Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes

Abstract

A rapidly inactivating K(+) current (A-type current; I(A)) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic I(A) resembles that of Kv4-derived currents. We examined the contribution of Kv4 alpha-subunits to I(A) in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd(2+) decreased peak I(A) and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La(3+). Colonic I(A) was sensitive to low micromolar concentrations of flecainide (IC(50) = 11 microM). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K(+) channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of I(A) in murine colonic myocytes.

Figure 1. Cadmium decreases peak colonic A-type current and shifts the voltage dependence of activation and inactivation to more depolarized potentials

A and B, whole-cell A-type currents recorded from a colonic myocyte before (A) and after (B) Cd²⁺ (500 μM). The membrane potential was stepped for 500 ms from −80 mV to potentials between −80 and +40 mV. C, difference currents obtained by digitally subtracting records in B from those in A. D, summarized data quantifying the effect of Cd²⁺ (500 μM) on peak and sustained current at a test potential of +20 mV. * Significant reduction in peak and sustained current amplitude after Cd²⁺ compared to control (P < 0.05; n = 7). E, voltage dependence of activation of A-type current K⁺ permeabilities. Peak K⁺ currents (at test potentials between −80 and +40 mV; not shown) were converted into permeabilities using the Goldman-Hodgkin-Katz current equation. Permeabilities were then normalized, plotted as a function of test potential and fitted with a Boltzmann function. F, voltage dependence of inactivation of A-type current. Normalized peak currents at +20 mV (I/I_max; not shown) are plotted as a function of the conditioning potential ranging from −80 to +20 mV for 3 s and fitted with a Boltzmann function.

Figure 2. Lanthanum decreases peak colonic A-type current and shifts the voltage dependence of activation and inactivation to more depolarized potentials

A and B, whole-cell A-type currents recorded from a colonic myocyte before (A) and after (B) La³⁺ (100 μM). The membrane potential was stepped for 500 ms from −80 mV to potentials between −80 and +40 mV. C, difference currents obtained by digitally subtracting records in B from those in A. D, summarized data quantifying the effect of La³⁺ (100 μM) on peak and sustained current at a test potential of +20 mV. * Significant reduction in peak current amplitude after La³⁺ compared to control (P < 0.05; n = 4). E, voltage dependence of activation of A-type current K⁺ permeabilities. Peak K⁺ currents (at test potentials between −80 and +40 mV; not shown) were converted into permeabilities using the Goldman-Hodgkin-Katz current equation. Permeabilities were then normalized, plotted as a function of test potential and fitted with a Boltzmann function. F, voltage dependence of inactivation of A-type current. Normalized peak currents at +20 mV (I/I_max; not shown) are plotted as a function of the conditioning potential (ranging from −80 to +20 mV for 3 s) and fitted with a Boltzmann function.

Figure 3. Inhibition of colonic A-type current by flecainide

A and B, whole-cell A-type currents recorded from a colonic myocyte before (A) and after (B) flecainide (10 μM). The membrane potential was stepped for 500 ms from −80 mV to potentials between −80 and +40 mV. C, whole-cell A-type currents recorded from a colonic myocyte before and after different concentrations of flecainide (concentrations indicated in figure). The membrane potential was stepped for 500 ms from −80 to 0 mV. D, dose-dependent inhibition of peak A-type current by flecainide. Normalized peak currents at 0 mV (I/I_max; not shown) were plotted as a function of flecainide concentration (ranging from 0.1 to 100 μM) and fitted with a variable slope logistic equation, from which an IC₅₀ of 11 ± 1 μM was determined.

Figure 4. Quantification of Kv4 transcripts in colon and jejunum

A and B, RT-PCR analysis of primer pairs used for real-time PCR in colon (A) and jejunum (B). From left to right: 100 bp marker; Kv4.1 (amplicon = 116 bp); Kv4.2 (amplicon = 111 bp); Kv4.3, long isoform (amplicon = 176 bp). Amplicon identity confirmed by DNA sequencing; see Table 1 for primer sequences. C and D, Kv4.1, Kv4.2 and Kv4.3 gene expression relative to β-actin in colon (C) and jejunum (D) as determined by real-time PCR. * Significantly greater expression of Kv4.3 transcripts relative to Kv4.1 or Kv4.2 within the same tissue (P < 0.05; n = 5).

Figure 5. Kv4.2- and Kv4.3-like immunoreactivity in the tunica muscularis of murine colon and jejunum

Haematoxylin counterstain. A and B, Kv4.2-like (A) and Kv4.3-like (B) immunoreactivity (in brown) throughout the circular (cm) and longitudinal (lm) muscle layers of the tunica muscularis in murine colon. Arrowheads indicate Kv4-like immunoreactivity found within myenteric ganglia. C and D, Kv4.2-like (C) and Kv4.3-like (D) immunoreactivity (in brown) throughout the circular (cm) and longitudinal (lm) layers of the tunica muscularis in murine jejunum. Scale bars, 20 μm.

Figure 6. Comparison of colonic and jejunal A-type currents

A and B, whole-cell A-type currents recorded from a colonic (A) and a jejunal (B) myocyte in the presence of TEA (10 mm). The membrane potential was stepped for 500 ms from −80 mV to potentials between −70 and +20 mV. Inset in B ahows representative traces demonstrating jejunal I_A recovery from inactivation. The membrane potential was stepped for 1 s from −80 to 0 mV followed by a repolarization to −80 mV. Recovery from inactivation was then determined by stepping the membrane potential back to 0 mV after incrementally (50 ms) increasing periods of time. C, peak current density (pA pF⁻¹) as a function of voltage in colonic and jejunal myocytes. * Significantly greater current density in colonic myocytes relative to jejunal myocytes (P < 0.05; n = 5). D, whole-cell A-type currents recorded from a jejunal myocyte before and after different concentrations of flecainide (concentrations indicated in figure). The membrane potential was stepped from −80 to 0 mV for 500 ms.

Figure 7. Quantification of KChIP transcripts in colon and jejunum

A and B, detection of KChIP transcripts in isolated colonic (A) and jejunal (B) myocytes and RT-PCR analysis of primer pairs used for real-time PCR. From left to right: 100 bp marker; KChIP1 (amplicon = 164 bp); KChIP2 (amplicon = 190 bp); KChIP3 (amplicon = 168 bp); and KChIP4 (amplicon = 186 bp). Amplicon identity confirmed by DNA sequencing; see Table 1 for primer sequences. C, KChIP1, KChIP2, KChIP3 and KChIP4 gene expression relative to β-actin in colon as determined by real-time PCR. * Significantly greater expression of KChIP1 transcripts relative to KChIP2, KChIP3 or KChIP4 (P < 0.05; n = 5); † significantly greater expression of KChIP4 transcripts relative to KChIP2 or KChIP3 (P < 0.05; n = 5). D, KChIP1, KChIP2, KChIP3 and KChIP4 gene expression relative to β-actin in jejunum as determined by real-time PCR. * Significantly greater expression of KChIP1 transcripts relative to KChIP2, KChIP3 or KChIP4 (P < 0.05; n = 5); † significantly greater expression of KChIP2 transcripts relative to KChIP3 or KChIP4 (P < 0.05; n = 5).