca(2+)-activated K+ channels (BK channels). The relative permeability of these channels to different ion species was in the order K+ (1.0) > Rb+ (0.8) > NH4+ (0.14) > Na+, Cs+ (< 0.05). 3. Curves relating open state probability to [Ca2+]i, for membrane potentials between -50 and +50 mV, were similar to those observed for BK channels of rat muscle. However, the maximum open state probability (100-1000 microM [Ca2+]i and 50 mV membrane potential) was 0.4-0.9, and always less than 1. 4. These channels had a short arithmetic mean open time ranging from 0.08 to 1.2 ms (0.08-0.5 ms in 88% of cases) and an arithmetic mean shut time ranging from 0.24 to 1.2 ms (10 microM [Ca2+]i and 50 mV membrane potential). The shut intervals were more sensitive to changes in [Ca2+]i and membrane potential than were the open intervals. 5. The distribution of individual open and shut intervals was fitted with the sum of exponential functions. Except for the slowest shut component, which only accounted for less than 1% of shut events, all other components had time constants shorter than 1 ms. As a result of these short open and shut intervals, the current trace had a flickery pattern rather than a burst-interburst pattern. 6. There was a rough correlation between unitary conductance and mean open time, i.e. channels with a large unitary conductance had a longer mean open time. 7. The sensitivity to [Ca2+]i of the Ca(2+)-activated K+ channel in goldfish hair cells was one to two orders of magnitude lower than that of BK channels in rat muscle. Channels with a longer mean open time had a higher Ca2+ sensitivity. 8. The stability of the single Ca(2+)-activated K+ channel kinetics was studied by measuring the 'moving' mean duration of open and shut intervals.(ABSTRACT TRUNCATED AT 400 WORDS)

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: Effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992–22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca 2+ and ryanodine, inhibition by Mg 2+ (mM) and ruthenium red (μM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 μM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

Fast and slow blockades of the inward-rectifier K+ channel by external divalent cations in guinea-pig cardiac myocytes

The present patch-clamp study shows that external Mg2+, Ca2+ and Sr2+ decrease the unit amplitude of inward current through the inward-rectifier K+ channel in a concentration-dependent manner. Sr2+ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr2+ binding site (delta) is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg2+ or Ca2+ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg2+ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs+ block (delta = 1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs+ and Sr2+ bind.

Arginine-vasopressin induces mode-2 gating in L-type Ca2+ channels (smooth muscle cells of the urinary bladder of the guinea-pig).

The effect of arginine-vasopressin (AVP, 0.1 microM) on elementary Ca2+ channel currents (L-type) was studied in cell-attached patches with 10 mM BaCl2 as the charge carrier. At a constant potential of -30 mV, bath applied AVP increased the channel openness (NPo) by a factor of 4.7 +/- 3.0 (mean +/- SD, n = 9), the effect resulted from an increase in the frequency of opening (factor 2.5 +/- 0.8) and from a longer mean open time. Under control, openings longer than 5 ms contributed only 4% of the total, however, with the application of AVP this contribution increased to 29%. Under control, the open times were distributed along a single exponential (tau o1 = 0.8 +/- 0.4 ms), a double exponential distribution was obtained during AVP (tau o1 = 0.8 +/- 0.5 ms, tau o2 = 7.5 +/- 0.7 ms). The Ca2+ agonist BAYk8644 (1 microM) changed the open time distribution similarly to AVP (tau o1 = 1.0 +/- 0.5 ms, tau o2 = 9 +/- 2.8 ms). With 1 microM BAYk8644 in the bath, AVP did not significantly increase the relative contribution of long openings, however, AVP increased the frequency of openings by a factor of 2.0 +/- 1 (n = 6). The results are compatible with the idea that AVP can change the gating of L-type Ca2+ channels from mode 1 to mode 2.

Functional properties of newly inserted acetylcholine receptors in embryonic Xenopus muscle cells

Single-channel properties of nicotinic acetylcholine receptors in Xenopus embryonic muscles were investigated by the patch clamp technique. Dissociated muscle cells were prepared from embryos in early stages of development (stages 18–19) before innervation takes place, and were cultured without neurons for 4–6 days. Despite the absence of innervation in their history, the cells displayed two classes of acetylcholine receptors, one characterized by a small channel conductance (32 pS), and the other by a large conductance (48 pS) (13 °C; agonist, suberyldicholine). The small conductance events had longer mean open times than the large conductance events. Both types of channels had reversal potentials near −15 mV. Hyperpolarization prolonged the open times of both channels; an e-fold change was produced by a 70–80 mV polarization. In order to characterize channel properties of newly inserted receptors, existing receptors were inactivated by α-bungarotoxin, and recordings were made over the next 4–8 h. These new receptors already exhibited the two classes of characteristics, which were similar to those of old acetylcholine receptors. These results suggest that innervation is not a prerequisite for expression of the two classes of acetylcholine receptors, and indicate that receptors become functionally mature soon after insertion into the plasma membrane. These results of ‘metabolically’ new receptors are in sharp contrast with the data of Leonard et al. ( Soc. Neurosci. Abstr., 9 (1983) 1180), who reported that ‘ontogenetically’ new acetylcholine receptors had much longer open times than old receptors.

Single channel currents activated by gamma-aminobutyric acid, muscimol, and (-)-pentobarbital in cultured mouse spinal neurons

The patch electrode technique was used to record single channel current pulses in tissue-cultured mouse spinal cord neurons. In agreement with earlier noise studies, channels activated by gamma-aminobutyric acid (GABA), muscimol, and (-)-pentobarbital were found to have equal unit conductances. The kinetics of channel closing were studied by analyzing the distributions of open state lifetimes. Channels activated by (-)-pentobarbital and muscimol had longer mean open times than channels activated by GABA. As a result, the kinetics of (-)-pentobarbital- and muscimol-activated channels could be studied in greater detail. Most observed open state lifetime distributions were not exponential but contained an excess of short duration events. A sum of two exponential functions gave a much better fit than a single exponential function to most observed open state lifetime distributions. A critical comparison of noise analysis with single channel recording shows that the fast process responsible for the rapid closures would be very difficult to detect in a noise experiment. The channel noise is dominated by the slower process, and as a result, the relaxation time of the slower kinetic component derived from single channel studies is close to the mean open state lifetime derived from noise measurements. The observation of a faster process points toward either an additional population of channels or a scheme for the channel closing transition which is not a simple first order process.