Light stimuli produce graded hyperpolarizations of the photoreceptor plasma membrane and an associated decrease in a voltagegated calcium channel conductance that mediates release of glutamate neurotransmitter. The Ca v1.4 channel is thought to be involved in this process. The CACNA1F gene encodes the poreforming subunit of the Cav1.4 channel and various mutations in CACNA1F cause X-linked incomplete congenital stationary night blindness (CSNB2). The molecular mechanism of the pathology underlying the CSNB2 phenotype remains to be established. Recent clinical investigations of a New Zealand family found a severe visual disorder that has some clinical similarities to, but is clearly distinct from, CSNB2. Here, we report investigations into the molecular mechanism of the pathology of this condition. Molecular genetic analyses identified a previously undescribed nucleotide substitution in CACNA1F that is predicted to encode an isoleucine to threonine substitution at CACNA1F residue 745. The I745T CACNA1F allele produced a remarkable approximately ؊30-mV shift in the voltage dependence of Cav1.4 channel activation and significantly slower inactivation kinetics in an expression system. These findings imply that substitution of this wild-type residue in transmembrane segment IIS6 may have decreased the energy required to open the channel. Collectively, these findings suggest that a gain-of-function mechanism involving increased Cav1.4 channel activity is likely to cause the unusual phenotype.

The entry of Ca 2ϩ through voltage-gated Ca 2ϩ channels has direct effects on muscle contraction, release of hormones and neurotransmitters, hearing, vision, gene expression, and other important physiological functions (2). The pore-forming ␣ 1 -subunits of voltage-gated Ca 2ϩ channels are composed of four homologous domains formed by six transmembrane segments (S1-S6) that are linked together on a single polypeptide (3). A membrane depolarization initiates channel openings (activation) and closures (inactivation). These events can be considered a multistep process consisting of a conformational change in the voltage sensor, a transmission of the signal to the pore region, the opening of the pore, and channel closure due to inactivation. The voltage-sensing machinery is formed by multiple charged amino acids located in segment S4 and adjacent structures of each domain (4). A large number of amino acids involved in Ca 2ϩ channel inactivation have been identified and several molecular mechanisms for this process have been proposed (for reviews see Refs. 5-7).The molecular mechanism of the voltage-dependent pore opening of Ca 2ϩ channels, however, is less studied and largely unknown. The first attempt to localize the structural elements in Ca 2ϩ channel ␣ 1 -subunits that are involved in channel activation was made by Tanabe et al. (8) who constructed chimeric channels in which sequence stretches of a slow activating ("skeletal muscle-like") Ca V 1.1 ␣ 1 -subunit were replaced by sequences from a fast activating ("cardiac-like") Ca V 1.2 ␣ 1 -subunit. The chimeras activated slowly if repeat I of the Ca V 1.2 ␣ 1 -subunit was replaced by the Ca V 1.1 ␣ 1 -sequence. In a later study, replacement of domains I, II, and III of the low voltage and fast activating Ca V 3.1 ␣ 1 -subunit with the corresponding domains of the high voltage-activated Ca V 1.2 ␣ 1 -subunit resulted in a high voltage-activated channel (9). An important role of domains I and III but not II and IV on midpoint voltage and time constants of activation was reported by Garcia et al. (10) who mutated the arginines in the S4 segments of all four domains of a chimeric channel to neutral or negative amino acids. The removal of prolines that are conserved in segments IS4 and IIIS4 of voltage-gated Ca 2ϩ channels resulted in shortening of channel open time, whereas introduction of extra prolines to corresponding positions of IIS4 and IVS4 lengthened the channel open time (11).Our present study was initiated by the recent finding that a novel retinal disorder is caused by a point mutation (I745T) in segment IIS6 of the Ca V 1.4 ␣ 1 -subunit that shifts the voltage dependence of Ca V 1.4 channel activation by approximately Ϫ30 mV (1, 12). As Ca V 1.4 channels express only at low density in mammalian cell lines (13) we have decided to study the functional roles of this residue and neighboring residues in segment IIS6 by introducing and characterizing mutations in the homologous Ca V 1.2 channel. Our findings demonstrate that residue Ile-781 and three neigh...

Evolution has created a large family of different classes of voltage‐gated Ca2+ channels and a variety of additional splice variants with different inactivation properties. Inactivation controls the amount of Ca2+ entry during an action potential and is, therefore, believed to play an important role in tissue‐specific Ca2+ signalling. Furthermore, mutations in a neuronal Ca2+ channel (Cav2.1) that are associated with the aetiology of neurological disorders such as familial hemiplegic migraine and ataxia cause significant changes in the process of channel inactivation. Ca2+ channels of a given subtype may inactivate by three different conformational changes: a fast and a slow voltage‐dependent inactivation process and in some channel types by an additional Ca2+‐dependent inactivation mechanism. Inactivation kinetics of Ca2+ channels are determined by the intrinsic properties of their pore‐forming α1‐subunits and by interactions with other channel subunits. This review focuses on structural determinants of Ca2+ channel inactivation in different parts of Ca2+ channel α1‐subunits, including pore‐forming transmembrane segments and loops, intracellular domain linkers and the carboxyl terminus. Inactivation is also affected by the interaction of the α1‐subunits with auxiliary β‐subunits and intracellular regulator proteins. The evidence shows that pore‐forming S6 segments and conformational changes in extra‐ (pore loop) and intracellular linkers connected to pore‐forming segments may play a principal role in the modulation of Ca2+ channel inactivation. Structural concepts of Ca2+ channel inactivation are discussed.

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The use of myoblast and fibroblast therapy represents a minimally invasive, safe and effective treatment for post-prostatectomy incontinence after a followup of 1 year.

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BackgroundIn an earlier pilot study with 10 women, we investigated a new approach for therapy of faecal incontinence (FI) due to obstetric trauma, involving ultrasound-guided injection of autologous skeletal muscle-derived cells (SMDC) into the external anal sphincter (EAS), and observed significant improvement. In the current study, we tested this therapeutic approach in an extended patient group: male and female patients suffering from FI due to EAS damage and/or atrophy. Furthermore, feasibility of lower cell counts and cryo-preserved SMDC was assessed.MethodsIn this single-centre, explorative, baseline-controlled clinical trial, each patient (n = 39; mean age 60.6 ± 13.81 years) received 79.4 ± 22.5 × 106 cryo-preserved autologous SMDC. Changes in FI parameters, Fecal Incontinence Quality of Life (FIQL), anorectal manometry and safety from baseline to 1, 6 and 12 months post implantation were evaluated.ResultsSMDC used in this trial contained a high percentage of myogenic-expressing (CD56+) and muscle stem cell marker-expressing (Pax7+, Myf5+) cells. Intervention was well tolerated without any serious adverse events. After 12 months, the number of weekly incontinence episodes (WIE, primary variable), FIQL and patient condition had improved significantly. In 80.6% of males and 78.4% of females, the WIE frequency decreased by at least 50%; Wexner scores and severity of FI complaints decreased significantly, independent of gender and cause of FI.ConclusionsInjection of SMDCs into the EAS effectively improved sphincter-related FI due to EAS damage and/or atrophy in males and females. When confirmed in a larger, placebo-controlled trial, this minimal invasive procedure has the potential to become first-line therapy for FI.Trial registrationEU Clinical Trials Register, EudraCT 2010-023826-19 (Date of registration: 08.11.2010).

Autologous skeletal-muscle-derived cells to treat obstetric anal incontinence resulted in sustained improvement in incontinence episodes, physiological measurements of anal function and quality of life at 5 years.