Altered expression of Ano1 variants in human diabetic gastroparesis.

Diabetes affects many organs including the stomach. Altered number and function of interstitial cells of Cajal (ICC), the gastrointestinal pacemaker cells, underlie a number of gastrointestinal motility disorders, including diabetic gastroparesis. In the muscle layers, ICC selectively express Ano1, thought to underlie classical Ca(2+)-activated Cl(-) currents. Mice homozygous for Ano1 knock-out exhibit abnormal ICC function and motility. Several transcripts for Ano1 are generated by alternative splicing of four exons. Here, we report expression levels of transcripts encoded by alternative splicing of Ano1 gene in gastric muscles of patients with diabetic gastroparesis and nondiabetic control tissues. Expression of mRNA from two alternatively transcribed exons are significantly different between patients and controls. Furthermore, patients with diabetic gastroparesis express mRNA for a previously unknown variant of Ano1. The 5' end of this novel variant lacks exons 1 and 2 and part of exon 3. Expression of this variant in HEK cells produces a decreased density of Ca(2+)-activated Cl(-) currents that exhibit slower kinetics compared with the full-length Ano1. These results identify important changes in expression and splicing of Ano1 in patients with diabetic gastroparesis that alter the electrophysiological properties of the channel. Changes in Ano1 expression in ICC may directly contribute to diabetic gastroparesis.

Mechanosensitivity of Nav1.5, a voltage-sensitive sodium channel.

The voltage-sensitive sodium channel Na(v)1.5 (encoded by SCN5A) is expressed in electromechanical organs and is mechanosensitive. This study aimed to determine the mechanosensitive transitions of Na(v)1.5 at the molecular level. Na(v)1.5 was expressed in HEK 293 cells and mechanosensitivity was studied in cell-attached patches. Patch pressure up to -50 mmHg produced increases in current and large hyperpolarizing shifts of voltage dependence with graded shifts of half-activation and half-inactivation voltages (V(1/2)) by ∼0.7 mV mmHg(-1). Voltage dependence shifts affected channel kinetics by a single constant. This suggested that stretch accelerated only one of the activation transitions. Stretch accelerated voltage sensor movement, but not rate constants for gate opening and fast inactivation. Stretch also appeared to stabilize the inactivated states, since recovery from inactivation was slowed with stretch. Unitary conductance and maximum open probability were unaffected by stretch, but peak current was increased due to an increased number of active channels. Stretch effects were partially reversible, but recovery following a single stretch cycle required minutes. These data suggest that mechanical activation of Na(v)1.5 results in dose-dependent voltage dependence shifts of activation and inactivation due to mechanical modulation of the voltage sensors.

Sodium channel mutation in irritable bowel syndrome: evidence for an ion channelopathy.

The SCN5A-encoded Na(v)1.5 Na(+) channel is expressed in interstitial cells of Cajal and smooth muscle in the circular layer of the human intestine. Patients with mutations in SCN5A are more likely to report gastrointestinal symptoms, especially abdominal pain. Twin and family studies of irritable bowel syndrome (IBS) suggest a genetic basis for IBS, but no genes have been identified to date. Therefore, our aims were to evaluate SCN5A as a candidate gene involved in the pathogenesis of IBS and to determine physiological consequences of identified mutations. Mutational analysis was performed on genomic DNA obtained from 49 subjects diagnosed with IBS who reported at least moderately severe abdominal pain. One patient hosted a loss-of-function missense mutation, G298S, that was not observed in >3,000 reference alleles derived from 1,500 healthy control subjects. Na(+) currents were recorded from the four common human SCN5A transcripts in transfected HEK-293 cells. Comparing Na(v)1.5 with G298S-SCN5A versus wild type in HEK cells, Na(+) current density was significantly less by 49-77%, and channel activation time was delayed in backgrounds that also contained the common H558R polymorphism. Single-channel measurements showed no change in Na(v)1.5 conductance. Mechanosensitivity was reduced in the H558/Q1077del transcript but not in the other three backgrounds. In conclusion, the G298S-SCN5A missense mutation caused a marked reduction of whole cell Na(+) current and loss of function of Na(v)1.5, suggesting SCN5A as a candidate gene in the pathophysiology of IBS.

The lens: local transport and global transparency.

The perception of the lens changed remarkably during the career of David Maurice. The early view was that it was an inert sack of protein that assisted the cornea in focusing light on the retina. As investigators looked more carefully, more and more complexity was revealed and today we know the lens is a living, dynamic organ that carries out a host of biochemical and physiological processes necessary for homeostasis. We have worked on the lens over this period and have provided a small part of the data on lens physiology. This paper is an overview of our own contributions, in the context of the ever evolving view of the lens. Given this is a brief tribute to the career of David Maurice, there is not enough space nor is it appropriate to provide a complete review of all the work that has contributed to this evolving

alpha(1C) (Ca(V)1.2) L-type calcium channel mediates mechanosensitive calcium regulation.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the alpha(1C) L-type calcium channel subunit (Ca(V)1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a beta(2) calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac alpha(1C) splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the alpha(1C) COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming alpha(1C)-subunit.

Single-cell electroporation.

Using modified patch-clamp methodology, we demonstrated that it is possible to insert genes or other compounds routinely into single cells by electroporation. When the cell is indented by a small-tipped microelectrode, a voltage of 10 V or less in the pipette is divided by the pipette resistance and the series resistance of the cleft between the pipette tip and the cell surface. The voltage at the cell membrane can be high enough to cause localized dielectric breakdown of the membrane and create pores that allow compounds in the pipette to enter the cell. Rectangular pulses from 20 micros to more than 300 ms are effective, as are frequencies from DC to 5 kHz. The most significant parameter was the total time for which the voltage was applied. Pipette voltages of 2-10 V were required, with larger genes requiring larger voltages. With optimal parameters, transfection rates in excess of 80% were also possible routinely. This approach offers an effective alternative to intracellular pressure injection and iontophoresis for placing genes, drugs, and other compounds in cells. Because of the small size of the electrode tips, substances can be inserted in cells from almost any location on their surfaces. In addition, the small tips electroporated only a limited area and so did little cell damage.

Sodium current in human jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Sodium channels are key regulators of neuronal and muscle excitability. However, sodium channels have not been definitively identified in gastrointestinal smooth muscle. The aim of the present study was to determine if a Na(+) current is present in human jejunal circular smooth muscle cells. METHODS: Currents were recorded from freshly dissociated cells using patch-clamp techniques. Complementary DNA (cDNA) libraries constructed from the dissociated cells were screened to determine if a message for alpha subunits of Na(+) channels was expressed. Smooth muscle cells were also collected using laser-capture microdissection and screened. RESULTS: A tetrodotoxin-insensitive Na(+) channel was present in 80% of cells patch-clamped. Initial activation was at -65 mV with peak inward current at -30 mV. Steady-state inactivation and activation curves revealed a window current between -75 and -60 mV. The Na(+) current was blocked by lidocaine and internal and external QX314. A cDNA highly homologous to SCN5A, the alpha subunit of the cardiac Na(+) channel, was present in the cDNA libraries constructed from dissociated cells and from smooth muscle cells collected using laser-capture microdissection. CONCLUSIONS: Human jejunal circular smooth muscle cells express a tetrodotoxin-insensitive Na(+) channel, probably SCN5A. Whether SCN5A plays a role in the pathophysiology of human gut dysmotilities remains to be determined.