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 Ca2+-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 Ca2+-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.

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 (delta V-1/2) by similar to 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.

Stress induced immunomodulation has evolved to confer maximum biological advantage to an organism exposed to varying environmental stressors. Stress is also implicated in the aetiology of Irritable Bowel Syndrome (IBS). Herein, we examined the immunophenotype of IBS patients for evidence of stress related immunomodulation. Methods: 24-IBS patients and 9-matched controls subjects were studied. Comprehensive clinical, psychological (e.g. somatisation) and symptomatic (e.g. Visceral Sensitivity Index (VSI)) evaluations were performed. Peripheral bloodmononuclear cells (PBMC's) were stimulated using lipopolysacharide (5-concentrations) in the presence or absence of exogenous corticotrophin releasing hormone (CRH). A broad panel of inflammatory markers were measured in the supernatant. A one-way ANOVA was performed and data reported as point estimates of difference, ±95%CI (IBS-Controls). Results: Levels of IL2 (-44.5 (-66,-22) p<0.001), IL4 (-32.5 (-54,11) p=0.0004), IL5 (-15.3 (-21, 8.9) p<0.001), IL10 (-1088 (685, 1491) p<0.001), IL13 (60.3 (-85, -36) p<0.001), TNFα (-5452 (-3577, -7327) p<0.001), and IFNγ (-1353 (-471, -2235) p=0.002) were lower in IBS compared to controls and negatively correlated with measures of symptom severity (e.g. IL-5 and Verbal Descriptor Visual Analogue Scale for symptom intensity (VDVAS-I) r=-0.65, p<0.001) and psychological co-morbidity (e.g. IL-5 and somatisation, r=-0.41, p=0.02). In contrast, IL6 (8431 (13160, 3703) p<0.001) and IL8 (14186 (17934, 10437) p<0.001) levels were higher in IBS and positively correlated with measures of symptom severity (e.g. IL-8 and VDVAS-I, r= 0.61, p<0.001) and psychological co-morbidity (e.g. IL-8 and somatisation, r=0.37, p=0.03). Co-incubation with CRH suppressed IFNγ levels in control subjects to the same level as IBS patients (254.1 (-491, 999) p=0.05). Conclusion: As seen in chronic stress, IBS patients suppress Th1 and Th2 cytokine release from stimulated PBMC's and this suppression is negatively correlated with increasing symptom severity and psychological co-morbidity. Elevation of IL-6, and suppression of IFN-γ, IL-2 and IL-4 may indicate a shift towards a TH17 immune profile. This immunophenotype would convey a biological advantage in terms of enhanced mucosal host defence but may also establish a pro-inflammatory mucosal cytokine profile which would promote gastrointestinal symptoms. The potential interaction between stress and TH17 immune function in IBS could represent a new insight in IBS pathophysiology.

Voltage-gated ion channels are often found in tissues where electrical and mechanical stimuli coexist. The mechanosensitive, voltage-gated sodium channel Nav1.5 (encoded by SCN5A) is expressed in two such electromechanical organs, the heart and the gastrointestinal tract. Mutations in SCN5A are frequently pathogenic and may affect mechanoelectrical coupling. The aim of this study was to assess mechanical sensitivity of Nav1.5 at the molecular level. SCN5A was expressed in HEK cells and studied using a pipette pulled and fire polished to ensure that a small number (2-50) of channels were reliably present in cell-attached micropatches. This allowed resolution of both single channel events and averaged behavior. Both positive and negative pressures (up to 50mmHg) produced visible patch distention, an increase in patch current at all voltages and large hyperpolarizing shifts in steady-state voltage-sensitivity of activation and inactivation. From voltage dependence of activation at rest (V1/2 = −30mV at 0mmHg), pressure resulted in graded shifts of V1/2 for activation and inactivation of −0.71mV/mmHg and −0.72mV/mmHg, respectively. Channel kinetics were predictably affected by the voltage shifts, but channel opening and fast inactivation were otherwise unaffected by pressure. Single channel traces showed that unitary conductance was unaffected, rather peak currents appeared to increase due to an increase in the number of active channels in the patch. These effects were minimally reversible for as long as 30 minutes after a single stretch stimulus. Patch excision resulted in an immediate shift of activation V1/2=-75mV and loss of stretch sensitivity. Application of the inhibitor of actin polymerization, cytochalasin D, diminished sensitivity to stretch (−0.42mV/mmHg). Our work demonstrates that mechanical stress at physiologically relevant levels affects voltage sensing of Nav1.5 channels, without affecting the pore, channel gate and fast inactivation. Supported by NIH DK52766.

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 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 view.

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 µs 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.

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.

Tetrodotoxin-resistant Na+currents are expressed in a variety of muscle cells including human jejunal circular smooth muscle (HJCSM) cells. The aim of this study was to determine the molecular identity of the pore-forming alpha-subunit of the HJCSM Na+ channel. Degenerate primers identified a cDNA fragment of 1.5 kb with 99% nucleotide homology with human cardiac SCN5A. The identified clone was also amplified from single smooth muscle cells by reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot analysis showed expression of full-length SCN5A. Laser capture microdissection was used to obtain highly purified populations of HJCSM cells. RT-PCR on the harvested cells showed that SCN5A was present in circular but not in longitudinal muscle. A similar result was obtained using a pan-Na+ channel antibody. The full-length sequence for SCN5A was obtained by combining standard polymerase chain reaction with 5' and 3' rapid amplification of cDNA end techniques. The intestinal SCN5A was nearly identical to the cardiac SCN5A. The data indicate that SCN5A is more widely distributed than previously thought and encodes the pore-forming alpha-subunit of the tetrodotoxin-resistant Na+ current in HJCSM 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.

Purpose. To determine the existence of inward rectifier (Kir2.1) sequences in mRNA from corneal epithelium and endothelium.Methods. cDNA library construction, cloning, PCR, patch clamp.Results. Both the corneal epithelium and endothelium contain mRNA for Kir2.1 inwardly rectifying potassium channels. When the cDNA is transfected into Chinese hamster ovary cells, the channel currents match those expected from Kir2.1 inward rectifiers.Conclusions. The mRNA for Kir2.1 potassium channels exists in corneal epithelium and endothelium. Therefore, Kir2.1 inwardly rectifying potassium channels probably make some contributions to the resting voltages of cornea epithelium and endothelium. Previous data, however, suggest that they are probably not the dominant contributors in these preparations.

Human Kv3.3/KCNC3 is a Shaw-type potassium channel that has been mapped to chromosome 19q13.3-13.4. Complete mouse and rat Kv3.3 cDNA coding sequences have been published, yet the human Kv3.3 cDNA has remained incomplete for years. We report here for the first time the amino acid sequence for hKv3.3 and the electrophysiological behavior of the encoded channel in transiently transfected mammalian cells. In addition, we report the occurrence of Kv3.3 message in rabbit corneal endothelial cells and the properties of the currents when the corneal channel is expressed. The hKv3.3 gene is highly GC-rich (69%) and contains numerous GC runs which made DNA sequencing and PCR amplification especially problematic. The full-length sequence contains two possible start codons. The encoded 757 amino acid hKv3. 3 protein is about 93% identical to mouse and rat Kv3.3 in the first 659 amino acids before the C-terminal domains diverge greatly as a result of alternative splicing. The rabbit cornea Kv3.3 is a close sequence match for hKv3.3 even in the C-terminal domain. However, we have not yet found a cornea sequence which contains the first potential start codon from hKv3.3. Electrophysiologically, the hKv3. 3 channel produces an A-current although expression of constructs which lack the 5' region of the first start codon inactivate much more slowly than full-length constructs. This short hKv3.3 construct also shows changes in activation.

We describe the cloning and characterization of the first human members, hKv9.1 and hKv9.3, of the electrically silent delayed-rectifying-like K+ channel subfamily. Their modulatory effects on the electrically active subfamily member hKv2.1 are also quantified. The hKv9 K+ channels were isolated from a human lens epithelium cDNA library, but both hKv9.1 mRNA and hKv9.3 mRNA were found to coexist with the mRNA for hKv2.1 in a large number of human tissues. The hKv9.1 gene is composed of a minimum of five exons, with at least two alternatively spliced exons in the 5'-untranslated region (UTR). In contrast, the hKv9.3 gene is intronless across the coding region, 3'-UTR, and all of the analyzed 5'-UTR. Radiation hybrid mapping localized the hKv9.1 gene to 20q12 and the hKv9.3 gene to 2p24. Each electrically silent subunit, when coexpressed with hKv2.l, slows deactivation and inactivation compared with hKv2. 1 expressed alone. In addition, each results in an increment in the single channel conductance.