James L. Rae

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

The voltage‐sensitive sodium channel Nav1.5 (encoded by SCN5A) is expressed in electromechanical organs and is mechanosensitive. This study aimed to determine the mechanosensitive transitions of Nav1.5 at the molecular level. Nav1.5 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 (ΔV1/2) 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 Nav1.5 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.

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 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.

Characterization of an outward potassium current in canine jejunal circular smooth muscle and its activation by fenamates.

1. A perforated patch clamp technique was used to study an outward potassium current in freshly dissociated circular smooth muscle cells of the canine jejunum. 2. A voltage‐dependent outward current was identified which was highly potassium selective, weakly holding voltage sensitive, increased its open probability at ‐65 mV, and reached unit open probability at +5 mV. 3. Quinidine (0.1‐1 mM) and tetraethylammonium ion (TEA) (10‐50 mM), blocked the potassium current in a dose‐dependent manner. Blockade of the outward potassium current was accompanied by membrane depolarization which reversed on removal of the blocker from the bathing solution. 4. Mefenamic and flufenamic acids, non‐steroidal anti‐inflammatory agents in the fenamate group, were potent activators of the current. Activation was accompanied by hyperpolarization of the membrane with a mean shift in the membrane voltage of 22 mV. 5. It was concluded that the outward potassium current is a major regulator of the resting membrane voltage in isolated circular smooth muscle cells of the canine jejunum. Fenamates activated this current with potentially profound effects on cellular excitability.

SCN5A is expressed in human jejunal circular smooth muscle cells.

Abstract u2002Tetrodotoxin‐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 α‐subunit of the HJCSM Na+channel. Degenerate primers identified a cDNA fragment of 1.5u2003kb 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 α‐subunit of the tetrodotoxin‐resistant Na+current in HJCSM cells.

Distribution of heme oxygenase and effects of exogenous carbon monoxide in canine jejunum

Carbon monoxide (CO) has been postulated to be a messenger in the gastrointestinal tract. The aims of this study were to determine the distribution of heme oxygenase (HO), the source for endogenous CO in the canine jejunum, and to determine the effects of CO on jejunal circular smooth muscle cells. HO-2 isoform was present in a population of myenteric and submucosal neuronal cell bodies, in nerve fibers innervating the muscle layers, and in smooth muscle cells. HO-1 isozyme was not detected in the canine jejunum. Exogenous CO increased whole cell current by 285 ± 86%, hyperpolarized the membrane potential by 8.5 ± 2.9 mV, and increased guanosine 3,5-cyclic monophosphate (cGMP) levels in smooth muscle cells. 8-Bromo- cGMP also increased the whole cell current. The data suggest that endogenous activity of HO-2 may be a source of CO in the canine jejunum and that exogenously applied CO can modulate intestinal smooth muscle electrical activity. It is therefore reasonable to suggest a role for endogenously produced CO as a messenger in the canine jejunum.

Patch Clamp Recordings from the Epithelium of the Lens Obtained using Glasses Selected for Low Noise and Improved Sealing Properties

We have measured currents from single ionic channels located in the apical membrane of lens epithelial cells from six species of animals. These cells require no enzyme treatment to prepare their surfaces for sealing. Because most of the channel types show fast flickering and some have small single channel conductances, we have spent considerable effort to lower electronic noise from the headstage and to investigate special glasses whose specifications predict low electrical noise when used as patchclamp electrodes.

Calcium currents in human and canine jejunal circular smooth muscle cells

BACKGROUND & AIMSnAlthough calcium plays an essential role in intestinal smooth muscle contractile activity, calcium entry pathways in canine and human small intestine are largely unknown. The goal of this study was to characterize calcium channels, a potential entry pathway for calcium, in isolated circular smooth muscle cells of canine and human jejunum.nnnMETHODSnSingle freshly dissociated human and canine jejunal circular smooth muscle cells were studied using single-channel and perforated whole-cell patch clamp recordings as well as fluorescence dual wavelength ratio imaging.nnnRESULTSnAn inward whole-cell current was identified that was carried by a 17 pS (80 mmol/L Ba2+) dihydropyridine-sensitive, barium-permeable channel. The current was potentiated by BayK 8644 (1 mumol/L; n = 3; 82% +/- 34%), acetylcholine (1 mumol/L; n = 8; 42% +/- 5%), and erythromycin (1 mumol/L; n = 9; 70% +/- 11%) and was completely blocked by nifedipine (1 mumol/L; n = 6) or diltiazem (200 mumol/L; n = 4). Application of BayK 8644 (1 mumol/L), acetylcholine (1 mumol/L), or erythromycin (1 mumol/L) to Fura-2-loaded smooth muscle cells bathed in Krebs solution containing 2.54 mmol/L calcium increased intracellular calcium levels.nnnCONCLUSIONSnA calcium entry pathway was identified in canine and human jejunal circular smooth muscle cells. The pathway was mediated by a dihydropyridine-sensitive calcium channel. The channel allowed the entry of significant amounts of calcium at physiological extracellular calcium concentration.

Effects of melatonin on ionic currents in cultured ocular tissues

The effects of melatonin on ionic conductances in a cultured mouse lens epithelial cell line (alpha-TN4) and in cultured human trabecular meshwork (HTM) cells were measured using the amphotericin perforated patch whole cell voltage-clamp technique. Melatonin stimulated a voltage-dependent Na+-selective current in lens epithelial cells and trabecular meshwork cells. The effects of melatonin were observed at 50 pM and were maximal at 100 microM. Melatonin enhanced activation and inactivation kinetics, but no change was observed in the voltage dependence of activation. The results are consistent with an increase in the total number of ion channels available for activation by membrane depolarization. Melatonin was also found to stimulate a K+-selective current at high doses (1 mM). Melatonin did not affect the inwardly rectifying K+ current or the delayed rectifier type K+ current that has been described in cultured mouse lens epithelial cells. The results show that melatonin specifically stimulated the TTX-insensitive voltage-dependent Na+ current by an apparently novel mechanism.The effects of melatonin on ionic conductances in a cultured mouse lens epithelial cell line (α-TN4) and in cultured human trabecular meshwork (HTM) cells were measured using the amphotericin perforated patch whole cell voltage-clamp technique. Melatonin stimulated a voltage-dependent Na+-selective current in lens epithelial cells and trabecular meshwork cells. The effects of melatonin were observed at 50 pM and were maximal at 100 μM. Melatonin enhanced activation and inactivation kinetics, but no change was observed in the voltage dependence of activation. The results are consistent with an increase in the total number of ion channels available for activation by membrane depolarization. Melatonin was also found to stimulate a K+-selective current at high doses (1 mM). Melatonin did not affect the inwardly rectifying K+ current or the delayed rectifier type K+ current that has been described in cultured mouse lens epithelial cells. The results show that melatonin specifically stimulated the TTX-insensitive voltage-dependent Na+ current by an apparently novel mechanism.

Technology of Patch-Clamp Electrodes

The extracellular patch voltage clamp technique has allowed the currents through single ionic channels to be studied from a wide variety of cells. In its early form (Neher and Sakmann, 1976), the resolution of this technique was limited by the relatively low (-50 MR) resistances that isolated the interior of the pipet from the bath. The high resolution that presently can be achieved with the patch-clamp technique originated with the discovery (Neher, 1981) that very highresistance (tens or even hundreds of GS2) seals can form between the cell membrane and the tip of a clean pipet when gentle suction is applied to the pipet interior. Although the precise mechanisms involved in this membrane-to-glass seal are still not fully understood, the importance of the GS2 seal is obvious. The high resistance of the seal ensures that almost all of the current from the membrane patch flows into the pipet and to the input of the current-sensitive headstage preamplifier. It also allows the small patch of membrane to be voltage-clamped rapidly and accurately via the pipet, and the mechanical stability of the seal is vital to the whole-cell voltage clamp technique. Of equal importance, the high resistance of the seal greatly reduces the noise it contributes to single-channel measurements. Although the seal can often represent only a small fraction of total patch-clamp noise (particularly as the bandwidth of recording increases), its importance should never be minimized. Without such high