Clinton J. Doering

The CACNA1F Gene Encodes an L-Type Calcium Channel with Unique Biophysical Properties and Tissue Distribution

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Cav1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Cav1.4 calcium channels. Cav1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel β subunits. Additionally, the channel supports a large window current and activates near -40 mV in 2 mM external calcium, making Cav1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Cav1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel–ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Scanning Mutagenesis of ω-Atracotoxin-Hv1a Reveals a Spatially Restricted Epitope That Confers Selective Activity against Insect Calcium Channels

We constructed a complete panel of alanine mutants of the insect-specific calcium channel blocker ω-atracotoxin-Hv1a. Lethality assays using these mutant toxins identified three spatially contiguous residues, Pro10, Asn27, and Arg35, that are critical for insecticidal activity against flies (Musca domestica) and crickets (Acheta domestica). Competitive binding assays using radiolabeled ω-atracotoxin-Hv1a and neuronal membranes prepared from the heads of American cockroaches (Periplaneta americana) confirmed the importance of these three residues for binding of the toxin to target calcium channels presumably expressed in the insect membranes. At concentrations up to 10 μm, ω-atracotoxin-Hv1a had no effect on heterologously expressed rat Cav2.1, Cav2.2, and Cav1.2 calcium channels, consistent with the previously reported insect selectivity of the toxin. 30 μm ω-atracotoxin-Hv1a inhibited rat Cav currents by 10-34%, depending on the channel subtype, and this low level of inhibition was essentially unchanged when Asn27 and Arg35, which appears to be critical for interaction of the toxin with insect Cav channels, were both mutated to alanine. We propose that the spatially contiguous epitope formed by Pro10, Asn27, and Arg35 confers specific binding to insect Cav channels and is largely responsible for the remarkable phyletic selectivity of ω-atracotoxin-Hv1a. This epitope provides a structural template for rational design of chemical insecticides that selectively target insect Cav channels.

Modified Cav1.4 Expression in the Cacna1fnob2 Mouse Due to Alternative Splicing of an ETn Inserted in Exon 2

The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1fnob2 mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1fnob2 mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.

Congenital Stationary Night Blindness in Mice - A Tale of Two Cacna1f Mutants

BACKGROUND Mutations in CACNA1F, which encodes the Ca(v)1.4 subunit of a voltage-gated L-type calcium channel, cause X-linked incomplete congenital stationary night blindness (CSNB2), a condition of defective retinal neurotransmission which results in night blindness, reduced visual acuity, and diminished ERG b-wave. We have characterized two putative murine CSNB2 models: an engineered null-mutant, with a stop codon (G305X); and a spontaneous mutant with an ETn insertion in intron 2 of Cacna1f (nob2). METHODS Cacna1f ( G305X ): Adults were characterized by visual function (photopic optokinetic response, OKR); gene expression (microarray) and by cell death (TUNEL) and synaptic development (TEM). Cacna1f ( nob2 ): Adults were characterized by properties of Cacna1f mRNA (cloning and sequencing) and expressed protein (immunoblotting, electrophysiology, filamin [cytoskeletal protein] binding), and OKR. RESULTS The null mutation in Cacna1f ( G305X ) mice caused loss of cone cell ribbons, failure of OPL synaptogenesis, ERG b-wave and absence of OKR. In Cacna1f ( nob2 ) mice alternative ETn splicing produced ~90% Cacna1f mRNA having a stop codon, but ~10% mRNA encoding a complete polypeptide. Cacna1f ( nob2 ) mice had normal OKR, and alternatively-spliced complete protein had WT channel properties, but alternative ETn splicing abolished N-terminal protein binding to filamin. CONCLUSIONS Ca(v)1.4 plays a key role in photoreceptor synaptogenesis and synaptic function in mouse retina. Cacna1f ( G305X ) is a true knockout model for human CSNB2, with prominent defects in cone and rod function. Cacna1f ( nob2 ) is an incomplete knockout model for CSNB2, because alternative splicing in an ETn element leads to some full-length Ca(v)1.4 protein, and some cones surviving to drive photopic visual responses.

FUNCTIONAL ANALYSIS OF CONGENITAL STATIONARY NIGHT BLINDNESS TYPE-2 CACNA1F MUTATIONS F742C, G1007R, AND R1049W

Congenital stationary night blindess-2 (incomplete congenital stationary night blindness (iCSNB) or CSNB-2) is a nonprogressive, X-linked retinal disease which can lead to clinical symptoms such as myopia, hyperopia, nystagmus, strabismus, decreased visual acuity, and impaired scotopic vision. These clinical manifestations are linked to mutations found in the CACNA1F gene which encodes for the Ca(v)1.4 voltage-gated calcium channel. To better understand the physiological effects of these mutations, three missense mutants, F742C, G1007R and R1049W, previously shown to be mutated in patients with CSNB-2, were transiently expressed in human embryonic kidney (HEK) tsA-201 cells and characterized using whole-cell patch clamp. The G1007R mutation is located in transmembrane segment 5 (S5) of domain III and R1049W is located in the extracellular linker between S5 and the P-loop of domain III. Both mutants produced full length proteins that targeted to the membrane but did not support ionic currents. In 20 mM Ba(2+), F742C (S6 domain II) produced a approximately 21 mV hyperpolarizing shift in half activation potential (V(a[1/2])) and a approximately 23 mV hyperpolarizing shift in half inactivation potential (V(h[1/2])). Additionally, F742C displayed slower inactivation kinetics and a smaller whole cell conductance (G(max)). In physiological 2 mM Ca(2+), F742C produced a approximately 19 mV hyperpolarizing shift in V(a[1/2]). These findings suggest that the pathology of CSNB-2 in patients with these missense mutations in the Ca(v)1.4 calcium channel is the result in either a gain of function (F742C) or a loss of function (G1007R, R1049W).

The Ca(v)1.4 calcium channel: more than meets the eye.

Cav1.4 channels are the latest calcium channels to be described in the literature. Originally identified in 1997 from the human genome project, several reports have since been published describing mutations in the CACNA1F gene encoding Cav1.4 channels, and implicated these mutations in human disorders such as X-linked rod-cone dystrophy (CORDX3) and incomplete X-linked congenital stationary night blindness type 2 (CSNB2). The gene was subsequently cloned and expressed in heterologous expression systems beginning in 2003, and many of the mutations linked to CSNB2 have been tested. Here, we review literature describing the discovery of the CACNA1F gene, its tissue expression profile, alternative splicing events, and biophysical and pharmacological characteristics of the channel in various expression systems. Channel biophysics are also compared to those obtained from recordings made from vertebrate photoreceptors, suggesting that these studies may have been describing Cav1.4 channels in native cells.

TEMPERATURE DEPENDENCE OF Cav1.4 CALCIUM CHANNEL GATING

The CACNA1F gene encodes the pore-forming subunit of the L-type Cav1.4 voltage-gated calcium channel (VGCC) and plays a central role in tonic vesicular release at photoreceptor ribbon synapses. The main objective of this study was to examine the effects of temperature on human Cav1.4 cDNA clone VGCCs. With 20 mM Ba2+ as charge carrier, increasing the temperature from 23 degrees C to 37 degrees C increases whole-cell conductance, shifts the voltage-dependence of activation to more hyperpolarized voltages, and accelerates the degree of recovery from inactivation over a given time, but does not significantly alter the half-inactivation potential (Vh). The window current for Cav1.4 was also shifted to more hyperpolarized voltages, observable from approximately -35 mV to +20 mV at 37 degrees C in 20 mM Ba2+. Several comparable results were observed when characterizing Cav1.2 at temperatures ranging from 23 degrees C to 37 degrees C. However, one difference between Cav1.4 and Cav1.2 was the temperature dependence of voltage-dependent inactivation kinetics. Increasing temperature from 23 degrees C to 37 degrees C accelerates Cav1.4 inactivation kinetics approximately 50-fold, whereas Cav1.2 only accelerates approximately 10-fold over the same temperature range. The time constant of inactivation (tauh) temperature coefficient (Q10) was 18.8 for Cav1.4 over a temperature range of 23 degrees to 33 degrees C (corresponding to an activation energy Ea=221 kJ/mol), compared with Cav1.2 with a Q10 of 3 (Ea=90 kJ/mol) recorded under identical conditions. In addition, Cav1.4 was also tested using 2 mM Ca2+ as a charge carrier and similar changes in current-voltage Boltzmann parameters and gating kinetics were observed. Hence, despite the accelerated inactivation kinetics of Cav1.4 channels observed at near physiological temperatures the window current is preserved and could allow for tonic glutamate release from photoreceptors in the retina during dark adapted conditions.

Syntaxin 1A is required for normal in utero development

We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life.

Molecular Pharmacology of Non-L-type Calcium Channels

Voltage-gated calcium channels are key sources of calcium entry into the cytosol. Mutations in calcium channels have been implicated in numerous disorders such as migraine, incomplete congenital X-linked stationary night blindness, epilepsy, and ataxia, and they are important therapeutic targets for the treatment of pain, stroke, hypertension, and epilepsy. Calcium channel antagonists can be broadly classified into three groups. 1) Inorganic ions typically nonselectively block the pore of most calcium channel subtypes, and in some cases, alter gating kinetics. 2) Peptides isolated from arachnids, cone snails, and snakes frequently selectively antagonize individual calcium channel subtypes by direct occlusion of the pore or altering gating kinetics. 3) Small organic molecules of various structure-activity-relationship (SAR) classes can mediate both selective and nonselective effects on individual calcium channel subtypes, and occlude the pore or reduce channel availability. Here, we provide an overview of classes of inhibitors of non-L-type calcium channels.