A mutation in telethonin alters Nav1.5 function

Abstract

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 Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-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 Na(v)1.5, 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 Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.

FIGURE 1.

Molecular characterization of TCAP mutation. a, normal (blue) and mutated (red) TCAP denaturing HPLC. b, sequencing chromatograms showing a heterozygous C to T base change in position 226 in exon 2 of the telethonin gene, TCAP, in the patient compared with normal. c, amino acid conservation analysis showing the highly conserved Arg in position 76 in different species. d, graphic map of the telethonin gene showing the localization of the identified mutation in the area of interaction with muscle LIM protein and titin. e, alignment of the amino acid sequences of native and mutated telethonin shows the resulting R76C codon change in the protein sequence (box).

FIGURE 2.

Expression of telethonin mRNA in human jejunum smooth muscle. a, expression of telethonin mRNA in muscle strips from human jejunum. RT-PCR using primers designed against the human telethonin gene showed the presence of telethonin mRNA in the jejunal smooth muscle strips isolated by dissection. RNA extracted from heart tissue was used as a positive control. No bands were amplified in the -RT negative controls shown in the lower panel. b, RT-PCR on a pool of 3–4 human jejunal smooth muscle single cells showing the presence of a correct size band for TCAP. The identity of the band was confirmed by sequence analysis. There were no products in negative controls containing water bath solution (4 μl aspirated just above a smooth muscle cell) and in controls performed by omitting the reverse transcriptase enzyme during the RT reaction. Arrowhead, 500-bp size band. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 3.

Expression of telethonin protein in mouse cardiac muscle. a, immunofluorescence on mouse heart showed the typical striated staining for telethonin (green). b, the staining for SCN5A (red) appeared to have a distribution similar to telethonin. c, merged image showing the overlapping distribution of the two proteins (yellow). Negative controls carried out by omitting the primary antibodies showed no staining. Immunofluorescence on mouse heart performed by omitting the primary antibodies showed an absence of the striated staining for telethonin (green) (d) and SCN5A (red) (e). Scale bars, 20 μm. f, immunoblotting using an antibody against telethonin showed the presence of this protein (arrowhead) in immunoprecipitates obtained from total homogenates of mouse heart tissue using a pan-sodium channel antibody (SP19).

FIGURE 4.

Telethonin mutation R76C alters activation and inactivation kinetics of SCN5A co-transfected in HEK cells. a, inward Na⁺ current at -50 mV recorded from HEK 293 cells co-transfected with SCN5A alone or with telethonin. b, steady state inactivation and activation curves for SCN5A alone or with telethonin, showing that telethonin did not alter activation or inactivation curves. c, telethonin did not delay the time to peak of activation of Na⁺ current in cells transfected with both SCN5A and telethonin compared with SCN5A alone. d, inward Na⁺ current at -50 mV for SCN5A alone or SCN5A with R76C telethonin. e, steady state inactivation and activation curves for SCN5A with R76C or SCN5A alone. Steady state activation of SCN5A with telethonin R76C shifted negative, resulting in a larger window current (inset) than SCN5A alone. f, telethonin R76C sped up the time to peak of activation of Na⁺ current in cells co-transfected with SCN5A as compared with those with SCN5A alone.

FIGURE 5.

Inactivation kinetics of SCN5A co-transfected in HEK cells in the presence of wild-type or mutant telethonin. Telethonin wild type did not affect the fast τ (a) or the slow τ (b) of inactivation of Na⁺ current in HEK cells co-transfected with SCN5A as compared with those with SCN5A alone. No significant differences were present at any voltages. Telethonin R76C significantly decreased the fast τ (c) but not the slow τ of inactivation (d).

FIGURE 6.

Expression and knockdown of endogenous telethonin in HEK 293 cells stably transfected with SCN5A. a, RT-PCR using primers designed against the human telethonin gene showed the presence of telethonin mRNA in HEK 293 cells. No bands were identified in the -RT negative controls (ctrl). b, representative current traces at -55 mV stepped from a holding potential of -100 mV, recorded from HEK 293 cells stably expressing SCN5A treated for 4 days with scrambled siRNA or with siRNA against telethonin. c, steady state inactivation and activation curves for sodium current in HEK 293 cells stably expressing SCN5A and transfected with either scrambled siRNA (n = 10) or TCAP siRNA (n = 11). Steady state activation of SCN5A after treatment with siRNA against TCAP was shifted positive with respect to cells treated with scrambled siRNA (^*, p < 0.05).