Gating of heteromeric retinal rod channels by cyclic AMP: role of the C-terminal and pore domains

Cyclic nucleotide-gated channels are tetramers composed of homologous alpha and beta subunits. C-terminal truncation mutants of the alpha and beta subunits of the retinal rod channel were expressed in Xenopus oocytes, and analyzed for cGMP- and cAMP-induced currents (single-channel records and macroscopic currents). When the alpha subunit truncated downstream of the cGMP-binding site (alpha D608stop) is co-injected with truncated beta subunits, the heteromeric channels present a drastic increase of cAMP sensitivity. A partial effect is observed with heteromeric alpha R656stop-containing channels, while alpha K665stop-containing channels behave like alpha wt/beta wt. The three truncated alpha subunits have wild-type activity when expressed alone. Heteromeric channels composed of alpha wt or truncated alpha subunits and chimeric beta subunits containing the pore domain of the alpha subunit have the same cAMP sensitivity as alpha-only channels. The results disclose the key role of two domains distinct from the nucleotide binding site in the gating of heteromeric channels by cAMP: the pore of the beta subunit, which has an activating effect, and a conserved domain situated downstream of the cGMP-binding site in the alpha subunit (I609-K665), which inhibits this effect.     

Probing the interactions between cAMP and cGMP in cyclic nucleotide-gated channels using covalently tethered ligands

Cyclic nucleotide-gated channels contain four ligand-binding subunits, and they are directly activated by the binding of cGMP or cAMP. Channels with different combinations of subunits are known to have different sensitivities to the two nucleotides. However, the consequences of mixed occupancy by cGMP and cAMP are not well understood, and may have important implications for understanding the functions of these channels in different cell types. We studied the activation of homomeric and heteromeric retinal rod cyclic nucleotide-gated channels with the four ligand-binding sites occupied by different combinations of cGMP (a strong agonist) and cAMP (a weak agonist). Control of occupancy was obtained by covalently tethering different numbers of cGMP moieties using the photoaffinity analogue 8-p-azidophenacylthio-cGMP; the remaining sites were then saturated with cAMP, or cGMP, for comparison. The fractional current activated by cAMP increased dramatically as the number of tethered cGMP moieties increased. In homomeric channels comprised of the alpha subunit, cAMP became an effective agonist only after three of the four sites were occupied by tethered cGMP moieties. In contrast, in heteromeric channels comprised of two alpha and two beta subunits, cAMP caused significant activation after two sites were occupied by tethered cGMP moieties. In agreement with earlier work, a single residue on the beta subunit, N1201, accounted for much of the increased efficacy of cAMP on heteromeric channels. The results are consistent with significant interactions between subunits, including the two types of subunits in heteromeric channels.     

Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.     

Second transmembrane domains of ENaC subunits contribute to ion permeation and selectivity

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.     

A distinct contribution of the delta subunit to acetylcholine receptor channel activation revealed by mutations of the M2 segment.

Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12' position of the M2 segment) were examined at the single-channel level. For all subunits (alpha, beta, epsilon, and delta), a 12'P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12'P mutations were co-expressed with alpha subunits having a binding site mutation that slows channel opening (alphaD200N). In these AChRs, a 12'P mutation in epsilon or beta slowed the closing rate constant approximately 6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12'P mutation in delta slowed the channel closing rate constant only approximately 2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12'P subunits indicates that mutations in epsilon and beta act nearly independently, but one in delta reduces the effect of a homologous mutation in epsilon or beta. The results suggest that a 12'P mutation in epsilon and beta has mainly local effects, whereas one in delta has both local and distributed effects that influence both agonist binding and channel gating.     

Proteolytic processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation

Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the alpha subunit and at a single extracellular site within the gamma subunit. Channels lacking furin processing of the alpha subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK(186)) in the gamma subunit. We also demonstrated that the tract alpha D206-R231, between the two furin cleavage sites in the alpha subunit, as well as the tract gamma E144-K186, between the furin and prostasin cleavage sites in the gamma subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the alpha and gamma subunit inhibitory tracts, respectively. We examined whether release of either thealpha orgamma inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the alpha subunit (alphaR205A,R208A,R231Abetagamma) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the proteolytic processing of thealpha subunit, amiloride-sensitive currents in oocytes expressing channels carrying gamma subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (alphabetagammaR143A,DeltaE144-K186 and alphaR205A,R208A,R231AbetagammaR143A, DeltaE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the alpha and gamma subunit inhibitory tracts, alpha subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in thegamma subunit (alphabetagammaR143A,DeltaE144-K186) showed a significant reduction in the efficacy of block by the syntheticalpha-26 inhibitory peptide representing the tract alphaD206-R231. Our data indicate that removal of the inhibitory tract from the gamma subunit, in the absence ofalpha subunit cleavage, results in nearly full activation of the channel.     

Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release

Electrophysiological studies of neurons reveal different Ca2+ currents designated L-, N-, P-, Q-, R-, and T-type. High-voltage-activated neuronal Ca2+ channels are complexes of a pore-forming alpha 1 subunit of about 190-250 kDa, a transmembrane, disulfide-linked complex of alpha 2 and delta subunits, and an intracellular beta subunit, similar to the alpha 1, alpha 2 delta, and beta subunits previously described for skeletal muscle Ca2+ channels. The primary structures of these subunits have all been determined by homology cDNA cloning using the corresponding subunits of skeletal muscle Ca2+ channels as probes. In most neurons, L-type channels contain alpha 1C or alpha 1D subunits, N-type contain alpha 1B subunits, P- and Q-types contain alternatively spliced forms of alpha 1A subunits, R-type contain alpha 1E subunits, and T-type contain alpha 1G or alpha 1H subunits. Association with different beta subunits also influences Ca2+ channel gating substantially, yielding a remarkable diversity of functionally distinct molecular species of Ca2+ channels in neurons.     

Differential effects of beta 1 and beta 2 subunits on BK channel activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.     

Mutating three residues in the bovine rod cyclic nucleotide-activated channel can switch a nucleotide from inactive to active

Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N(1)-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the alpha subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N(1)-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod alpha subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N(1)-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences.     

Incomplete incorporation of tandem subunits in recombinant neuronal nicotinic receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking alpha (alpha2-alpha4, alpha6) and beta (beta2, beta4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (beta4_alpha2, beta4_alpha3, beta4_alpha4, and beta2_alpha4) when coexpressed with the corresponding beta monomer subunit. All tandems expressed when injected alone, except for beta4_alpha3, which produced functional channels only together with beta4 monomer and was chosen for further characterization. These channels produced from beta4_alpha3 tandem constructs plus beta4 monomer were identical with receptors expressed from monomer alpha3 and beta4 constructs in acetylcholine sensitivity and in the number of alpha and beta subunits incorporated in the channel gate. However, separately mutating the beta subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of beta subunits from the tandem and one from the beta monomer construct, whereas the rest incorporated two or three beta monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily.     

Potentiation of neuronal L calcium channels by IGF-1 requires phosphorylation of the alpha1 subunit on a specific tyrosine residue

Insulin-like growth factor 1 (IGF-1) rapidly potentiates N and L calcium channel currents in cerebellar granule neurons by an unknown mechanism. Here, we show that the L channel alpha1C subunit is tyrosine phosphorylated in response to IGF-1. Moreover, expression of kinase-dead c-Src in neurons or acute block of Src family kinases with a cell-permeable inhibitor specifically blocks L channel potentiation. Purified Src kinase phosphorylates tyrosine residue Y2122 of the C terminus of neuronal alpha1C in vitro, and c- and v-Src directly bind the C terminus. When expressed in neuroblastoma cells, point mutation of Y2122 prevents both tyrosine phosphorylation of alpha1C and IGF-1 potentiation. Our data provide a biochemical mechanism whereby phosphorylation of a single specific tyrosine residue rapidly modifies ion channel physiology.     

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Efficient coupling of ligand binding to channel opening by the binding domain of a modulatory (beta) subunit of the olfactory cyclic nucleotide-gated channel.

CNG channels in vivo are heteromers of homologous alpha and beta subunits that each contain a six-transmembrane segment domain and a COOH-terminal cytoplasmic cyclic nucleotide binding domain (BD). In heterologous expression systems, heteromeric alphabeta channels activate with greater sensitivity to ligand than do homomeric alpha channels; however, ligand-gating of channels containing only beta subunit BDs has never been studied because beta subunits cannot form functional homomeric CNG channels. To characterize directly the contribution of the beta subunit BD to ligand-gating, we constructed a chimeric subunit, X-beta, whose BD sequence was that of the beta subunit CNG5 from rat, but whose sequence outside the BD was derived from alpha subunits. For comparison, we constructed another chimera, X-alpha, whose sequence outside the BD was identical to that of X-beta, but whose BD sequence was that of the alpha subunit CNG2 from catfish. When expressed in Xenopus oocytes, X-beta and X-alpha each formed functional homomeric channels activated by both cAMP and cGMP. This is the first demonstration that the beta subunit BD can couple ligand binding to activation in the absence of alpha subunit BD residues. Notably, both agonists activate X-beta more effectively than X-alpha (higher opening efficacy and lower K(1/2)). The BD is believed to comprise two functionally distinct subdomains: (1) the roll subdomain (beta-roll and flanking A- and B-helices) and (2) the C-helix subdomain. Opening efficacy was previously believed to be controlled primarily by the C-helix, but when we made additional chimeras by exchanging the subdomains between X-beta and X-alpha, we found that both subdomains contain significant determinants of efficacy and agonist selectivity. In particular, only channels containing the roll subdomain of the beta subunit had high efficacy. Thermodynamic linkage analysis shows that interaction between the two subdomains accounts for a significant portion of their contribution to activation energetics.     

Identification of an amino acid defining the distinct properties of murine beta1 and beta3 subunit-containing GABA(A) receptors

Murine gamma-aminobutyric acid (GABA) type A homomeric receptors made of beta1 subunits are profoundly different, when expressed in Xenopus oocytes, from beta3 homomeric receptors. Application of the intravenous general anesthetic pentobarbital, etomidate, or propofol to beta3 homomeric receptors allows current flow. In contrast, beta1 homomers do not respond to any of these agents. Through construction of chimeric beta1/beta3 receptors, we identified a single amino acid that determines the pharmacological difference between the two beta subunits. When the serine residue present in the wild-type nonresponsive beta1 subunit is replaced by an asparagine found in the same position in the beta3 subunit, the resulting point-mutated beta1S265N forms receptors responsive to intravenous general anesthetics, like the wild-type beta3 subunits. Conversely, after mutation of the wild-type beta3 to beta3N265S, the homomeric receptor loses its ability to respond to these same general anesthetics. Wild-type-to-mutant titration experiments showed that the nonresponsive phenotype is dominant: A single nonresponsive residue within a pentameric receptor is sufficient to render the receptor nonresponsive. In alpha1betax or alpha1betaxgamma2 heteromeric receptors, the same residue manifests as a partial determinant of the degree of potentiation of the GABA-induced current by some general anesthetics. The location of this amino acid at the extracellular end of the second transmembrane segment, its influence in both homomeric and heteromeric receptor function, and its dominant behavior suggest that this residue of the beta subunit is involved in an allosteric modulation of the receptor.     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

Identification of sites responsible for potentiation of type 2.3 calcium currents by acetyl-beta-methylcholine

To address mechanisms for the differential sensitivity of voltage-gated Ca2+ channels (Cav) to agonists, channel activity was compared in Xenopus oocytes coexpressing muscarinic M(1) receptors and different Cav alpha1 subunits, all with beta1B,alpha2/delta subunits. Acetyl-beta-methylcholine (MCh) decreased Cav 1.2c currents, did not affect 2.1 or 2.2 currents, but potentiated Cav 2.3 currents. Phorbol 12-myristate 13-acetate (PMA) did not affect Cav 1.2c or 2.1 currents but potentiated 2.2 and 2.3 currents. Comparison of the amino acid sequences of the alpha1 subunits revealed a set of potential protein kinase C phosphorylation sites in common between the 2.2 and 2.3 channels that respond to PMA and a set of potential sites unique to the alpha1 2.3 subunits that respond to MCh. Quadruple Ser --> Ala mutation of the predicted MCh sites in the alpha1 2.3 subunit (Ser-888, Ser-892, and Ser-894 in the II-III linker and Ser-1987 in the C terminus) caused loss of the MCh response but not the PMA response. Triple Ser --> Ala mutation of just the II-III linker sites gave similar results. Ser-888 or Ser-892 was sufficient for the MCh responsiveness, whereas Ser-894 required the presence of Ser-1987. Ser --> Asp substitution of Ser-888, Ser-892, Ser-1987, and Ser-892/Ser-1987 increased the basal current and decreased the MCh response but did not alter the PMA response. These results reveal that sites unique to the II-III linker of alpha1 2.3 subunits mediate the responsiveness of Cav 2.3 channels to MCh. Because Cav 2.3 channels contribute to action potential-induced Ca2+ influx, these sites may account for M1 receptor-mediated regulation of neurotransmission at some synapses.     

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.     

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.