Potassium channel gating in the absence of the highly conserved glycine of the inner transmembrane helix

Potassium channel activation regulates cellular excitability, such as in neuronal and cardiac cells. Regulation of ion channel activity relies on a switching mechanism between two major conformations, the open and closed states, known as gating. It has been suggested that potassium channels are generally gated via a pivoted mechanism the pore-lining helix (TM2) in the proximity of a glycine that is conserved in about 80% of potassium channels, even though about 20% of the channels lack a glycine at this position. Yet, as we show in G-protein gated potassium (Kir3) channels that lack a glycine at this position, the betagamma subunits of G-proteins can still stimulate channel activity. Our results suggest that the effect of mutation of the central glycine (at position 175 in Kir3.4) on betagamma-induced whole-cell currents is related to the extent of the interaction between residues located at the position of the central glycine and two residues, one located in the signature sequence of the selectivity filter (T149 in Kir3.4) and the other in the pore helix (E147 in Kir3.4). Our results also suggest that interactions with position 149 are more detrimental to channel function than interactions with position 147. The ability of Gbetagamma to overcome such restraining interactions is likely to depend on a combination of characteristics specific to each residue.     

Implications of threonine hydrogen bonding in the glycophorin A transmembrane helix dimer

The transmembrane helix of glycophorin A contains a seven-residue motif, LIxxGVxxGVxxT, that mediates protein dimerization. Threonine is the only polar amino acid in this motif with the potential to stabilize the dimer through hydrogen-bonding interactions. Polarized Fourier transform infrared spectroscopy is used to establish a robust protocol for incorporating glycophorin A transmembrane peptides into membrane bilayers. Analysis of the dichroic ratio of the 1655-cm(-1) amide I vibration indicates that peptides reconstituted by detergent dialysis have a transmembrane orientation with a helix crossing angle of <35 degrees. Solid-state nuclear magnetic resonance spectroscopy is used to establish high resolution structural restraints on the conformation and packing of Thr-87 in the dimer interface. Rotational resonance measurement of a 2.9-A distance between the gamma-methyl and backbone carbonyl carbons of Thr-87 is consistent with a gauche- conformation for the chi1 torsion angle. Rotational-echo double-resonance measurements demonstrate close packing (4.0 +/- 0.2 A) of the Thr-87 gamma-methyl group with the backbone nitrogen of Ile-88 across the dimer interface. The short interhelical distance places the beta-hydroxyl of Thr-87 within hydrogen-bonding range of the backbone carbonyl of Val-84 on the opposing helix. These results refine the structure of the glycophorin A dimer in membrane bilayers and highlight the complementary role of small and polar residues in the tight association of transmembrane helices in membrane proteins.     

Highly conserved serine in the third transmembrane helix of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor activation

The elucidation of the role of highly conserved polar amino acids in the transmembrane helices of G-protein-coupled receptors (GPCRs) is important in understanding the mechanism of receptor activation. To this end, the significance of a highly conserved serine residue in the third transmembrane alpha-helix (TM3) of the luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) in regulating receptor activation was examined. Results showed that mutation of serine 431 to alanine (S431A) decreased the ability of the receptor to mediate cAMP production in response to hCG, suggesting that S431 stabilizes the active state of the receptor. Homology with other GPCRs suggests that S431 may participate in the coordination of a Na(+) ion. Since Na(+) has been found to stabilize the active state of the receptor in the presence of hCG, the possibility that S431 promotes receptor activation by mediating the effects of Na(+) was explored. Results showed that the regulation of hormone-induced receptor activation by S431 was independent of Na(+). A rhodopsin-based homology model of the TM region of the LH/hCGR was developed to identify other amino acids that might mediate the effects of Na(+) on receptor function. Results indicate that substitution of an Asp at position 556 with Tyr alters the ability of Na(+) to regulate receptor activation. The homology model is used to explain this result as well as to identify a mechanism through which S431 may regulate receptor signaling. Taken together, these studies provide novel insights into the mechanism of LH/hCG receptor activation.     

Mutation of a conserved glycine in the SH1-SH2 helix affects the load-dependent kinetics of myosin

The ATP hydrolysis rate and shortening velocity of muscle are load-dependent. At the molecular level, myosin generates force and motion by coupling ATP hydrolysis to lever arm rotation. When a laser trap was used to apply load to single heads of expressed smooth muscle myosin (S1), the ADP release kinetics accelerated with an assistive load and slowed with a resistive load; however, ATP binding was mostly unaffected. To investigate how load is communicated within the motor, a glycine located at the putative fulcrum of the lever arm was mutated to valine (G709V). In the absence of load, stopped-flow and laser trap studies showed that the mutation significantly slowed the rates of ADP release and ATP binding, accounting for the ~270-fold decrease in actin sliding velocity. The load dependence of the mutant's ADP release rate was the same as that of wild-type S1 (WT) despite the slower rate. In contrast, load accelerated ATP binding by ~20-fold, irrespective of loading direction. Imparting mechanical energy to the mutant motor partially reversed the slowed ATP binding by overcoming the elevated activation energy barrier. These results imply that conformational changes near the conserved G709 are critical for the transmission of mechanochemical information between myosin's active site and lever arm.     

Identification of amino acid residues lining the pore of a gap junction channel

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.     

Conformational dynamics of the alphaM3 transmembrane helix during acetylcholine receptor channel gating

Muscle acetylcholine receptors are synaptic ion channels that "gate" between closed- and open-channel conformations. We used Phi-value analysis to probe the transition state of the diliganded gating reaction with regard to residues in the M3, membrane-spanning helix of the muscle acetylcholine receptor alpha-subunit. Phi (a fraction between 1 and 0) parameterizes the extent to which a mutation changes the opening versus the closing rate constant and, for a linear reaction mechanism, the higher the Phi-value, the "earlier" the gating motion. In the upper half of alphaM3 the gating motions of all five tested residues were temporally correlated (Phi approximately 0.30) and serve to link structural changes occurring at the middle of the M2, pore-lining helix with those occurring at the interface of the extracellular and transmembrane domains. alphaM3 belongs to a complex and diverse set of synchronously moving parts that change structure relatively late in the channel-opening process. The propagation of the gating Brownian conformational cascade has a complex spatial distribution in the transmembrane domain.     

Conformational changes in a pore-lining helix coupled to cystic fibrosis transmembrane conductance regulator channel gating

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.     

Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.     

Ductin – a proton pump component,a gap junction channel and a neurotransmitter release channel

Ductin is the highest conserved membrane protein yet found in eukaryotes. It is multifunctional, being the subunit c or proteolipid component of the vacuolar H⁺-ATPase and at the same time the protein component of a form of gap junction in metazoan animals. Analysis of its structure shows it to be a tandem repeat of two 8-kDa domains derived from the subunit c of the F₀ proton pore from the F₁F₀ ATPase. Each domain contains two transmembrane α-helices, which together may form a four-helix bundle. In both the V-ATPase and gap junction channel, ductin is probably arranged as a hexamer of subunits forming a central channel of gap junction-like proportions. The two functions appear to be seggregated by ductin having two orientations in the bilayer. Ductin is also the major component of the mediatophore, a protein complex which may aid in the release of neurotransmitters across the pre-synaptic membrane. It is also a target for a class of poorly understood viral polypeptides. These polypeptides are small and highly hydrophobic and some have oncogenic activity. Ductin thus appears to be at the crossroads of a number of biological processes.     

A Calpha model for the transmembrane alpha helices of gap junction intercellular channels

Gap junction channels connect the cytoplasms of apposed cells via an intercellular conduit formed by the end-to-end docking of two hexameric hemichannels called connexons. We used electron cryomicroscopy to derive a three-dimensional density map at 5.7 angstroms in-plane and 19.8 angstroms vertical resolution, allowing us to identify the positions and tilt angles for the 24 alpha helices within each hemichannel. The four hydrophobic segments in connexin sequences were assigned to the alpha helices in the map based on biochemical and phylogenetic data. Analyses of evolutionary conservation and compensatory mutations in connexin evolution identified the packing interfaces between the helices. The final model, which specifies the coordinates of Calpha atoms in the transmembrane domain, provides a structural basis for understanding the different physiological effects of almost 30 mutations and polymorphisms in terms of structural deformations at the interfaces between helices, revealing an intimate connection between molecular structure and disease.     

Molecular characterization and functional expression of the human cardiac gap junction channel

Gap junctions permit the passage of ions and chemical mediators from cell to cell. To identify the molecular genetic basis for this coupling in the human heart, we have isolated clones from a human fetal cardiac cDNA library which encode the full-length human cardiac gap junction (HCGJ) mRNA. The predicted amino acid sequence is homologous to the rat cardiac gap junction protein, connexin43 (Beyer, E. D., D. Paul, and D. A. Goodenough. 1987. J. Cell Biol. 105:2621-2629), differing by 9 of 382 amino acids. HCGJ mRNA is detected as early as fetal week 15 and persists in adult human cardiac samples. Genomic DNA analysis suggests the presence of two highly homologous HCGJ loci, only one of which is functional. Stable transfection of the HCGJ cDNA into SKHep1 cells, a human hepatoma line which is communication deficient, leads to the formation of functional channels. Junctional conductance in pairs of transfectants containing 10 copies of the HCGJ sequence is high (approximately 20 nS). Single channel currents are detectable in this expression system and correspond to conductances of approximately 60 pS. These first measurements of the HCGJ channel are similar to the junctional conductance recorded between pairs of rat or guinea pig cardiocytes.     

Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA(K) lines a solvent-accessible channel

The staphylococcal TetA(K) tetracycline exporter is classified within the major facilitator superfamily of transport proteins and contains 14 alpha-helical transmembrane segments (TMS). Using cysteine-scanning mutagenesis, 27 amino acid residues across and flanking putative TMS 10 of the TetA(K) transporter were individually replaced with cysteine. The level of solvent accessibility to each of the targeted amino acid positions was determined as a measure of fluorescein maleimide reactivity and demonstrated that TMS 10 of TetA(K) has a cytoplasmic boundary at G313 and is likely to extend from at least V298 on the periplasmic side. TMS 10 was found to be amphiphilic containing at least partially solvent accessible amino acid residues along the length of one helical face, suggesting that this helix may line a solvent-exposed channel. Functional analyses of these cysteine mutants demonstrated a significant role for a number of amino acid residues, including a predominance of glycine residues which were further analyzed by alanine substitution. These residues are postulated to allow interhelical interactions between TMS 10 and distal parts of TetA(K) that are likely to be required for the tetracycline transport mechanism in TetA(K) and may be a general feature required by bacterial tetracycline transporters for activity.     

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation. Direct CaM-Cx interaction at junctional and cytoplasmic spots was demonstrated by confocal immunofluorescence microscopy in HeLa cells transfected with Cx32 and in cryosectioned mouse liver. This was confirmed in HeLa cells coexpressing Cx32-GFP (green) and CaM-RFP (red) or Cx32-CFP (cyan) and CaM-YFP (yellow) fusion proteins. Significantly, these cells did not form gap junctions. In contrast, HeLa cells expressing only one of the two fusion proteins (Cx32-GFP, Cx32-CFP, CaM-RFP or CaM-YFP) revealed both junctional and non-junctional fluorescent spots. In these cells, CaM-Cx32 colocalization was demonstrated by secondary immunofluorescent labeling of Cx32 in cells expressing CaM-YFP or CaM in cells expressing Cx32-GFP. CaM-Cx colocalization was further demonstrated at rat liver gap junctions by Freeze-fracture Replica Immunogold Labeling (FRIL).     

Temperature dependence of embryonic cardiac gap junction conductance and channel kinetics

We have investigated the effects of temperature on the conductance and voltage-dependent kinetics of cardiac gap junction channels between pairs of seven-day embryonic chick ventricle myocytes over the range of 14–26°C. Records of junctional conductance (G _j ) and steady-state unit junctional channel activity were made using the whole-cell double patch-clamp technique while the bath temperature was steadily changed at a rate of about 4°C/min. The decrease inG _j upon cooling was biphasic with a distinct break at 21°C. In 12 cell pairs,Q ₁₀ was 2.2 from 26 to 21°C, while between 21 and 14°C it was 6.5. The meanG _j at 22°C (G _j22 ) was 3.0±2.1 nS, ranging in different preparations from 0.24 to 6.4 nS. At room temperature, embryonic cardiac gap junctions contain channels with conductance states near 240, 200, 160, 120, 80 and 40 pS. In the present study, we demonstrate that cooling decreases the frequency of channel openings at all conductance levels, and at temperatures below 20°C shifts the prevalence of openings from higher to lower conductance states: all 240 pS openings disappear below 20°C; 200 pS openings are suppressed at 17°C; below 16°C 160 and 120 pS events disappear and only 80 and 40 pS states are seen. Temperature also affected the voltage-dependent kinetics of the channels. Application of a 6 sec, 80 mV voltage step across the junction (V _j80 ) caused a biexponential decay in junctional conductance. Decay was faster at lower temperatures, whereas the rate of recovery ofG _j after returning toV _j0 was slowed. Cooling reduced the fast decay time constant, increased both recovery time constants, and decreased the magnitude of GitG_j decay, thus leaving a 10–16% larger residual conductance (G _ss/G _init,±80 mVV _j ) at 18 than at 22°C. From these results we propose that embryonic chick cardiac gap junctions contain at least two classes of channels with different conductances and temperature sensitivities.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of gap junction channel attenuates the migration of breast cancer cells

Gap junction provides intercellular communications that play a critical role in invasion of metastatic cancer cells. However, the effects of inhibiting this pathway in breast cancer cell migration have not been investigated. Here, we present data demonstrating that functional blockade of gap junctions during the formation of monolayer decreased the levels of aligned fibers of actin between neighboring breast cancer cells. Furthermore, gap junction inhibitors attenuated the invasion ability of highly metastatic MDA-MB-231 cells, but had no significant effects on less invasive MCF-7 cells, which caused by shRANKL. Our work is the first to demonstrate the inhibitory effect of gap junction channel inhibitors on the migration of highly invasive breast cancer cells.