Exploring HCN channels as novel drug targets

Nature Reviews Drug Discovery, 2011, doi:10.1038/nrd3576, published on 18.11.2011
Nature Reviews Drug Discovery, online article
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of heart rate and neuronal excitability. Ivabradine is the first compound acting on HCN channels to be clinically approved for the treatment of angina pectoris. HCN channels may offer excellent opportunities for the development of novel anticonvulsant, anaesthetic and analgesic drugs. In support of this idea, some well-established drugs that act on the central nervous system — including lamotrigine, gabapentin and propofol — have been found to modulate HCN channel function. This Review gives an up-to-date summary of compounds acting on HCN channels, and discusses strategies to further explore the potential of these channels for therapeutic intervention.


Full View

The Role of HCN Channels in Ventricular Repolarization

Trends in Cardiovascular Medicine, 2012, http://dx.doi.org/10.1016/j.tcm.2012.05.013, Volume 21, Issue 8, Pages 216–220 published on 15.11.2011
Trends in Cardiovascular Medicine, online article
Hyperpolarization-activated cyclic nucleotide gated (HCN) channels pass a cationic current (Ih/If) that crucially contributes to the slow diastolic depolarization (SDD) of sinoatrial pacemaker cells and, hence, is a key determinant of cardiac automaticity and the generation of the heartbeat. However, there is growing evidence that HCN channels are not restricted to the spontaneously active cells of the sinoatrial node and the conduction system but are also present in ventricular cardiomyocytes that produce an action potential lacking SDD. This observation raises the question of the principal function(s) of HCN channels in working myocardium. Our recent analysis of an HCN3-deficient (HCN3–/–) mouse line has shed new light on this central question. We propose that HCN channels contribute to the ventricular action potential waveform, specifically during late repolarization. In this review, we outline this new concept.



Full View

Spatial constraints dictate glial territories at murine neuromuscular junctions

The Journal of Cell Biology, 2011, doi: 10.1083/jcb.201108005, vol. 195 no. 2 293-305, published on 17.10.2011
The Journal of Cell Biology, online article
Schwann cells (SCs), the glial cells of the peripheral nervous system, cover synaptic terminals, allowing them to monitor and modulate neurotransmission. Disruption of glial coverage leads to axon degeneration and synapse loss. The cellular mechanisms that establish and maintain this coverage remain largely unknown. To address this, we labeled single SCs and performed time-lapse imaging experiments. Adult terminal SCs are arranged in static tile patterns, whereas young SCs dynamically intermingle. The mechanism of developmental glial segregation appears to be spatial competition, in which glial–glial and axonal–glial contacts constrain the territory of single SCs, as shown by four types of experiments: laser ablation of single SCs, which led to immediate territory expansion of neighboring SCs; axon removal by transection, resulting in adult SCs intermingling dynamically; axotomy in mutant mice with blocked axon fragmentation in which intermingling was delayed; and activity blockade, which had no immediate effects. In summary, we conclude that glial cells partition synapses by competing for perisynaptic space.

Misgeld_Brill et al JCB 2011_500

Full View

HCN3 Contributes to the Ventricular Action Potential Waveform in the Murine Heart

Circulation Research, 2011, doi: 10.1161/​CIRCRESAHA.111.246173, published on 09.09.2011
Human Molecular Genetics, online article
Rationale: The hyperpolarization-activated current Ih that is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) plays a key role in the control of pacemaker activity in sinoatrial node cells of the heart. By contrast, it is unclear whether Ih is also relevant for normal function of cardiac ventricles. Objective: To study the role of the HCN3-mediated component of ventricular Ih in normal ventricular function. Methods and Results: To test the hypothesis that HCN3 regulates the ventricular action potential waveform, we have generated and analyzed a HCN3-deficient mouse line. At basal heart rate, mice deficient for HCN3 displayed a profound increase in the T-wave amplitude in telemetric electrocardiographic measurements. Action potential recordings on isolated ventricular myocytes indicate that this effect was caused by an acceleration of the late repolarization phase in epicardial myocytes. Furthermore, the resting membrane potential was shifted to more hyperpolarized potentials in HCN3-deficient mice. Cardiomyocytes of HCN3-deficient mice displayed approximately 30% reduction of total Ih. At physiological ionic conditions, the HCN3-mediated current had a reversal potential of approximately −35 mV and displayed ultraslow deactivation kinetics. Conclusions: We propose that HCN3 together with other members of the HCN channel family confer a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization. In conclusion, our data indicate that HCN3 plays an important role in shaping the cardiac action potential waveform.


Full View

Chemo- and Thermosensory Responsiveness of Grueneberg Ganglion Neurons Relies on Cyclic Guanosine Monophosphate Signaling Elements

Neurosignals, 2011, DOI: 10.1159/000329333, published on 31.08.2011
Neurosignals, online article
Neurons of the Grueneberg ganglion (GG) in the anterior na- sal region of mouse pups respond to cool temperatures and to a small set of odorants. While the thermosensory reactiv- ity appears to be mediated by elements of a cyclic guanosine monophosphate (cGMP) cascade, the molecular mecha- nisms underlying the odor-induced responses are unclear. Since odor-responsive GG cells are endowed with elements of a cGMP pathway, specifically the transmembrane guany- lyl cyclase subtype GC-G and the cyclic nucleotide-gated ion channel CNGA3, the possibility was explored whether these cGMP signaling elements may also be involved in chemo- sensory GG responses. Experiments with transgenic mice deficient for GC-G or CNGA3 revealed that GG responsive- ness to given odorants was significantly diminished in these knockout animals. These findings suggest that a cGMP cas- cade may be important for both olfactory and thermosen- sory signaling in the GG. However, in contrast to the thermo- sensory reactivity, which did not decline over time, the che- mosensory response underwent adaptation upon extended stimulation, suggesting that the two transduction processes only partially overlap.


Full View

Dendritic coding of multiple sensory inputs in single cortical neurons in vivo

PNAS, 2011, doi: 10.1073/pnas.1112355108, published on 29.08.2011
PNAS, online article
Single cortical neurons in the mammalian brain receive signals arising from multiple sensory input channels. Dendritic integration of these afferent signals is critical in determining the amplitude and time course of the neurons’ output signals. As of yet, little is known about the spatial and temporal organization of converging sensory inputs. Here, we combined in vivo two-photon imaging with whole-cell recordings in layer 2 neurons of the mouse vibrissal cortex as a means to analyze the spatial pattern of subthreshold dendritic calcium signals evoked by the stimulation of different whiskers. We show that the principle whisker and the surrounding whiskers can evoke dendritic calcium transients in the same neuron Distance-dependent attenuation of dendritic calcium transients and the corresponding subthreshold depolarization suggest feed-forward activation. We found that stimulation of different whiskers produced multiple calcium hotspots on the same dendrite. Individual hotspots were activated with low probability in a stochastic manner. We show that these hotspots are generated by calcium signals arising in dendritic spines. Some spines were activated uniquely by singlewhiskers, butmany spineswere activated bymultiple whiskers. These shared spines indicate the existence of presynaptic feeder neurons that integrate and transmit activity arising frommultiple whiskers. Despite the dendritic overlap of whisker-specific and shared inputs, different whiskers are represented by a unique set of activation patterns within the dendritic field of each neuron.

konnerth_varga_et al_pnas_online_2011_500

Full View

Development of Direction Selectivity in Mouse Cortical Neurons

Neuron, 2011, doi:10.1016/j. neuron.2011.06.013., Volume 71, Issue 3, 425-432, published on 11.08.2011
Neuron, online article
Previous studies of the ferret visual cortex indicate that the development of direction selectivity requires visual experience. Here,weused two-photon calcium imaging to study the development of direction selectivity in layer 2/3 neurons of the mouse visual cortex in vivo. Surprisingly, just after eye opening nearly all orientation-selective neurons were also direction selective. During later development, the number of neurons responding to drifting gratings increased in parallel with the fraction of neurons that were orientation, but not direction, selective. Our experiments demonstrate that direction selectivity develops normally in dark-reared mice, indicating that the early development of direction selectivity is independent of visual experience. Furthermore, remarkable functional similarities exist between the development of direction selectivity in cortical neurons and the previously reported development of direction selectivity in the mouse retina. Together, these findings provide strong evidence that the development of orientation and direction selectivity in the mouse brain is distinctly different from that in ferrets.

konnerth_rochefort_et al_neuron_2011_500

Full View

CNGA3: A Target of Spinal Nitric Oxide/cGMP Signaling and Modulator of Inflammatory Pain Hypersensitivity

The Journal of Neuroscience, 2011, doi: 10.1523/​JNEUROSCI.6159-10.2011, 11184-11192; published on 03.08.2011
The Journal of Neuroscience, online article
A large body of evidence indicates that nitric oxide (NO) and cGMP contribute to central sensitization of pain pathways during inflammatory pain. Here, we investigated the distribution of cyclic nucleotide-gated (CNG) channels in the spinal cord, and identified the CNG channel subunit CNGA3 as a putative cGMP target in nociceptive processing. In situ hybridization revealed that CNGA3 is localized to nhibitory neurons of the dorsal horn of the spinal cord, whereas its distribution in dorsal root ganglia is restricted to non-neuronal cells. CNGA3 expression is upregulated in the superficial dorsal horn of the mouse spinal cord and in dorsal root ganglia following hindpaw inflammation evoked by zymosan. Mice lacking CNGA3 (CNGA3 -/- mice) exhibited an increased nociceptive behavior in models of inflammatory pain, whereas their behavior in models of acute or neuropathic pain was normal. Moreover, CNGA3 -/- mice developed an exaggerated pain hypersensitivity induced by intrathecal administration of cGMP analogs or NO donors. Ourm results provide evidence that CNGA3 contributes in an inhibitory manner to the central sensitization of pain pathways during inflammatory pain as a target of NO/cGMP signaling.



Full View

Functional mapping of single spines in cortical neurons in vivo

Nature, 2011, doi:10.1038/nature10193, 475, 501–505 published on 26.06.2011
Nature, online article
The individual functional properties and spatial arrangement of afferent synaptic inputs on dendrites have a critical role in the processing of information by neurons in the mammalian brain. Although recent work has identified visually-evoked local dendritic calcium signals in the rodent visual cortex, sensory-evoked signalling on the level of dendritic spines, corresponding to individual afferent excitatory synapses, remains unexplored6. Here we used a new variant of high-resolution two-photon imaging7 to detect sensory-evoked calcium transients in single dendritic spines of mouse cortical neurons in vivo. Calcium signals evoked by sound stimulation required the activation of NMDA (N-methyl-D-aspartate) receptors. Active spines are widely distributed on basal and apical dendrites and pure-tone stimulation at different frequencies revealed both narrowly and widely tuned spines. Notably, spines tuned for different frequencies were highly interspersed on the same dendrites: even neighbouring spines were mostly tuned to different frequencies. Thus, our results demonstrate thatNMDA-receptor-dependent single-spine synaptic inputs to the same dendrite are highly heterogeneous. Furthermore, our study opens the way for in vivo mapping of functionally defined afferent sensory inputs with single-synapse resolution.

konnerth_chen_et al_nature_2011_500

Full View

Near-infrared branding efficiently correlates light and electron microscopy

Nature Methods, 2011, doi:10.1038/nmeth.1622, 568–570 (2011) published on 05.06.2011
Nature Methods, online article
The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.


Full View

In vivo imaging reveals a phase-specific role of STAT3 during central and peripheral nervous system axon regeneration

PNAS, 2011, doi: 10.1073/pnas.1015239108, vol. 108, no.15, 6282-6287 published on 12.04.2011
PNAS, online article
In the peripheral nervous system (PNS), damaged axons regenerate successfully, whereas axons in the CNS fail to regrow. In neurons of the dorsal root ganglia (DRG), which extend branches to both the PNS and CNS, only a PNS lesion but not a CNS lesion induces axonal growth. How this differential growth response is regulated in vivo is only incompletely understood. Here, we combine in vivo timelapse fluorescence microscopy with genetic manipulations in mice to reveal how the transcription factor STAT3 regulates axonal regeneration. We show that selective deletion of STAT3 in DRG neurons of STAT3-floxed mice impairs regeneration of peripheral DRG branches after a nerve cut. Further, overexpression of STAT3 induced by viral gene transfer increases outgrowth and collateral sprouting of central DRG branches after a dorsal column lesion by more than 400%. Notably, repetitive in vivo imaging of individual fluorescently labeled PNS and CNS axons reveals that STAT3 selectively regulates initiation but not later perpetuation of axonal growth. With STAT3, we thus identify a phase-specific regulator of axonal outgrowth. Activating STAT3 might provide an opportunity to “jumpstart” regeneration, and thus prime axons in the injured spinal cord for application of complementary therapies that improve axonal elongation.

misgeld_Bareyre et al_Misgeld_500

Full View

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

Nature Medicine, 2011, doi:10.1038/nm.2324, 495–499 (2011) published on 27.03.2011
Nature Medicine, online article
In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits1,2. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed ‘focal axonal degeneration’ (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view2–6, demyelination—a hallmark of multiple sclerosis—is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.


Full View

Attenuated Abeta42 responses to low potency gamma-secretase modulators can be overcome for many pathogenic presenilin mutants by second-generation compounds

The Journal of Biological Chemistry, 2010, doi: 10.1074/jbc.M110.213587, published on 25.02.2011
The Journal of Biological Chemistry, online article
Sequential processing of the beta-amyloid precursor protein (APP) by beta- and gamma - secretase generates the amyloid beta - peptide (Abeta), which is widely believed to play a causative role in Alzheimer´s disease (AD). Selective lowering of the pathogenic 42 amino acid variant of Abeta by gamma-secretase modulators (GSMs) is a promising therapeutic strategy. Here we report that mutations in presenilin (PS), the catalytic subunit of gamma-secretase, display differential responses to nonsteroidal anti-inflammatory drug (NSAID) - type GSMs and more potent second-generation compounds. While many pathogenic PS mutations resisted lowering of Abeta42 generation by the NSAID sulindac sulfide, the potent NSAID-like second-generation compound GSM-1 was capable of lowering Abeta42 for many but not all mutants. We further found that mutations at homologous positions in PS1 and PS2 can elicit differential Abeta42 responses to GSM-1 suggesting that a positive GSM-1 response depends on the spatial environment in gamma-secretase. The aggressive pathogenic PS1 L166P mutation was one of the few pathogenic mutations that resisted GSM-1, and L166 was identified as a critical residue with respect to the Abeta42-lowering response of GSM-1. Finally, we found that GSM-1 responsive and resistant PS mutants behave very similarly towards other potent second-generation compounds of different structural class than GSM-1. Taken together, our data show that a positive Abeta42 response for PS mutants depends both on the particular mutation and the GSM used, and that attenuated Abeta42 responses to low potency GSMs can be overcome for many PS mutants by second generation GSMs.



Full View

The cGMP-Dependent Protein Kinase II Is an Inhibitory Modulator of the Hyperpolarization-Activated HCN2 Channel

PLoS ONE, 2011, doi:10.1371/journal.pone.0017078, published on 14.02.2011
PLoS ONE, online article
Opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated by direct binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus. Here, we show for the first time that in the HCN2 channel cGMP can also exert an inhibitory effect on gating via cGMP-dependent protein kinase II (cGKII)-mediated phosphorylation. Using coimmunoprecipitation and immunohistochemistry we demonstrate that cGKII and HCN2 interact and colocalize with each other upon heterologous expression as well as in native mouse brain. We identify the proximal C-terminus of HCN2 as binding region of cGKII and show that cGKII phosphorylates HCN2 at a specific serine residue (S641) in the C-terminal end of the CNBD. The cGKII shifts the voltage-dependence of HCN2 activation to 2–5 mV more negative voltages and, hence, counteracts the stimulatory effect of cGMP on gating. The inhibitory cGMP effect can be either abolished by mutation of the phosphorylation site in HCN2 or by impairing the catalytic domain of cGKII. By contrast, the inhibitory effect is preserved in a HCN2 mutant carrying a CNBD deficient for cGMP binding. Our data suggest that bidirectional regulation of HCN2 gating by cGMP contributes to cellular fine-tuning of HCN channel activity.


Full View

Rescue of Progranulin Deficiency Associated with Frontotemporal Lobar Degeneration by Alkalizing Reagents and Inhibition of Vacuolar ATPase

The Journal of Neuroscience, 2010, doi:10.1523/JNEUROSCI.5757-10.2011, 1885-1894 published on 02.02.2011
The Journal of Neuroscience, online article
Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR–DNAbinding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation thatGRN has neurotrophic properties, pharmacological stimulation ofGRNproduction is a promising approach to rescue GRNhaploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescueGRNdeficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.



Full View

mGluR1/TRPC3-mediated Synaptic Transmission and Calcium Signaling in Mammalian Central Neurons

Cold Spring Harbor Perspectives in Biology, 2011, doi: 10.1101/cshperspect.a006726, published on 26.01.2011
Cold Spring Harbor Perspectives in Biology, online article
Metabotropic glutamate receptors type 1 (mGluR1s) are required for a normal function of the mammalian brain. They are particularly important for synaptic signaling and plasticity in the cerebellum. Unlike ionotropic glutamate receptors that mediate rapid synaptic transmission, mGluR1s produce in cerebellar Purkinje cells a complex postsynaptic response consisting of two distinct signal components, namely a local dendritic calcium signal and a slow excitatory postsynaptic potential. The basic mechanisms underlying these synaptic responses were clarified in recent years. First, the work of several groups established that the dendritic calcium signal results from IP3 receptor-mediated calcium release from internal stores. Second, it was recently found that mGluR1-mediated slow excitatory postsynaptic potentials are mediated by the transient receptor potential channel TRPC3. This surprising finding established TRPC3 as a novel postsynaptic channel for glutamatergic synaptic transmission.

konnerth_hartmann_et_al_csh perspectives_2011_500

Full View

A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa

HMG, 2011, doi:10.1093/hmg/ddq539, published on 05.01.2011
Human Molecular Genetics, online article
The rd1 natural mutant is one of the first and probably the most commonly studied mouse model for retinitis pigmentosa (RP), a severe and frequently blinding human retinal degeneration. In several decades of research, the link between the increase in photoreceptor cGMP levels and the extremely rapid cell death gave rise to a number of hypotheses. Here, we provide clear evidence that the presence of cyclic nucleotide gated (CNG) channels in the outer segment membrane is the key to rod photoreceptor loss. In Cngb1−/− × rd1 double mutants devoid of regular CNG channels, cGMP levels are still pathologically high, but rod photoreceptor viability and outer segment morphology are greatly improved. Importantly, cone photoreceptors, the basis for high-resolution daylight and colour vision, survived and remained functional for extended periods of time. These findings strongly support the hypothesis of deleterious calcium (Ca2+)-influx as the cause of rapid rod cell death and highlight the importance of CNG channels in this process. Furthermore, our findings suggest that targeting rod CNG channels, rather than general Ca2+-channel blockade, is a most promising symptomatic approach to treat otherwise incurable forms of cGMP-related RP.


Full View

In vivo two-photon imaging of sensory-evoked dendritic calcium signals in cortical neurons

Nature Protocols, 2011, doi:10.1038/nprot.2010.169, 6, 28–35 (2011), published on 03.01.2011
Nature Protocols, online article
Neurons in cortical sensory regions receive modality-specific information through synapses that are located on their dendrites. Recently, the use of two-photon microscopy combined with whole-cell recordings has helped to identify visually evoked dendritic calcium signals in mouse visual cortical neurons in vivo. The calcium signals are restricted to small dendritic domains (‘hotspots’) and they represent visual synaptic inputs that are highly tuned for orientation and direction. This protocol describes the experimental procedures for the recording and the analysis of these visually evoked dendritic calcium signals. The key points of this method include delivery of fluorescent calcium indicators through the recording patch pipette, selection of an appropriate optical plane with many dendrites, hyperpolarization of the membrane potential and two-photon imaging. The whole protocol can be completed in 5–6 h, including 1–2 h of two-photon calcium imaging in combination with stable whole-cell recordings.


konnerth_Jia_etal_Nat Prot_2011_500

Full View

HCN2 channels in local inhibitory interneurons constrain LTP in the hippocampal direct perforant path

Cellular and Molecular Life Sciences, 2011, doi:10.1007/s00018-010-0446-z, published on 10.07.2010
Cell. Mol. Life Sci., online article
Neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to modulate spontaneous activity, resting membrane potential, input resistance, afterpotential, rebound activity, and dendritic integration. To evaluate the role of HCN2 for hippocampal synaptic plasticity, we recorded long-term potentiation (LTP) in the direct perforant path (PP) to CA1 pyramidal cells. LTP was enhanced in mice carrying a global deletion of the channel (HCN2−/−) but not in a pyramidal neuron-restricted knockout. This precludes an influence of HCN2 located in postsynaptic pyramidal neurons. Additionally, the selective HCN blocker zatebradine reduced the activity of oriens-lacunosum moleculare interneurons in wild-type but not HCN2−/− mice and decreased the frequency of spontaneous inhibitory currents in postsynaptic CA1 pyramidal cells. Finally, we found amplified LTP in the PP of mice carrying an interneuron-specific deletion of HCN2. We conclude that HCN2 channels in inhibitory interneurons modulate synaptic plasticity in the PP by facilitating the GABAergic output onto pyramidal neurons.


Full View

The Glutamic Acid-Rich Protein Is a Gating Inhibitor of Cyclic Nucleotide-Gated Channels

The Journal of Neuroscience, 2011, doi:10.1523/​JNEUROSCI.4735-10.2011, published on 05.01.2010
The Journal of Neuroscience, online article
The cyclic nucleotide-gated (CNG) cation channel of rod photoreceptors is a heterotetramer consisting of homologous CNGA1 and CNGB1a subunits. While CNGA1 is indispensable for channel activation, the specific role of CNGB1a in this process has remained elusive. Here, we show that the N-terminal glutamic acid-rich protein (GARP) domain of CNGB1a and soluble GARP2, which corresponds to the proximal portion of the GARP domain, act as autoinhibitory domains that decrease the opening probability of the CNG channel. In the presence of mutations that structurally impair the cyclic nucleotide-binding domain (CNBD) of CNGB1a, the GARP domain completely abolishes channel activity. In agreement with an inhibitory function of GARP, the activity of mutant CNG channels could be fully restored by deletion of the GARP domain. We identified two sequences within the GARP domain that confer most of the inhibitory effect and demonstrate that the profound inhibition imposed by the GARP domain is caused by direct and autonomous protein–protein interaction with the CNG channel complex. In wild-type rod CNG channels, this inhibitory effect can be relieved by binding of cGMP to the CNBD of CNGB1a. In conclusion, we propose that the N terminus of CNGB1a and soluble GARPs act as molecular gate keepers that control the activation of heteromeric rod CNG channels. Our results suggest that the GARP domain has evolved in rod photoreceptors to reduce current noise resulting from openings of CNG channels in the absence of cGMP.


Full View

TU München
Helmholz Muenchen
MPI of Neurobiology
MPI of Biochemistry