Molecular evidence for a role of Shaw (Kv3) potassium channel subunits in potassium currents of dog atrium

Abstract

We previously described an ultrarapid delayed rectifier current in dog atrial myocytes (IKur,d) with properties resembling currents reported for Kv3.1 channels in neural tissue; however, there was no direct molecular evidence for Shaw subfamily (Kv3) subunit expression in the heart. To identify the molecular basis of IKur,d, we cloned a full-length cDNA (dKv3.1) from canine atrium with homology-based reverse transcription (RT)- polymerase chain reaction (PCR) cloning techniques. A 1755 bp full-length cDNA (dKv3.1) was obtained, with 94.2 % homology to rat brain Kv3.1 (rbKv3.1). The deduced amino acid sequence had 99.3 % homology with rbKv3.1. Heterologous expression of dKv3.1 in Xenopus oocytes produced currents with activation voltage dependence, rectification, and activation and deactivation kinetics that strongly resemble native IKur,d. Like IKur,d, dKv3.1 was found to be highly sensitive to extracellular 4-aminopyridine (4-AP) and tetraethylammonium (TEA). RNase protection assays, Western blots and immunohistochemical studies demonstrated the presence of dKv3.1 transcripts and proteins in dog atrial preparations and isolated canine atrial myocytes. Protein corresponding to the Kv1.5 subunit, which can also carry ultrarapid delayed rectifier current, was absent. Unlike neural tissues, which express two splice variants (Kv3.1a and Kv3.1b), canine atrium showed only Kv3.1b transcripts. Whole-cell patch-clamp studies showed that IKur,d is absent in canine ventricular myocytes, and immunohistochemical and Western blot analysis demonstrated the absence of dKv3.1 protein in canine ventricle. We conclude that the Shaw-type channel dKv3.1 is present in dog atrium, but not ventricle, and is the likely molecular basis of canine atrial IKur,d.

Figure 1. Deduced amino acid sequence of dKv3.1 compared with that of rat brain Kv3.1 (RBKv3.1)

Dots indicate identical units. The transmembrane domains and pore regions are indicated by boxes.

Figure 2. Evaluation of the presence of dKv3.1 mRNA in canine atrium

A, RPA with a probe for Kv3.1b. Lane 1, probe. Lane 2, negative control (tRNA). Lanes 3 and 4, RNA from one dog atrium in each lane (DA1, DA2). Lane 5, RNA extracted from isolated canine atrial myocytes (DA3). Lane 6, RNA from rat brain (RB). B, RPA with a probe for Kv3.1a and Kv3.1b. Lane 1, probe. Lane 2, tRNA. Lane 3, RNA from dog atrium. Lane 4, rat brain sample.

Figure 3. Protein studies in dog atrium

A, Western blots of membrane proteins prepared from dog atrium (lane 1, DA1) and isolated dog atrial myocytes (lane 2, DA2) probed with a Kv3.1 antibody. Lane 3, dog atrial membrane preparation after pre-incubation of antibody with Kv3.1 peptide (Pre-inc). B, Western blots with Kv1.5 antibody obtained with human atrial (HA) and dog atrial (DA) tissues.

Figure 4. Immunohistochemical evidence for the presence of dKv3.1 in dog atrial myocytes

A, immunocytochemical images of canine atrial myocytes prepared with primary antibody pre-incubated (a) and not pre-incubated (b-d) with the Kv3.1 peptide against which the antibody had been raised. B, tissue sections of dog atrium: a is a typical immunohistochemical image prepared with Kv3.1 antibody and b is a bright-field micrograph of the same section; c was prepared in the same fashion as a, but with the Kv3.1 antibody pre-incubated with Kv3.1 peptide, and d is a bright-field image of the section shown in c.

Figure 5. Comparison between currents carried by dKv3.1 and I_Kur,d

A, currents recorded from an oocyte 20 h after dKv3.1 cRNA injection. B, currents in a dog atrial myocyte. C, mean (±s.e.m.) current-voltage relation of step and tail currents recorded from oocytes (n = 8). D, mean current density-voltage relation of step and tail currents recorded from myocytes (n = 8). TP, test potential.

Figure 6. Voltage- and time-dependent properties of dKv3.1 currents in oocytes

A, currents elicited by 20 ms pulses from a V_h of −80 mV. Tail currents were recorded upon repolarization to −30 mV. B, normalized tail currents fitted by a Boltzmann distribution (n = 8). C, currents elicited by 60 s conditioning pulses to conditioning potentials (CP) between −100 and +50 mV followed by a 1100 ms test pulse to +50 mV. D, inactivation curve with conditioning pulses of 1, 10 and 60 s (n = 8 for each). E, voltage-dependent activation and deactivation time constants (n = 8). F, time-dependent inactivation of dKv3.1 during a 100 s pulse to +40 mV and biexponential fit.

Figure 7. Response of dKv3.1 to 4-AP and TEA

A, original recordings from one oocyte under control conditions and after exposure to varying concentrations of 4-AP. B, dose-response curve for 4-AP effects on tail currents following an activating pulse to +30 mV (n = 8). C, original recordings from another oocyte under control conditions and after exposure to varying concentrations of TEA. D, dose-response curve for TEA inhibition of dKv3.1 tail currents following an activating pulse to +30 mV (n = 6).

Figure 8. Evaluation of dKv3.1 protein and I_Kur,d current expression in dog ventricle

A, Western blots of dog atrial (DA) and ventricular (DV) tissues with Kv3.1 antibody. B, immunohistochemical image obtained with Kv3.1 antibody (left) and bright-field image (right) on a dog ventricular tissue section. C, currents recorded from a dog ventricular myocyte with the same voltage protocol and conditions as used to record I_Kur,d in atrium (Fig. 5B). Currents were recorded before (left) and after (right) superfusion with 50 μm 4-AP.