Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels.

Cyclic nucleotide-gated (CNG) ion channels of olfactory sensory neurons contain three types of homologue subunits, two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit. Each subunit carries an intracellular cyclic nucleotide binding domain (CNBD) whose occupation by up to four cyclic nucleotides evokes channel activation. Thereby, the subunits interact in a cooperative fashion. Here we studied 16 concatamers with systematically disabled, but still functional, binding sites and quantified channel activation by systems of intimately coupled state models transferred to 4D hypercubes, thereby exploiting a weak voltage dependence of the channels. We provide the complete landscape of free energies for the complex activation process of heterotetrameric channels, including 32 binding steps, in both the closed and open channel, as well as 16 closed-open isomerizations. The binding steps are specific for the subunits and show pronounced positive cooperativity for the binding of the second and the third ligand. The energetics of the closed-open isomerizations were disassembled to elementary subunit promotion energies for channel opening, [Formula: see text], adding to the free energy of the closed-open isomerization of the empty channel, E0. The [Formula: see text] values are specific for the four subunits and presumably invariant for the specific patterns of liganding. In conclusion, subunit cooperativity is confined to the CNBD whereas the subunit promotion energies for channel opening are independent.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Corrigendum: Quantifying the cooperative subunit action in a multimeric membrane receptor.

This corrects the article DOI: 10.1038/srep20974.

Quantifying the cooperative subunit action in a multimeric membrane receptor.

In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 µM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.

Exon 11 skipping of SCN10A coding for voltage-gated sodium channels in dorsal root ganglia.

The voltage-gated sodium channel Na(V)1.8 (encoded by SCN10A) is predominantly expressed in dorsal root ganglia(DRG) and plays a critical role in pain perception. We analyzed SCN10A transcripts isolated from human DRGs using deep sequencing and found a novel splice variant lacking exon 11, which codes for 98 amino acids of the domain I/II linker. Quantitative PCR analysis revealed an abundance of this variant of up to 5­10% in human, while no such variants were detected in mouse or rat. Since no obvious functional differences between channels with and without the exon-11 sequence were detected, it is suggested that SCN10A exon 11 skipping in humans is a tolerated event.

Mechanism and molecular basis for the sodium channel subtype specificity of µ-conopeptide CnIIIC.

BACKGROUND AND PURPOSE: Voltage-gated sodium channels (Na(V) channels) are key players in the generation and propagation of action potentials, and selective blockade of these channels is a promising strategy for clinically useful suppression of electrical activity. The conotoxin µ-CnIIIC from the cone snail Conus consors exhibits myorelaxing activity in rodents through specific blockade of skeletal muscle (Na(V) 1.4) Na(V) channels. EXPERIMENTAL APPROACH: We investigated the activity of µ-CnIIIC on human Na(V) channels and characterized its inhibitory mechanism, as well as the molecular basis, for its channel specificity. KEY RESULTS: Similar to rat paralogs, human Na(V) 1.4 and Na(V) 1.2 were potently blocked by µ-CnIIIC, the sensitivity of Na(V) 1.7 was intermediate, and Na(V) 1.5 and Na(V) 1.8 were insensitive. Half-channel chimeras revealed that determinants for the insensitivity of Na(V) 1.8 must reside in both the first and second halves of the channel, while those for Na(V) 1.5 are restricted to domains I and II. Furthermore, domain I pore loop affected the total block and therefore harbours the major determinants for the subtype specificity. Domain II pore loop only affected the kinetics of toxin binding and dissociation. Blockade by µ-CnIIIC of Na(V) 1.4 was virtually irreversible but left a residual current of about 5%, reflecting a 'leaky' block; therefore, Na(+) ions still passed through µ-CnIIIC-occupied Na(V) 1.4 to some extent. TTX was excluded from this binding site but was trapped inside the pore by µ-CnIIIC. CONCLUSION AND IMPLICATIONS: Of clinical significance, µ-CnIIIC is a potent and persistent blocker of human skeletal muscle Na(V) 1.4 that does not affect activity of cardiac Na(V) 1.5.

Molecular determinants for the subtype specificity of µ-conotoxin SIIIA targeting neuronal voltage-gated sodium channels.

Voltage-gated sodium channels (Na(V) channels) play a pivotal role in neuronal excitability; they are specifically targeted by µ-conotoxins from the venom of marine cone snails. These peptide toxins bind to the outer vestibule of the channel pore thereby blocking ion conduction through Na(V) channels. µ-Conotoxin SIIIA from Conus striatus was shown to be a potent inhibitor of neuronal sodium channels and to display analgesic effects in mice, albeit the molecular targets are not unambiguously known. We therefore studied recombinant Na(V) channels expressed in mammalian cells using the whole-cell patch-clamp method. Synthetic µSIIIA slowly and partially blocked rat Na(V)1.4 channels with an apparent IC(50) of 0.56 ± 0.29 µM; the block was not complete, leaving at high concentration a residual current component of about 10% with a correspondingly reduced single-channel conductance. At 10 µM, µSIIIA potently blocked rat Na(V)1.2, rat and human Na(V)1.4, and mouse Na(V)1.6 channels; human Na(V)1.7 channels were only inhibited by 58.1 ± 4.9%, whereas human Na(V)1.5 as well as rat and human Na(V)1.8 were insensitive. Employing domain chimeras between rNa(V)1.4 and hNa(V)1.5, we located the determinants for µSIIIA specificity in the first half of the channel protein with a major contribution of domain-2 and a minor contribution of domain-1. The latter was largely accounted for by the alteration in the TTX-binding site (Tyr401 in rNa(V)1.4, Cys for Na(V)1.5, and Ser for Na(V)1.8). Introduction of domain-2 pore loops of all tested channel isoforms into rNa(V)1.4 conferred the µSIIIA phenotype of the respective donor channels highlighting the importance of the domain-2 pore loop as the major determinant for µSIIIA's subtype specificity. Single-site substitutions identified residue Ala728 in rNa(V)1.4 as crucial for its high sensitivity toward µSIIIA. Likewise, Asn889 at the homologous position in hNa(V)1.7 is responsible for the channel's reduced µSIIIA sensitivity. These results will pave the way for the rational design of selective Na(V)-channel antagonists for research and medical applications.

A subtle alternative splicing event of the Na(V)1.8 voltage-gated sodium channel is conserved in human, rat, and mouse.

The voltage-gated sodium channel subtype Na(V)1.8 (SCN10A) is exclusively expressed in dorsal root ganglia (DRG) and plays a critical role in pain perception. We isolated mRNA from human, rat, and mouse DRGs and screened for alternatively spliced isoforms of the SCN10A mRNA using 454 sequencing. In all three species, we found an event of subtle alternative splicing at a NAGNAG tandem acceptor that results in isoforms including or lacking glutamine 1030 (Na(V)1.8+Q and Na(V)1.8-Q, respectively) within the cytoplasmic loop between domains II and III. The relative amount of Na(V)1.8-Q mRNA in adult DRG was measured with 14.1 +/- 0.1% in humans and 11.2 +/- 0.2% in rats. This is in contrast to an abundance of 64.3 +/- 0.3% in mouse DRG. Thus, the NAGNAG tandem acceptor in SCN10A is conserved among rodents and humans but its alternative usage apparently occurs with species-specific abundance. Analysis of human Na(V)1.8+Q and -Q isoforms in whole-cell patch-clamp experiments after heterologous expression in the neuroblastoma cell line Neuro-2A revealed no obvious impact of the splicing event on channel function.