Molecular determinants of a Ca2+-binding site in the pore of cyclic nucleotide-gated channels: S5/S6 segments control affinity of intrapore glutamates.

Cyclic nucleotide-gated (CNG) channels play an important role in Ca2+ signaling in many cells. CNG channels from various tissues differ profoundly in their Ca2+ permeation properties. Using the voltage-dependent Ca2+ blockage of monovalent current in wild-type channels, chimeric constructs and point mutants, we have identified structural elements that determine the distinctively different interaction of Ca2+ with CNG channels from rod and cone photoreceptors and olfactory neurons. Segments S5 and S6 and the extracellular linkers flanking the pore region are the only structural elements that account for the differences between channels. Ca2+ blockage is strongly modulated by external pH. The different pH dependence of blockage suggests that the pKa of intrapore glutamates and their protonation pattern differ among channels. The results support the hypothesis that the S5-pore-S6 module, by providing a characteristic electrostatic environment, determines the protonation state of pore glutamates and thereby controls Ca2+ affinity and permeation in each channel type.     

Functionally important calmodulin-binding sites in both NH2- and COOH-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.     

Voltage and calcium use the same molecular determinants to inactivate calcium channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.     

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.     

Mechanism of beta4 subunit modulation of BK channels

Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.     

Voltage-dependence of ion permeation in cyclic GMP-gated ion channels is optimized for cell function in rod and cone photoreceptors

The kinetics of the photocurrent in both rod and cone retinal photoreceptors are independent of membrane voltage over the physiological range (-30 to -65 mV). This is surprising since the photocurrent time course is regulated by the influx of Ca(2+) through cGMP-gated ion channels (CNG) and the force driving this flux changes with membrane voltage. To understand this paradigm, we measured Pf, the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact, isolated photoreceptors. To measure Pf we activated CNG channels by suddenly increasing free 8-Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+) binding dye, Fura-2, also loaded into the cells. We determined Pf under physiological solutions at various holding membrane voltages between -65 and -25 mV. Pf is larger in cones than in rods, but in both photoreceptor types its value is independent of membrane voltage over the range tested. This biophysical feature of the CNG channels offers a functional advantage since it insures that the kinetics of the phototransduction current are controlled by light, and not by membrane voltage. To explain our observation, we developed a rate theory model of ion permeation through CNG channels that assumes the existence of two ion binding sites within the permeation pore. To assign values to the kinetic rates in the model, we measured experimental I-V curves in membrane patches of rods and cones over the voltage range -90 to 90 mV in the presence of simple biionic solutions at different concentrations. We optimized the fit between simulated and experimental data. Model simulations describe well experimental photocurrents measured under physiological solutions in intact cones and are consistent with the voltage-independence of Pf, a feature that is optimized for the function of the channel in photoreceptors.     

Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.     

Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

The Ca-activated Cl channel and its control in rat olfactory receptor neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was approximately 30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were approximately 8 and 62 micro m-2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.     

Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.     

Cellular processing of cone photoreceptor cyclic GMP-gated ion channels: a role for the S4 structural motif

We examined cellular protein processing and functional expression of photoreceptor cyclic nucleotide-gated (CNG) ion channels. In a mammalian cell line, wild type bovine cone photoreceptor channel alpha subunits (bCNGA3) convert from an unglycosylated state, at 90 kDa, to two glycosylated states at 93 and 102 kDa as they transit within the cell to their final location at the plasma membrane. Glycosylation per se is not required to yield functional channels, yet it is a milestone that distinguishes sequential steps in channel protein maturation. CNG ion channels are not gated by membrane voltage although their structure includes the transmembrane S4 motif known to function as the membrane voltage sensor in all voltage-gated ion channels. S4 must be functionally important because its natural mutation in cone photoreceptor CNG channels is associated with achromatopsia, a human autosomal inherited loss of cone function. Point mutation of specific, not all, charged and neutral residues within S4 cause failure of functional channel expression. Cellular channel protein processing fails in every one of the non-functional S4 mutations we studied. Mutant proteins do not reach the 102-kDa glycosylated state and do not arrive at the plasma membrane. They remain trapped within the endoplasmic reticulum and fail to transit out to the Golgi apparatus. Coexpression of cone CNG beta subunit (CNGB3) does not rescue the consequence of S4 mutations in CNGA3. It is likely that an intact S4 is required for proper protein folding and/or assembly in the endoplasmic reticulum membrane.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Activation of the BK (SLO1) potassium channel by mallotoxin

Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.     

Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero-oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S-202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit-containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.     

Multimerization of the ligand binding domains of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.     

Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.