Peter R. Strege

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.

SCN5A is expressed in human jejunal circular smooth muscle cells.

Abstract  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 α‐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 α‐subunit of the tetrodotoxin‐resistant Na+current in HJCSM cells.

A mutation in telethonin alters Nav1.5 function.

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na+ channels in excitable cells results in influx of Na+ and cellular depolarization. The function of Nav1.5, an Na+ channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming α-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na+ channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Nav1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Nav1.5 kinetics and may play a role in intestinal pseudo-obstruction.

Inhibition of Cell Proliferation by a Selective Inhibitor of the Ca2+-activated Cl− Channel, Ano1

BACKGROUND Ion channels play important roles in regulation of cellular proliferation. Ano1 (TMEM16A) is a Ca(2+)-activated Cl(-) channel expressed in several tumors and cell types. In the muscle layers of the gastrointestinal tract Ano1 is selectively expressed in interstitial cells of Cajal (ICC) and appears to be required for normal gastrointestinal slow wave electrical activity. However, Ano1 is expressed in all classes of ICC, including those that do not generate slow waves suggesting that Ano1 may have other functions. Indeed, a role for Ano1 in regulating proliferation of tumors and ICC has been recently suggested. Recently, a high-throughput screen identified a small molecule, T16A(inh)-A01 as a specific inhibitor of Ano1. AIM To investigate the effect of the T16A(inh)-A01 inhibitor on proliferation in ICC and in the Ano1-expressing human pancreatic cancer cell line CFPAC-1. METHODS Inhibition of Ano1 was demonstrated by whole cell voltage clamp recordings of currents in cells transfected with full-length human Ano1. The effect of T16A(inh)-A01 on ICC proliferation was examined in situ in organotypic cultures of intact mouse small intestinal smooth muscle strips and in primary cell cultures prepared from these tissues. ICC were identified by Kit immunoreactivity. Proliferating ICC and CFPAC-1 cells were identified by immunoreactivity for the nuclear antigen Ki67 or EdU incorporation, respectively. RESULTS T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents by 60% at 10μM in a voltage-independent fashion. Proliferation of ICC was significantly reduced in primary cultures from BALB/c mice following treatment with T16A(inh)-A01. Proliferation of the CFPAC-1 human cell-line was also reduced by T16A(inh)-A01. In organotypic cultures of smooth muscle strips from mouse jejunum, the proliferation of ICC was reduced but the total number of proliferating cells/confocal stack was not affected, suggesting that the inhibitory effect was specific for ICC. CONCLUSIONS The selective Ano1 inhibitor T16A(inh)-A01 inhibited Ca(2+)-activated Cl(-) currents, reduced the number of proliferating ICC in culture and inhibited proliferation in the pancreatic cancer cell line CFPAC-1. These data support the notion that chloride channels in general and Ano1 in particular are involved in the regulation of proliferation.

Ranolazine Decreases Mechanosensitivity of the Voltage-Gated Sodium Ion Channel NaV1.5 A Novel Mechanism of Drug Action

Background— NaV1.5 is a mechanosensitive voltage-gated sodium-selective ion channel responsible for the depolarizing current and maintenance of the action potential plateau in the heart. Ranolazine is a NaV1.5 antagonist with antianginal and antiarrhythmic properties. Methods and Results— Mechanosensitivity of NaV1.5 was tested in voltage-clamped whole cells and cell-attached patches by bath flow and patch pressure, respectively. In whole cells, bath flow increased peak inward current in both murine ventricular cardiac myocytes (24±8%) and human embryonic kidney 293 cells heterologously expressing NaV1.5 (18±3%). The flow-induced increases in peak current were blocked by ranolazine. In cell-attached patches from cardiac myocytes and NaV1.5-expressing human embryonic kidney 293 cells, negative pressure increased NaV peak currents by 27±18% and 18±4% and hyperpolarized voltage dependence of activation by −11 mV and −10 mV, respectively. In human embryonic kidney 293 cells, negative pressure also increased the window current (250%) and increased late open channel events (250%). Ranolazine decreased pressure-induced shift in the voltage dependence (IC50 54 &mgr;mol/L) and eliminated the pressure-induced increases in window current and late current event numbers. Block of NaV1.5 mechanosensitivity by ranolazine was not due to the known binding site on DIVS6 (F1760). The effect of ranolazine on mechanosensitivity of NaV1.5 was approximated by lidocaine. However, ionized ranolazine and charged lidocaine analog (QX-314) failed to block mechanosensitivity. Conclusions— Ranolazine effectively inhibits mechanosensitivity of NaV1.5. The block of NaV1.5 mechanosensitivity by ranolazine does not utilize the established binding site and may require bilayer partitioning. Ranolazine block of NaV1.5 mechanosensitivity may be relevant in disorders of mechanoelectric dysfunction.

Hydrogen sulfide is a partially redox-independent activator of the human jejunum Na+ channel, Nav1.5

Hydrogen sulfide (H(2)S) is produced endogenously by L-cysteine metabolism. H(2)S modulates several ion channels with an unclear mechanism of action. A possible mechanism is through reduction-oxidation reactions attributable to the redox potential of the sulfur moiety. The aims of this study were to determine the effects of the H(2)S donor NaHS on Na(V)1.5, a voltage-dependent sodium channel expressed in the gastrointestinal tract in human jejunum smooth muscle cells and interstitial cells of Cajal, and to elucidate whether H(2)S acts on Na(V)1.5 by redox reactions. Whole cell Na(+) currents were recorded in freshly dissociated human jejunum circular myocytes and Na(V)1.5-transfected human embryonic kidney-293 cells. RT-PCR amplified mRNA for H(2)S enzymes cystathionine β-synthase and cystathionine γ-lyase from the human jejunum. NaHS increased native Na(+) peak currents and shifted the half-point (V(1/2)) of steady-state activation and inactivation by +21 ± 2 mV and +15 ± 3 mV, respectively. Similar effects were seen on the heterologously expressed Na(V)1.5 α subunit with EC(50)s in the 10(-4) to 10(-3) M range. The reducing agent dithiothreitol (DTT) mimicked in part the effects of NaHS by increasing peak current and positively shifting steady-state activation. DTT together with NaHS had an additive effect on steady-state activation but not on peak current, suggesting that the latter may be altered via reduction. Pretreatment with the Hg(2+)-conjugated oxidizer thimerosal or the alkylating agent N-ethylmaleimide inhibited or decreased NaHS induction of Na(V)1.5 peak current. These studies show that H(2)S activates the gastrointestinal Na(+) channel, and the mechanism of action of H(2)S is partially redox independent.

Ranolazine decreases mechanosensitivity of the voltage-gated sodium ion channel Na(v)1.5: a novel mechanism of drug action.

Background— NaV1.5 is a mechanosensitive voltage-gated sodium-selective ion channel responsible for the depolarizing current and maintenance of the action potential plateau in the heart. Ranolazine is a NaV1.5 antagonist with antianginal and antiarrhythmic properties. Methods and Results— Mechanosensitivity of NaV1.5 was tested in voltage-clamped whole cells and cell-attached patches by bath flow and patch pressure, respectively. In whole cells, bath flow increased peak inward current in both murine ventricular cardiac myocytes (24±8%) and human embryonic kidney 293 cells heterologously expressing NaV1.5 (18±3%). The flow-induced increases in peak current were blocked by ranolazine. In cell-attached patches from cardiac myocytes and NaV1.5-expressing human embryonic kidney 293 cells, negative pressure increased NaV peak currents by 27±18% and 18±4% and hyperpolarized voltage dependence of activation by −11 mV and −10 mV, respectively. In human embryonic kidney 293 cells, negative pressure also increased the window current (250%) and increased late open channel events (250%). Ranolazine decreased pressure-induced shift in the voltage dependence (IC50 54 &mgr;mol/L) and eliminated the pressure-induced increases in window current and late current event numbers. Block of NaV1.5 mechanosensitivity by ranolazine was not due to the known binding site on DIVS6 (F1760). The effect of ranolazine on mechanosensitivity of NaV1.5 was approximated by lidocaine. However, ionized ranolazine and charged lidocaine analog (QX-314) failed to block mechanosensitivity. Conclusions— Ranolazine effectively inhibits mechanosensitivity of NaV1.5. The block of NaV1.5 mechanosensitivity by ranolazine does not utilize the established binding site and may require bilayer partitioning. Ranolazine block of NaV1.5 mechanosensitivity may be relevant in disorders of mechanoelectric dysfunction.

Lysophosphatidyl choline modulates mechanosensitive L-type Ca2+ current in circular smooth muscle cells from human jejunum.

The L-type Ca2+ channel expressed in gastrointestinal smooth muscle is mechanosensitive. Direct membrane stretch and shear stress result in increased Ca2+ entry into the cell. The mechanism for mechanosensitivity is not known, and mechanosensitivity is not dependent on an intact cytoskeleton. The aim of this study was to determine whether L-type Ca2+ channel mechanosensitivity is dependent on tension in the lipid bilayer in human jejunal circular layer myocytes. Whole cell currents were recorded in the amphotericin-perforated-patch configuration, and lysophosphatidyl choline (LPC), lysophosphatidic acid (LPA), and choline were used to alter differentially the tension in the lipid bilayer. Shear stress (perfusion at 10 ml/min) was used to mechanostimulate L-type Ca2+ channels. The increase in L-type Ca2+ current induced by shear stress was greater in the presence of LPC (large head-to-tail proportions), but not LPA or choline, than in the control perfusion. The increased peak Ca2+ current also did not return to baseline levels as in control conditions. Furthermore, steady-state inactivation kinetics were altered in the presence of LPC, leading to a change in window current. These findings suggest that changes in tension in the plasmalemmal membrane can be transmitted to the mechanosensitive L-type Ca2+ channel, leading to altered activity and Ca2+ entry in the human jejunal circular layer myocyte.