The absence of cyclic nucleotide-gated (CNG) channels in cone photoreceptor outer segments leads to achromatopsia, a severely disabling disease associated with the complete lack of cone photoreceptor ...function. In a common form, loss of the CNGA3 subunit disrupts visual transduction in cones and causes progressive degeneration. Here, we show that adeno-associated viral vector-mediated gene replacement therapy added the lacking sensual quality, cone-mediated vision, in the CNGA3−/− mouse model of the human disease. The functional rescue of cone vision was assessed at different sites along the visual pathway. In particular, we show electrophysiologically that treated CNGA3−/− mice became able to generate cone-mediated responses and to transfer these signals to bipolar and finally ganglion cells. In support, we found morphologically that expression of CNGA3 delayed cone cell death. Finally, we show in a behavioral test that treated mice acquired photopic vision suggesting that achromatopsia patients may as well benefit from gene replacement therapy.
The activation of cyclic nucleotide‐gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The ...cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide‐binding site is connected to the last transmembrane segment of the channel by a linker peptide (C‐linker) of ∼90 amino acids. Here we report that replacement of three amino acids in the cone C‐linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild‐type cone channel, the mutated channel exhibited similar single‐channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.
Cardiac pacemaking is controlled by a mixed Na(+)/K(+) current named I(f), which is activated by hyperpolarized membrane potentials. Recently, a family of hyperpolarization-activated cyclic ...nucleotide-gated cation (HCN) channels has been cloned. The members of this family exhibit the general features of I(f) channels. This review describes the molecular diversity of the HCN channel family and the structural determinants of channel function including activation by voltage, modulation by cyclic nucleotides and ion permeation. The relationships between cloned HCN channel types and native cardiac I(f) currents are explored.
It has been suggested that HCN1 is primarily expressed in hippocampus, however little is known about its effects on spatial learning and memory. In the present study, we investigated the effects of ...non-specific HCN1 blocker CsCl on spatial learning and memory by using Morris water maze and in situ hybridization in mice. The results showed CsCl 160 mg/kg ip for 4 days, and the mean escape latency was 34 s longer than that of normal control (P<0.01). In hippocampal tissues, staining for the HCN1 mRNA was stronger in the DG and CA1 region of the hippocampus (P <0.05, P<0.05, when CsCl-administration group was compared with normal group). Our results suggested that CsCl could significantly affect the spatial learning and memory in mice, and HCN channel is involved in the process of learning and memory.
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 Ca
2+
-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.
To study the electrophysiological and pharmacological properties of the L-type Ca(2+) channel (LTCC) Ca(v)1.4alpha1 (alpha1F) subunit from mouse retina and assess their contributions to the native ...retinal channel.
The full-length cDNA of Ca(v)1.4alpha1 was cloned from murine retina in an RT-PCR approach. Ca(v)1.4alpha1 was expressed alone or together with the auxiliary alpha2delta1 and beta2a or beta3 subunits in HEK293 cells. The electrophysiological and pharmacological characteristics of L-type Ca(2+) and Ba(2+) inward currents (I(Ca) and I(Ba)) induced by Ca(v)1.4alpha1 were determined by the whole-cell configuration of the patch-clamp method and compared with currents induced by the cardiac and smooth muscle-type Ca(v)1.2alpha1 (alpha1C) channel.
Ca(v)1.4alpha1-mediated I(Ba) was observed only when the alpha2delta1 and beta subunits were coexpressed. Current densities were approximately two times higher with beta2a than with beta3. I(Ba) activated faster and revealed much slower time-dependent inactivation than I(Ba) induced by Ca(v)1.2alpha1. Unlike in Ca(v)1.2alpha1, inactivation was not accelerated with Ca(2+) as the charge carrier, indicating the absence of Ca(2+)-dependent inactivation in Ca(v)1.4alpha1. Ca(v)1.4alpha1 exhibited voltage-dependent inactivation. The dihydropyridine (DHP) antagonist isradipine blocked Ca(v)1.4alpha1 with approximately 20-fold lower sensitivity than Ca(v)1.2alpha1. The agonistic DHP BayK 8644 stimulated maximum I(Ba) approximately sixfold. Ca(v)1.4alpha1 revealed only moderate sensitivities to L- and D-cis-diltiazem, with IC(50) in the micromolar range. Both enantiomers unexpectedly blocked Ca(v)1.4alpha1 with almost equal IC(50).
The data indicate that Ca(v)1.4alpha1 subunit constitutes the major molecular correlate of retinal L-type Ca(2+) current. Its intrinsic biophysical properties, in particular its unique inactivation properties, enable Ca(v)1.4alpha1 to provide a sustained I(Ca) over a voltage range such as required for tonic glutamate release at the photoreceptor synapse.
The stably expressed Ca
2+
channel
α
1C‐a
and
α
1C‐b
subunit were used to investigate the molecular basis for Ca
2+
‐dependent inactivation of the L‐type current. The Ba
2+
current (
I
Ba
) of both ...channels had similar kinetics and inactivated with one time constant of about 400 ms at +20 mV, whereas the Ca
2+
current (
I
Ca
) could be fitted only with a bi‐exponential function. The fast (
τ
f
) and the slow (
τ
s
) time constant were about 20 ms and 400 ms, respectively. The inactivation of
I
Ca
strongly depended on the entry of Ca
2+
as shown by prepulses and variation of the intracellular Ca
2+
chelator. Coexpression of the
α
1C
subunits with the auxiliary
α
2
/
δ
and β subunits accelerated the voltage‐dependent but not the Ca
2+
‐dependent inactivation of the channels. These results suggest that the
α
1C
unit of L‐type Ca
2+
channels itself mediates the Ca
2+
‐dependent inactivation of the current.
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