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1.
The α1 subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α2δ and β subunits. α1E channels directed with the expression of Ba2+ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α1E with α2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α1E with β2a shifted the peak current relationship by −10 mV, and strongly reduced Ba2+ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba2+ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β2a and the neuronal α2bδ subunits increased by ≈10-fold whole-cell Ba2+ currents although coinjection with either β2a or α2bδ alone failed to significantly increase α1E peak currents. Coexpression with β2a and α2bδ yielded Ba2+ currents with inactivation kinetics similar to the β2a induced currents, indicating that the neuronal α2bδ subunit has little effect on α1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit
genes. The slower inactivation was unique to α1E/β2a currents. Coexpression with β1a, β1b, β3, and β4, yielded faster-inactivating Ba2+ currents than currents recorded from the α1E subunit alone. Furthermore, α1E/α2bδ/β1a; α1E/α2bδ/β1b; α1E/α2bδ/β3; α1E/α2bδ/β4 channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The
β subunit-induced changes in the properties of α1E channel were comparable to modulation effects reported for α1C and α1A channels with β3≈β1b > β1a≈β4≫β2a inducing fastest to slowest rate of whole-cell inactivation.
Received: 27 March 1997/Revised: 10 July 1997 相似文献
2.
G.W. Zamponi 《The Journal of membrane biology》1999,167(2):183-192
Piperidines are a relatively novel class of calcium channel blockers which act at a unique receptor site associated with
the calcium channel α1 subunit. Calcium channel blocking affinities ranging from subnanomolar to several hundred micromolar have been reported in
the literature, suggesting that piperidine block is highly sensitive to the cellular environment experienced by the channel.
Here, I have investigated some of the cytoplasmic determinants of haloperidol block of N-type calcium channels expressed in
human embryonic kidney cells. In perforated patch clamp recordings, haloperidol blocks N-type calcium channels with an inhibition
constant of 120 μM. Upon internal dialysis with chloride containing pipette solution, the blocking affinity increases by 40-fold.
This effect could be attributed in part to the presence of internal chloride ions, as replacement of intracellular chloride
with methanesulfonate reduced haloperidol blocking affinity by almost one order of magnitude. Tonic inhibition of N-type channels
by Gβγ subunits further enhanced the blocking effects of haloperidol, suggesting the possibility of direct effects of Gβγ binding on the local environment of the piperidine receptor site. Overall, depending on the cytoplasmic environment experienced
by the channel, the blocking affinity of N-type calcium channels for haloperidol may vary by more than two orders of magnitude.
Thus, absolute blocking affinities at the piperidine receptor site must be interpreted cautiously and in the context of the
particular experimental setting.
Received: 23 July 1998/Revised: 19 October 1998 相似文献
3.
A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent
calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green
mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α1
C
) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics,
or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum
inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity
with an IC50 value of 88 nm. Three other types of high voltage activated channels tested (α1
A
, α1
B
, and α1
E
) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore,
the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized
test potentials, calcicludine actually upregulated current activity of (N-type) α1
B
channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α1
C
and α1
E
revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall,
calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action.
Received: 22 September 1999/Revised: 1 December 1999 相似文献
4.
5.
M.L. Tierney B. Birnir N.P. Pillai J.D. Clements S. M. Howitt G. B. Cox P.W. Gage 《The Journal of membrane biology》1996,154(1):11-21
The conserved leucine residues at the 9′ positions in the M2 segments of α1 (L264) and β1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant α1 subunits were co-expressed with normal or mutant β1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a
higher ``resting' chloride conductance than cells expressing wild-type α1β1 receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β1 with mutated α1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type
α1β1 receptors. In cells expressing mutated β1 subunits with wild-type or mutated α1 subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9′T mutation in the β1 subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an
open-channel state of the GABAA receptor in the absence of ligand.
Received: 17 April 1996/Revised: 5 July 1996 相似文献
6.
B. Birnir M.L. Tierney J.E. Dalziel G.B. Cox P.W. Gage 《The Journal of membrane biology》1997,155(2):157-166
Functional properties of the α1β1 GABAA receptor changes in a subunit-specific manner when a threonine residue in the M2 region at the 12′ position was mutated to
glutamine. The rate and extent of desensitization increased in all mutants but the rate of activation was faster in the β1 mutants. A negligible plateau current and abolition of potentiation by pentobarbitone of the GABA-activated current depended
on the Thr 12′ Gln mutation being present in the β1 subunit. The Hill coefficient of the peak current response to GABA was reduced to less than one also in a β1 subunit-specific manner. It was concluded that the β1 subunit dominated conformational changes activated by GABA.
Received: 18 July 1996/Revised: 30 September 1996 相似文献
7.
Using the whole-cell patch-clamp technique, the selectivity and pharmacology of 8-Br-cGMP-stimulated currents in the human
alveolar cell line A549 was compared to 8-Br-cGMP-stimulated currents in HK293 cells transfected with hαCNC1. Whole cell currents
stimulated by 8-Br-cGMP in HK293 cells transfected with hαCNC1 or A549 cells are carried by inward sodium and outward potassium
with nearly the same selectivity. The whole-cell inward currents that are stimulated by 8-Br-cGMP in HK293 cells transfected
with hαCNC1 are inhibited by l-cis-diltiazem with an IC50 of 154 μm, by 2′,4′-dichlorobenzamil with an IC50 of 50 μm and by amiloride with an IC50 of 133 μm. The whole-cell inward currents in A549 cells that are stimulated by 8-Br-cGMP, are inhibited by l-cis-diltiazem with an
IC50 of 87 μm, by 2′4′-dichlorobenzamil with an IC50 of 38 μm and by amiloride with an IC50 of 32 μm suggesting that these airway cells contain cyclic nucleotide-gated cation channels. RT-PCR data suggest that mRNA of both
αCNC1 and βCNC subunits are present in A549 cells and the presence of the βCNC subunit, may as previously reported, increase
the affinity of these channel blockers compared to the hαCNC1 subunit alone. The mRNA of two other isoforms of this channel,
CNC2 and CNC3, are also expressed in the A549 cell line. This study documents the IC50 of externally applied channel blockers that can be used for in vitro or in vivo experiments to document sodium absorption
via cyclic nucleotide-gated cation channels in airway cells.
Received: 24 February/Revised: 28 May 1999 相似文献
8.
Desensitization of ligand-gated receptor channels is an intrinsic feedback mechanism and prevents the receptor/channels from
becoming overly activated thereby maintaining biological function of the nervous system. Desensitization also plays an important
role in neuronal plasticity. By taking advantage of biophysical and pharmacological diversities of GABA β2 subunits from the brain and ρ1 subunits from the retina, structural determinants that confer agonist-induced desensitization were identified. A synthetic
chimeric receptor/channel was created from the β2 and ρ1 subunits for this investigation. The chimera was constructed from the extracellular N-domain of the β2 subunit, extending from the amino terminus to the beginning region of the M1 transmembrane segment, and from the C-domain
of the ρ1 subunit extending from the M1 transmembrane segment to the carboxyl terminus. The C-domain region included the M1 to M4 transmembrane
regions and the large intracellular loop between the M3 and M4 transmembrane segments. Homo-oligomeric GABA β2, ρ1, and β2/ρ1 chimeric receptor/channels were individually expressed in Xenopus oocytes, and the desensitization characteristics attributable to each type of subunit were compared. Results from the present
study reveal that motifs in the amino-terminal and carboxyl-terminal domains of the β2 subunit conferred the agonist-induced desensitization; chloroform modulation was linked to specific phases of the GABA-activated
current decay.
Received: 2 April 1997/Revised: 27 March 1998 相似文献
9.
To study interaction of specific antibodies with the GABA receptor/channel, antisera were raised against the extracellular
domains of the GABAA receptor/channel β2 subunit, γ2 subunit and the GABAC receptor/channel ρ1 subunit. The specificity of the antibodies was characterized by immunocytochemistry and by Western blotting of transfected
FDC-P1 cells expressing recombinant GABA receptor/channel subunits. The effects of the antibodies on whole-cell currents in
Xenopus laevis oocytes expressing homomeric recombinant GABA receptor/channel β2, γ2, and ρ1 were studied using two-microelectrode voltage clamp. In the absence of GABA, anti-α2, anti-γ2, and anti-ρ1 antisera elicited whole-cell currents in oocytes expressing β2, γ2, and ρ1 subunits, respectively. The effect of antibody on channel activation was concentration-dependent. The whole-cell currents
induced by anti-β2 and anti-γ2 were several-fold greater than those induced by application of 100 μm GABA. In Xenopus oocytes expressing recombinant ρ1 subunits, GABA-induced whole-cell currents were inhibited by the anti-ρ1 antibody. In contrast, the GABA-induced whole-cell currents were potentiated several-fold by anti-β2 and anti-γ2 antibodies in Xenopus oocytes expressing homomeric β2 and γ2 subunits. Our studies indicate that antibodies specific to the N-terminal domain of GABA receptor/channel subunits can modulate
the neurotransmitter receptor function.
Received: 2 February 2001/Revised: 11 April 2001 相似文献
10.
S.I. Ortiz-Miranda J.A. Lasalde P.A. Pappone M.G. McNamee 《The Journal of membrane biology》1997,158(1):17-30
We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type α418Cys
residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold,
respectively. The mutation of a homologous cysteine in the β subunit (β447Trp) had similar but smaller effects on mean open
time. Coexpression of α418Trp and β447Trp had the largest effect on channel open time, increasing mean open time 58-fold.
No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested.
However, the coexpression of the α418Trp and β447Trp mutated subunits also produced channels with at least two additional
conductance levels. Block by acetylcholine was apparent in the current records from α418Trp mutants. Burst analysis of the
α418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate
and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the
α- and β subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating,
and that mutations in multiple subunits act additively to increase channel open time.
Received: 27 September 1996/Revised: 28 January 1997 相似文献
11.
Thomas A. Gorr Barbara K. Mable Traute Kleinschmidt 《Journal of molecular evolution》1998,47(4):471-485
Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences.
Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were
compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated α chains into αA and αD types, which are present in all reptiles except crocodiles, where only αA chains are expressed. The occurrence of the αD chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards
and snakes also express two types of β chains (βI and βII), while only one type of β chain is present in birds and crocodiles.
Reconstructed hemoglobin trees for both α and β sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon+ squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be
due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based
on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that
relative rates for squamate αA and αD chains and crocodilian β chains are at least twice as high as those of the rest of the chains considered. In contrast to
these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination
of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded
1.7 (αA) and 3.3 (β) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation
events within snakes and for the squamate ↔ sphenodontid split. This suggests that hemoglobin chains of squamate reptiles
evolved ∼3.5 (αA) or ∼1.7 times (β) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some
intrasquamate divergence times.
Received: 15 September 1997 / Accepted: 4 February 1998 相似文献
12.
GABA-activated Cl− current was expressed in Xenopus oocytes after injecting cRNA that had been transcribed in vitro from complementary DNA (cDNA) coding for a single GABA ρi-subunit cloned from human retina. The expressed current was insensitive to 100 μm bicuculline, but was activated by the GABA analogue trans-4-aminocrontonic acid (TACA). Anion-selective permeability of the
expressed ρ1-subunit was determined by isotonically replacing the extracellular Cl− with different anions. The anion permeability was very similar to the native GABAA receptor/channel following a sequence of SCN− > I− > NO3
− > Br−≥ Cl−. Halogenated fatty acids, such as chlorotrifluoroethylene (CTFE) and perfluorinated oligomer acids inhibited the GABA-induced
current in oocytes expressing the human retinal GABA ρ1-subunit or rat brain GABAA receptor α1,β2,γ2 subunits. The inhibitory effect of halogenated fatty acids demonstrated a carbon chain length-dependent manner of: C10 > C8 > C6 > C4. Perfluorinated C8-oligomer acid (PFOA) was less effective at blocking this channel than the C8-CTFE oligomer acid. Radiolabeled GABA binding assay indicated that CTFE oligomer acids do not interfere at the GABA binding
site of the receptor. Furthermore, the C8-CTFE oligomer fatty acid did not compete with picrotoxin for binding sites within the pore of the channel. These studies
demonstrated that the heterologous expression system is useful for studying the molecular interaction between potential neurotoxic
agents and neuroreceptors. Our results provide detailed information that should contribute to our understanding of the structure
and function of retinal GABA receptors.
Received: 12 June 1995/Revised: 21 September 1995 相似文献
13.
Hong Xue 《Journal of molecular evolution》1998,47(3):323-333
The gene superfamily of ligand-gated ion channel (LGIC) receptors is composed of members of excitatory LGIC receptors (ELGIC)
and inhibitory LGIC receptors (ILGIC), all using amino acids as ligands. The ILGICs, including GABAA, Gly, and GluCl receptors, conduct Cl− when the ligand is bound. To evaluate the phylogenetic relationships among ILGIC members, 90 protein sequences were analyzed
by both maximum-parsimony and distance matrix-based methods. The strength of the resulting phylogenetic trees was evaluated
by means of bootstrap. Four major phylogenetic branches are recognized. Branch I, called BZ, for the majority of the members
are known to be related to benzodiazepine binding, is subdivided into IA, composed of all GABAA receptor α subunits, and IB, composed of the γ and ε subunits, which are shown to be tightly linked. Branch II, named NB
for non–benzodiazepine binding, and consisting of GABAA receptor β, δ, π, and ρ subunits, is further subdivided into IIA, containing β subunits; IIB, containing δ, and π subunits;
and IIC, containing ρ subunits. Branch IIIA, composed of vertebrate Gly receptors, is loosely clustered with Branch IIIB,
composed of invertebrate GluCl receptors, to form Branch III, which is designated NA for being non–GABA responsive. Branch
IV is called UD for being undefined in specificity. The existence of primitive forms of GABAA receptor non-β subunits in invertebrates is first suggested by the present analysis, and the identities of sequences p25123
from Drosophila melanogaster, s34469 from Lymnaea stagnalis, and u14635 and p41849 from C. aenorhabditis elegans are determined to be different from their previously given annotations. The proposed branching classification of ILGICs provides
a phylogenetic map, based on protein sequences, for tracing the evolutionary pathways of ILGIC receptor subunits and determining
the identities of newly discovered subunits on the basis of their protein sequences.
Received: 15 April 1997 / Accepted: 11 March 1998 相似文献
14.
Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels 总被引:4,自引:0,他引:4
Demetrios K. Vassilatis Keith O. Elliston Philip S. Paress Michel Hamelin Joseph P. Arena James M. Schaeffer Lex H.T. Van der Ploeg Doris F. Cully 《Journal of molecular evolution》1997,44(5):501-508
Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin
class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as
well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of
related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined
the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these
channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic
analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several
intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position.
Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily
and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties
similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride
channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination
with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel
family that may represent genes orthologous to the vertebrate glycine channels.
Received: 30 September 1996 / Accepted: 15 November 1996 相似文献
15.
Single cardiac ATP-sensitive K+ channels and, comparatively, two other members of the inwardly rectifying K+ channel family, cardiac K+
(ir) and K+
(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical
K+ gradient for open-state kinetics of these K+ channels.
The conductive state of K+
(ATP) channels was defined as a function of the electrochemical K+ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be
largely excluded as the underlying mechanism for two reasons: (i) τopen proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K+
(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of
τopen. Since gating continued to operate at E
K
, i.e., in the absence of K+ permeation through the pore, K+ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar
to Rb+ whose interference with an externally located binding site stabilizes the open state. In K+
(ir) and K+
(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the
electrochemical K+ gradient. Thus, K+
(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their
gating apparatus.
Received: 24 March 1997/Revised: 24 April 1998 相似文献
16.
GABAA receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised
of only α and β subunits. The pore of GABAA receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at
the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β
subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues
may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and
the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found
that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ,
with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors
remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized
potentials only (ECl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the
γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the
open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study
show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics. 相似文献
17.
We have previously shown that epithelial Na+ channels in mouse mandibular gland duct cells are controlled by cytosolic Na+ and Cl−, acting, respectively, via G
o
and G
i
proteins. Since we found no evidence for control of epithelial Na+ channels by extracellular Na+ ([Na+]
o
), our findings conflicted with the long-held belief that Na+ channel activators, such as sulfhydryl reagents, like para-chloromercuriphenylsulfonate (PCMPS), and amiloride analogues,
like benzimidazolylguanidinium (BIG) and 5-N-dimethylamiloride (DMA), induce their effects by blocking an extracellular channel
site which otherwise inhibits channel activity in response to increasing [Na+]
o
. Instead, we now show that PCMPS acts by rendering epithelial Na+ channels refractory to inhibition by activated G proteins, thereby eliminating the inhibitory effects of cytosolic Na+ and Cl− on Na+ channel activity. We also show that BIG, DMA, and amiloride itself, when applied from the cytosolic side of the plasma membrane,
block feedback inhibition of Na+ channels by cytosolic Na+, while leaving inhibition by cytosolic Cl− unaffected. Since the inhibitory effects of BIG and amiloride are overcome by the inclusion of the activated α-subunit of
G
o
in the pipette solution, we conclude that these agents act by blocking a previously unrecognized intracellular Na+ receptor.
Received: 1 October 1997/Revised: 24 December 1997 相似文献
18.
The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis
and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions
and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating
kinetics. The rate of channel locking increased from 1.05 × 10−3 sec−1 to 58.7 × 10−3 sec−1 when AMP-PNP concentration was raised from 0.5 to 5 mm in the presence of 1 mm MgATP and 180 nm protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of P
o
or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence
of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high [AMP-PNP] is
evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce P
o
and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed
to PKC alone. AMP-PNP increased P
o
at temperatures above 30°C but did not cause locking, evidence that the stabilizing interactions between domains, which have
been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating
by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q
10= 9.6) than the closing rate (Q
10= 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.
Received: 28 August 1997/Revised: 4 February 1998 相似文献
19.
The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits,
α and β, and tissue-specific isoforms exist for each of these, α1, α2 and α3 and β1, β2 and β3. We have proposed that an additional
α isoform, α4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively
in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative
α4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in α4 isoform
cDNA transfected 3T3 cells. Using an α4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected
for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence
of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K
D
values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.
Received: 4 December 1998/Revised: 1 February 1999 相似文献