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D M Papazian

Publications and source records attributed to D M Papazian.

At least 19 recordsLinked to original sources
PubMed2005

CACNA1A mutations causing episodic and progressive ataxia alter channel trafficking and kinetics.

BACKGROUND: CACNA1A encodes CaV2.1, the pore-forming subunit of P/Q-type voltage-gated calcium channel complexes. Mutations in CACNA1A cause a wide range of neurologic disturbances variably associated with cerebellar degeneration. Functional studies to date focus on electrophysiologic defects that do not adequately explain the phenotypic findings. OBJECTIVE: To investigate whether some missense mutations might interfere with protein folding and trafficking, eventually leading to protein aggregation and neuronal injury. METHODS: The authors studied the functional consequences of two pore missense mutations, C287Y and G293R, in two families with EA2, one newly discovered and the other previously reported. Both mutations caused episodic and interictal ataxia. The biophysical properties of mutant and wild type calcium channels were examined by whole-cell patch-clamp recordings in transfected COS-7 cells. The plasma membrane targeting was visualized by confocal fluorescence imaging on CaV2.1 tagged with green fluorescent protein. RESULTS: The mutant channels exhibited a marked reduction in current expression and deficiencies in plasma membrane targeting. CONCLUSIONS: In addition to altered channel function, the deficiency in protein misfolding and trafficking associated with the C287Y and G293R mutants may contribute to the slowly progressive cerebellar ataxia.

Adolescent↗
PubMed2001

Loss-of-function EA2 mutations are associated with impaired neuromuscular transmission.

OBJECTIVE: To examine the functional consequences of episodic ataxia type 2 (EA2)-causing nonsense and missense mutations in vitro and to characterize the basis of fluctuating weakness in patients with E2A. BACKGROUND: Mutations in CACNA1A encoding the Ca(v)2.1 calcium channel subunit cause EA2 through incompletely understood mechanisms. Although the Ca(v)2.1 subunit is important for neurotransmission at the neuromuscular junction, weakness has not been considered a feature of EA2. METHODS: The disease-causing mutations in three unrelated patients with EA2 and fluctuating weakness were identified by mutation screening and sequencing. Mutant constructs harboring mutations R1281X, F1406C, R1549X were transfected into COS7 cells and expressed for patch clamp studies. Single-fiber electromyography (SFEMG) was performed in patients to examine synaptic transmission at the neuromuscular junction. RESULTS: Functional studies in COS7 cells of nonsense and missense EA2 mutants demonstrated markedly decreased current densities compared with wild type. SFEMG demonstrated jitter and blocking in these patients with EA2, compared with normal subjects and three patients with SCA-6. CONCLUSION: EA2-causing missense and nonsense mutations in CACNA1A produced mutant channels with diminished whole cell calcium channel activity in vitro due to loss of function. Altered biophysical properties or reduced efficiency of plasma membrane targeting of mutant channels may contribute to abnormal neuromuscular transmission, manifesting as myasthenic syndrome.

Calcium Channels↗
PubMed2001

Glycosylation increases potassium channel stability and surface expression in mammalian cells.

N-linked glycosylation is not required for the cell surface expression of functional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607-5613). We have now investigated whether glycosylation increases the stability, cell surface expression, and proper folding of Shaker protein expressed in mammalian cells. The turnover rates of wild-type protein and an unglycosylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In contrast, the unglycosylated mutant was rapidly degraded (t(1/2) = approximately 18 h). Lactacystin slowed the degradation of the mutant protein, implicating cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive degradation could be conferred on wild-type Shaker by a glycosylation inhibitor. Expression of the unglycosylated mutant on the cell surface, assessed using immunofluorescence microscopy and biotinylation, was dramatically reduced compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfide-bonded adducts was quantitatively similar in the wild-type and unglycosylated mutant proteins. Our results indicate that glycosylation increases the stability and cell surface expression of Shaker protein but has little effect on acquisition of the native structure.

Acetylcysteine↗
PubMed2000

Voltage-dependent structural interactions in the Shaker K(+) channel.

Using a strategy related to intragenic suppression, we previously obtained evidence for structural interactions in the voltage sensor of Shaker K(+) channels between residues E283 in S2 and R368 and R371 in S4 (Tiwari-Woodruff, S.K., C.T. Schulteis, A.F. Mock, and D. M. Papazian. 1997. Biophys. J. 72:1489-1500). Because R368 and R371 are involved in the conformational changes that accompany voltage-dependent activation, we tested the hypothesis that these S4 residues interact with E283 in S2 in a subset of the conformational states that make up the activation pathway in Shaker channels. First, the location of residue 283 at hyperpolarized and depolarized potentials was inferred by substituting a cysteine at that position and determining its reactivity with hydrophilic, sulfhydryl-specific probes. The results indicate that position 283 reacts with extracellularly applied sulfhydryl reagents with similar rates at both hyperpolarized and depolarized potentials. We conclude that E283 is located near the extracellular surface of the protein in both resting and activated conformations. Second, we studied the functional phenotypes of double charge reversal mutations between positions 283 and 368 and between 283 and 371 to gain insight into the conformations in which these positions approach each other most closely. We found that combining charge reversal mutations at positions 283 and 371 stabilized an activated conformation of the channel, and dramatically slowed transitions into and out of this state. In contrast, charge reversal mutations at positions 283 and 368 stabilized a closed conformation, which by virtue of the inferred position of 368 corresponds to a partially activated (intermediate) closed conformation. From these results, we propose a preliminary model for the rearrangement of structural interactions of the voltage sensor during activation of Shaker K(+) channels.

Animals↗
PubMed2000

Extracellular Mg(2+) modulates slow gating transitions and the opening of Drosophila ether-à-Go-Go potassium channels.

We have characterized the effects of prepulse hyperpolarization and extracellular Mg(2+) on the ionic and gating currents of the Drosophila ether-à-go-go K(+) channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg(2+) dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg(2+) modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg(2+) slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg(2+) modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg(2+). We have also identified mutations in the S3-S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg(2+), indicating an important role for this region in the voltage-dependent activation of eag.

Animals↗
PubMed2000

Mg(2+) modulates voltage-dependent activation in ether-à-go-go potassium channels by binding between transmembrane segments S2 and S3.

Extracellular Mg(2+) directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg(2+) presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg(2+)-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K(+) channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg(2+) on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg(2+)-sensitivity and mimicked the slowing of activation kinetics caused by Mg(2+) binding to the wild-type channel. These results suggest that Mg(2+) modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg(2+), indicating that D284 and D319 do not mediate the slowing of activation caused by Mg(2+) binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg(2+)-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg(2+) sensitivity but is in the vicinity of the binding site. We conclude that Mg(2+) binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K(+) channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.

Amino Acid Sequence↗
PubMed1999

Calnexin associates with Shaker K+ channel protein but is not involved in quality control of subunit folding or assembly.

Calnexin is part of an ER chaperone system that monitors and promotes the proper folding and assembly of glycosylated membrane proteins. To investigate the role of calnexin in the biogenesis of the voltage-dependent Shaker K+ channel, wild-type and mutant Shaker proteins were expressed in mammalian cells. Association with calnexin was assayed by coimmunoprecipitation. Calnexin interacted transiently with wild-type Shaker protein in the ER. In contrast, calnexin failed to associate with an unglycosylated Shaker mutant that makes active, cell surface channels. Therefore, glycosylation of Shaker protein is required for association with calnexin, but calnexin is not required for the proper folding and assembly of Shaker channels. We also investigated whether calnexin is involved in the ER retention of mutant Shaker proteins defective in subunit folding, assembly, or pore formation. Each of the mutant proteins associated transiently with calnexin during biogenesis. Calnexin dissociated from wild-type and mutant proteins with similar time courses. Thus, non-native Shaker proteins escape the folding sensor of the calnexin chaperone system. Furthermore, stable association with calnexin is not the mechanism by which these mutant proteins are retained in the ER. Our results indicate that calnexin is not involved in the quality control of subunit folding, assembly, or pore formation in Shaker K+ channels.

Calcium-Binding Proteins↗
PubMed1998

Subunit folding and assembly steps are interspersed during Shaker potassium channel biogenesis.

In the voltage-dependent Shaker K+ channel, distinct regions of the protein form the voltage sensor, contribute to the permeation pathway, and recognize compatible subunits for assembly. To investigate channel biogenesis, we disrupted the formation of these discrete functional domains with mutations, including an amino-terminal deletion, Delta97-196, which is likely to disrupt subunit oligomerization; D316K and K374E, which prevent proper folding of the voltage sensor; and E418K and C462K, which are likely to disrupt pore formation. We determined whether these mutant subunits undergo three previously identified assembly events as follows: 1) tetramerization of Shaker subunits, 2) assembly of Shaker (alpha) and cytoplasmic beta subunits, and 3) association of the amino and carboxyl termini of adjacent Shaker subunits. Delta97-196 subunits failed to establish any of these quaternary interactions. The Delta97-196 deletion also prevented formation of the pore. The other mutant subunits assembled into tetramers and associated with the beta subunit but did not establish proximity between the amino and carboxyl termini of adjacent subunits. The results indicate that oligomerization mediated by the amino terminus is required for subsequent pore formation and either precedes or is independent of folding of the voltage sensor. In contrast, the amino and carboxyl termini of adjacent subunits associate late during biogenesis. Because subunits with folding defects oligomerize, we conclude that Shaker channels need not assemble from pre-folded monomers. Furthermore, association with native subunits can weakly promote the proper folding of some mutant subunits, suggesting that steps of folding and assembly alternate during channel biogenesis.

Animals↗
PubMed1998

Shaker and ether-à-go-go K+ channel subunits fail to coassemble in Xenopus oocytes.

Members of different voltage-gated K+ channel subfamilies usually do not form heteromultimers. However, coassembly between Shaker and ether-à-go-go (eag) subunits, members of two distinct K+ channel subfamilies, was suggested by genetic and functional studies (Zhong and Wu. 1991. Science. 252: 1562-1564; Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We investigated whether Shaker and eag form heteromultimers in Xenopus laevis oocytes using electrophysiological and biochemical approaches. Coexpression of Shaker and eag subunits produced K+ currents that were virtually identical to the sum of separate Shaker and eag currents, with no change in the kinetics of Shaker inactivation. According to the results of dominant negative and reciprocal coimmunoprecipitation experiments, the Shaker and eag proteins do not interact. We conclude that Shaker and eag do not coassemble to form heteromultimers in Xenopus oocytes.

Animals↗
PubMed1997

Potassium channel alpha and beta subunits assemble in the endoplasmic reticulum.

We have characterized the maturation of Shaker K+ channel protein and the cellular site of assembly of pore-forming alpha and cytoplasmic beta subunits in a transfected mammalian cell line. Shaker protein is made as a partially glycosylated, immature precursor that is converted to a fully glycosylated, mature product. Shaker protein did not mature when transport from the endoplasmic reticulum (ER) to the Golgi apparatus was blocked. Consistent with this finding, only the immature form was sensitive to digestion with endoglycosidase H. These results indicate that the immature protein is core-glycosylated in the ER, whereas the oligosaccharides of the mature protein have been further processed in the Golgi compartment. After inhibiting ER-to-Golgi transport, the oligomeric state of Shaker subunits was assessed by cross-linking in intact cells or by solubilization and sucrose gradient sedimentation. The results indicate that Shaker subunits assemble with each other in the ER. When co-expressed, the Kvbeta2 subunit also associated with Shaker in the ER. Assembly with the beta2 subunit did not increase the rate or extent of Shaker protein maturation. Our results indicate that the biogenesis of Shaker K+ channels in vivo involves core glycosylation and subunit assembly in the ER, followed by efficient transfer to the Golgi apparatus where the oligosaccharides are modified.

Animals↗
PubMed1997

Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits.

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.

Animals↗
PubMed1997

Transfer of voltage independence from a rat olfactory channel to the Drosophila ether-à-go-go K+ channel.

The S4 segment is an important part of the voltage sensor in voltage-gated ion channels. Cyclic nucleotide-gated channels, which are members of the superfamily of voltage-gated channels, have little inherent sensitivity to voltage despite the presence of an S4 segment. We made chimeras between a voltage-independent rat olfactory channel (rolf) and the voltage-dependent ether-à-go-go K+ channel (eag) to determine the basis of their divergent gating properties. We found that the rolf S4 segment can support a voltage-dependent mechanism of activation in eag, suggesting that rolf has a potentially functional voltage sensor that is silent during gating. In addition, we found that the S3-S4 loop of rolf increases the relative stability of the open conformation of eag, effectively converting eag into a voltage-independent channel. A single charged residue in the loop makes a significant contribution to the relative stabilization of the open stage in eag. Our data suggest that cyclic nucleotide-gated channels such as rolf contain a voltage sensor which, in the physiological voltage range, is stabilized in an activated conformation that is permissive for pore opening.

Amino Acid Sequence↗
PubMed1996

Intersubunit interaction between amino- and carboxyl-terminal cysteine residues in tetrameric shaker K+ channels.

Shaker potassium (K+) channels normally lack intrasubunit and intersubunit disulfide bonds. However, disulfide bonds are formed between Shaker subunits in intact cells exposed to oxidizing conditions. Upon electrophoresis under nonreducing conditions, intersubunit disulfide bond formation was detected by the presence of four high molecular weight adducts of Shaker protein. This result suggests that intracellular cysteine residues are in sufficiently close proximity in the native structure of the Shaker channel to form intersubunit disulfide bonds. To test this hypothesis, wild-type and mutant Shaker proteins were exposed to oxidizing conditions in intact cells. Intersubunit disulfide bond formation was eliminated upon serine substitution of either C96 in the amino terminal or C505 in the carboxyl terminal of the protein. In contrast, disulfide bond formation was not eliminated upon serine substitution of both C301 and C308 in the cytoplasmic loop between transmembrane segments S2 and S3. Exposure of Shaker-expressing cells to oxidizing conditions did not significantly alter the amplitude, kinetics, or voltage dependence of the Shaker current, demonstrating that the native tertiary and quaternary structures of the channel were maintained under oxidizing conditions. These results indicate that intersubunit disulfide bonds form between C96 and C505, providing evidence that the amino- and carboxyl-terminal regions of adjacent subunits are in proximity in the native structure of the channel. The disulfide-bonded adducts were found to represent a dimer, a trimer, and two forms of tetramer, one linear and one circular, containing one, two, three, or four disulfide bonds, respectively. These results provide a direct biochemical demonstration that Shaker K+ channels contain four pore-forming subunits.

Animals↗
PubMed1996

Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel.

The activation of Shaker K+ channels is steeply voltage dependent. To determine whether conserved charged amino acids in putative transmembrane segments S2, S3, and S4 contribute to the gating charge of the channel, the total gating charge movement per channel was measured in channels containing neutralization mutations. Of eight residues tested, four contributed significantly to the gating charge: E293, an acidic residue in S2, and R365, R368, and R371, three basic residues in the S4 segment. The results indicate that these residues are a major component of the voltage sensor. Furthermore, the S4 segment is not solely responsible for gating charge movement in Shaker K+ channels.

Animals↗
PubMed1995

Conserved cysteine residues in the shaker K+ channel are not linked by a disulfide bond.

Many voltage-activated K+ channels contain two conserved cysteine residues in putative transmembrane segments S2 and S6. It has been proposed that these cysteines form an intrasubunit disulfide bond [Guy, H.R., & Conti, F. (1990) Trends Neurosci. 13, 201-206]. This proposal was tested using site-directed mutagenesis followed by electrophysiological and biochemical analysis of the Shaker B K+ channel. Each Shaker B subunit contains seven cysteine residues, including the conserved residues C286 and C462 and a less conserved cysteine, C245. Each cysteine in the Shaker B protein can be mutated individually without eliminating functional activity, indicating that the protein does not contain a disulfide bond that is essential for protein folding or the assembly of active channels. To determine whether there is a nonessential disulfide bond, Shaker B protein was subjected to limited proteolysis. Fragments were analyzed by electrophoresis under reducing and nonreducing conditions followed by immunoblotting. The results indicate that the two conserved residues C286 and C462 do not form a disulfide bond with each other or with C245. In addition, the subunits are not linked by disulfide bonds. In HEK293T cells, Shaker B protein is first made as an incompletely glycosylated precursor that is converted to the fully glycosylated mature protein. Glycosylation occurs at two positions in the S1-S2 loop.

Amino Acid Sequence↗
PubMed1995

Electrostatic interactions of S4 voltage sensor in Shaker K+ channel.

The S4 segment comprises part of the voltage sensor in Shaker K+ channels. We have used a strategy similar to intragenic suppression, but without a genetic selection, to identify electrostatic interactions of the S4 segment that may be important in the mechanism of voltage-dependent activation. The S4 neutralization mutations K374Q and R377Q block maturation of the protein, suggesting that they prevent proper folding. K374Q is specifically and efficiently rescued by the second site mutations E293Q and D316N, located in putative transmembrane segments S2 and S3, respectively. These results suggest that K374, E293, and D316 form a network of strong, local, electrostatic interactions that stabilize the structure of the channel. Some other double mutant combinations result in inefficient suppression, identifying weak, presumably long-range electrostatic interactions. A simple structural hypothesis is proposed to account for the effects of the rescued double mutant combinations on the relative stabilities of open and closed channel conformations.

Animals↗