Water transport in human aquaporin-4: molecular dynamics (MD) simulations

Aquaporin-4 (AQP4) is the predominant water channel in the central nervous system, where it has been reported to be involved in many pathophysiological roles including water transport. In this paper, the AQP4 tetramer was modeled from its PDB structure file, embedded in a palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer, solvated in water, then minimized and equilibrated by means of molecular dynamics simulations. Analysis of the equilibrated structure showed that the central pore along the fourfold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-195, Leu-191 and Leu-75, form the narrowest part of the pore. Therefore water molecules are not expected to transport through the central pore, which was confirmed by MD simulations. Each monomer of the AQP4 tetramers forms a channel whose walls consist mostly of hydrophilic residues. There are eight water molecules in single file observed in each of the four channels, transporting through the selectivity filter containing Arg-216, His-201, Phe-77, Ala-210, and the two conserved Asn-Pro-Ala (NPA) motifs containing Asn-213 and Asn-97. By using Brownian dynamics fluctuation–dissipation-theorem (BD-FDT), the overall free-energy profile was obtained for water transporting through AQP4 for the first time, which gives a complete map of the entire channel of water permeation.     

Distinct sites regulating grayanotoxin binding and unbinding to D4S6 of Na(v)1.4 sodium channel as revealed by improved estimation of toxin sensitivity

Grayanotoxin (GTX) exerts selective effects on voltage-dependent sodium channels by eliminating fast sodium inactivation and causing a hyperpolarizing shift in voltage dependence of channel activation. In this study, we adopted a newly developed protocol that provides independent estimates of the binding and unbinding rate constants of GTX (k(on) and k(off)) to GTX sites on the sodium channel protein, important in the molecular analysis of channel modification. Novel GTX sites were determined in D2S6 (Asn-784) and D3S6 (Ser-1276) by means of site-directed mutagenesis; the results suggested that the GTX receptor consists of the S6 transmembrane segments of four homologous domains facing the ion-conducting pore. We systematically introduced at two sites in D4S6 (Na(v)1.4-Phe-1579 and Na(v)1.4-Tyr-1586) amino acid substituents with residues containing hydrophobic, aromatic, charged, or polar groups. Generally, substitutions at Phe-1579 increased both k(on) and k(off), resulting in no prominent change in dissociation constant (K(d)). It seems that the smaller the molecular size of the residue at Na(v)1.4-Phe-1579, the larger the rates of k(on) and k(off), indicating that this site acts as a gate regulating access of toxin molecules to a receptor site. Substitutions at Tyr-1586 selectively increased k(off) but had virtually no effect on k(on), thus causing a drastic increase in K(d). At position Tyr-1586, a hydrophobic or aromatic amino acid side chain was required to maintain normal sensitivity to GTX. These results suggest that the residue at position Tyr-1586 has a more critical role in mediating GTX binding than the one at position Phe-1579. Here, we propose that the affinity of GTX to Na(v)1.4 sodium channels might be regulated by two residues (Phe and Tyr) at positions Phe-1579 and Tyr-1586, which, respectively, control access and binding of GTX to its receptor.     

Physicochemical features of the HERG channel drug binding site

Blockade of hERG K(+) channels in the heart is an unintentional side effect of many drugs and can induce cardiac arrhythmia and sudden death. It has become common practice in the past few years to screen compounds for hERG channel activity early during the drug discovery process. Understanding the molecular basis of drug binding to hERG is crucial for the rational design of medications devoid of this activity. We previously identified 2 aromatic residues, Tyr-652 and Phe-656, located in the S6 domain of hERG, as critical sites of interaction with structurally diverse drugs. Here, Tyr-652 and Phe-656 were systematically mutated to different residues to determine how the physicochemical properties of the amino acid side group affected channel block by cisapride, terfenadine, and MK-499. The potency for block by all three drugs was well correlated with measures of hydrophobicity, especially the two-dimensional approximation of the van der Waals hydrophobic surface area of the side chain of residue 656. For residue 652, an aromatic side group was essential for high affinity block, suggesting the importance of a cation-pi interaction between Tyr-652 and the basic tertiary nitrogen of these drugs. hERG also lacks a Pro-Val-Pro motif common to the S6 domain of most other voltage-gated K(+) channels. Introduction of Pro-Val-Pro into hERG reduced sensitivity to drugs but also altered channel gating. Together, these findings assign specific residues to receptor fields predicted by pharmacophore models of hERG channel blockers and provide a refined molecular understanding of the drug binding site.     

Structure of the amino terminus of a gap junction protein

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.     

Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of the subunit C (Vma5p) of the yeast vacuolar H+-ATPase.

Subunit C is a V(1) sector subunit found in all vacuolar H(+)-ATPases (V-ATPases) that may be part of the peripheral stalk connecting the peripheral V(1) sector with the membrane-bound V(0) sector of the enzyme (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804--31810). To elucidate subunit C function, we performed random and site-directed mutagenesis of the yeast VMA5 gene. Site-directed mutations in the most highly conserved region of Vma5p, residues 305--325, decreased catalytic activity of the V-ATPase by up to 48% without affecting assembly. A truncation mutant (K360stop) identified by random mutagenesis suggested a small region near the C terminus of the protein (amino acids 382--388) might be important for subunit stability. Site-directed mutagenesis revealed that three aromatic amino acids in this region (Tyr-382, Phe-385, and Tyr-388) in addition to four other conserved aromatic amino acids (Phe-260, Tyr-262, Phe-296, Phe-300) are essential for stable assembly of V(1) with V(0), although alanine substitutions at these positions support some activity in vivo. Surprisingly, three mutations (F260A, Y262A, and F385A) greatly decrease the stability of the V-ATPase in vitro but increase its k(cat) for ATP hydrolysis and proton transport by at least 3-fold. The peripheral stalk of V-ATPases must balance the stability essential for productive catalysis with the dynamic instability involved in regulation; these three mutations may perturb that balance.     

A gating mutation in the internal pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.     

Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.     

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

Crystal Structure of a Yeast Aquaporin at 1.15 ? Reveals a Novel Gating Mechanism

Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.     

Aromatic Residues in the Substrate Cleft of RPE65 Protein Govern Retinol Isomerization and Modulate Its Progression

Previously, we showed that mutating RPE65 residue Phe-103 preferentially produces 13-cis-retinol instead of 11-cis-retinol, supporting a carbocation/radical cation mechanism of retinol isomerization. We asked whether this modulation of specificity can occur with residues other than Phe-103 and what role it plays in substrate binding and isomerization. We modeled the substrate-binding cleft of RPE65 to identify residues lining its surface. Many are phenylalanines and tyrosines, including three Phe residues (Phe-61, Phe-312, and Phe-526) forming an arch-like arrangement astride the cleft and Tyr-338. Also, Phe-418 sits at the neck of the cleft, lending a bend to the volume enclosed by the cleft. All mutations of Phe-61, Phe-312, and Phe-418 result in severely impaired or inactive enzyme. However, mutation of Phe-526 and Tyr-338, like Phe-103, decreases 11-cis-retinol formation, whereas increasing the 13-cis isomer. Significantly, 2 of these 3 residues, Phe-103 and Tyr-338, are located on putatively mobile interstrand loops. We propose that residual densities located in the binding cleft of the RPE65 structure represents a post-cleavage snapshot consistent not only with a fatty acid product, as originally modeled, but also an 11-cis-retinol product. Substrate docking simulations permit 11-cis- or 13-cis-retinyl ester binding in this relatively closed cleft, with the latter favored in F103L, F526A, and Y338A mutant structures, but prohibit binding of all-trans-retinyl ester, suggesting that isomerization occurs early in the temporal sequence, with O-alkyl ester cleavage occurring later. These findings provide insight into the mechanism of isomerization central to the visual cycle.     

Mutagenic analysis of the a subunit of the F1F0 ATP synthase in Escherichia coli: Gln-252 through Tyr-263.

The a subunit of F1F0 ATP synthase contains a highly conserved region near its carboxyl terminus which is thought to be important in proton translocation. Cassette site-directed mutagenesis was used to study the roles of four conserved amino acids Gln-252, Phe-256, Leu-259, and Tyr-263. Substitution of basic amino acids at each of these four sites resulted in marked decreases in enzyme function. Cells carrying a subunit mutations Gln-252-->Lys, Phe-256-->Arg, Leu-259-->Arg, and Tyr-263-->Arg all displayed growth characteristics suggesting substantial loss of ATP synthase function. Studies of both ATP-driven proton pumping and proton permeability of stripped membranes indicated that proton translocation through F0 was affected by the mutations. Other mutations, such as the Phe-256-->Asp mutation, also resulted in reduced enzyme activity. However, more conservative amino acid substitutions generated at these same four positions produced minimal losses of F1F0 ATP synthase. The effects of mutations and, hence, the relative importance of the amino acids for enzyme function appeared to decrease with proximity to the carboxyl terminus of the a subunit. The data are most consistent with the hypothesis that the region between Gln-252 and Tyr-263 of the a subunit has an important structural role in F1F0 ATP synthase.     

Novel plastocyanin containing phenylalanine-83 from the gymnosperm Ginkgo biloba

Plastocyanin was purified from the gymnosperm Ginkgo biloba L., and its complete amino acid sequence was determined. The protein was shown to contain Phe-83 instead of Tyr-83 conserved in other land plant plastocyanins. This is the first report of the characterization and complete amino acid sequence of a gymnosperm plastocyanin.     

Determinants of gating polarity of a connexin 32 hemichannel

There is good evidence supporting the view that the transjunctional voltage sensor (V(j)-sensor) of Cx32 and other Group 1 connexins is contained within a segment of the N-terminus that contributes to the formation of the channel pore. We have shown that the addition of negatively charged amino acid residues at several positions within the first 10 amino acid residues reverses the polarity of V(j)-gating and proposed that channel closure is initiated by the inward movement of this region. Here, we report that positive charge substitutions of the 2nd, 5th, and 8th residues maintain the negative polarity of V(j)-gating. These data are consistent with the original gating model. Surprisingly, some channels containing combinations of positive and/or negative charges at the 2nd and 5th positions display bipolar V(j)-gating. The appearance of bipolar gating does not correlate with relative orientation of charges at this position. However, the voltage sensitivity of bipolar channels correlates with the sign of the charge at the 2nd residue, suggesting that charges at this position may have a larger role in determining gating polarity. Taken together with previous findings, the results suggest that the polarity V(j)-gating is not determined by the sign of the charge lying closest to the cytoplasmic entry of the channel, nor is it likely to result from the reorientation of an electrical dipole contained in the N-terminus. We further explore the mechanism of polarity determination by utilizing the one-dimensional Poisson-Nernst-Plank model to determine the voltage profile of simple model channels containing regions of permanent charge within the channel pore. These considerations demonstrate how local variations in the electric field may influence the polarity and sensitivity of V(j)-gating but are unlikely to account for the appearance of bipolar V(j)-gating.     

Conserved low-affinity nickel-binding amino acids are essential for the function of the nickel permease NixA of Helicobacter pylori

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.     

Paragonimus westermani: a cytosolic glutathione S-transferase of a sigma-class in adult stage

We purified cytosolic glutathione S-transferase (GST) of adult Paragonimus westermani monitoring its activity with 1-chloro-2,4-dinitrobenzene (CDNB). The enzyme was purified 18.4-fold to electrophoretic homogeneity with 21% recovery rate through a three-step procedure. The purified enzyme (Pw28GST) has a subunit molecular weight of 28 kDa with an isoelectric point at 4.6. Monoclonal antibody (anti-Pw28GST) against Pw28GST did not cross-react with GSTs from other helminths. cDNA library was constructed in lambdaZAP II bacteriophage and screened with anti-Pw28GST. The corresponding gene containing a single open reading frame of 804 bp encoded 211 amino acids. The predicted amino acid sequence exhibited a higher homology with catalytic domain near N-terminus of class sigma GSTs (58%) than with schistosome 28-kDa GSTs (45-41%) or with class sigma GSTs themselves (33-31%). The sequence contained both Tyr-6 and Tyr-10 that are highly conserved in mammalian and helminth GSTs. The apparent K(m) value of a recombinant enzyme was 0.78 mM. Both native and recombinant enzymes showed the highest activity against CDNB, relatively weak activity against ethacrynic acid and reactive carbonyls, and no activity against epoxy-3-(p-nitrophenoxy)-propane. The activities were inhibited by bromosulfophthalein, cibacron blue, and albendazole, but not by praziquantel. These findings indicate that adult P. westermani has a class sigma GST.     

A type-1 metacaspase from Acanthamoeba castellanii

The complete sequence of a type-1 metacaspase from Acanthamoeba castellanii is reported comprising 478 amino acids. The metacaspase was recovered from an expression library using sera specific for membrane components implicated in stimulating encystation. A central domain of 155 amino acid residues contains the Cys/His catalytic dyad and is the most conserved region containing at least 30 amino acid identities in all metacaspases. The Acanthamoeba castellanii metacaspase has the most proline-rich N-terminus so far reported in type-1 metacaspases with over 40 prolines in the first 150 residues. Ala–Pro–Pro is present 11 times. Phylogenies constructed using only the conserved proteolytic domains or the complete sequences show identical branching patterns, differing only in the rates of change.     

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.     

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.     

Amino acid residues outside of the pore region contribute to N-type calcium channel permeation

It is widely believed that the selectivity of voltage-dependent calcium channels is mainly controlled by amino acid residues contained within four p-loop motifs forming the pore of the channel. An examination of the amino acid sequences of high voltage-activated calcium channels reveals that their domain III S5-H5 regions contain a highly conserved motif with homology to known EF hand calcium binding proteins, hinting that this region may contribute to channel permeation. To test this hypothesis, we used site-directed mutagenesis to replace three conserved negatively charged residues in the N-type calcium channel alpha1B subunit (Glu-1321, Asp-1323, and Glu-1332) with positively charged amino acids (lysine and arginine) and studied their effect on ion selectivity using whole cell and single channel patch clamp recordings. Whereas the wild type channels conducted barium much more effectively than calcium, the mutant displayed nearly equal permeabilities for these two ions. Individual replacement of residue 1332 or a double substitution of residues 1321 and 1323 with lysine and arginine, respectively, were equally effective. Disruption of the putative EF hand motif through replacement of the central glycine residue (1326) with proline resulted in a similar effect, indicating that the responses observed with the triple mutant were not due to changes in the net charge of the channel. Overall, our data indicate that residues outside of the narrow region of the pore have the propensity to contribute to calcium channel permeation. They also raise the possibility that interactions of calcium ions with a putative calcium binding domain at the extracellular side of the channel may underlie the differential permeabilities of the channel for barium and calcium ions.