Regulation of the Kv2.1 potassium channel by MinK and MiRP1

Abstract

Kv2.1 is a voltage-gated potassium (Kv) channel alpha-subunit expressed in mammalian heart and brain. MinK-related peptides (MiRPs), encoded by KCNE genes, are single-transmembrane domain ancillary subunits that form complexes with Kv channel alpha-subunits to modify their function. Mutations in human MinK (KCNE1) and MiRP1 (KCNE2) are associated with inherited and acquired forms of long QT syndrome (LQTS). Here, coimmunoprecipitations from rat heart tissue suggested that both MinK and MiRP1 form native cardiac complexes with Kv2.1. In whole-cell voltage-clamp studies of subunits expressed in CHO cells, rat MinK and MiRP1 reduced Kv2.1 current density three- and twofold, respectively; slowed Kv2.1 activation (at +60 mV) two- and threefold, respectively; and slowed Kv2.1 deactivation less than twofold. Human MinK slowed Kv2.1 activation 25%, while human MiRP1 slowed Kv2.1 activation and deactivation twofold. Inherited mutations in human MinK and MiRP1, previously associated with LQTS, were also evaluated. D76N-MinK and S74L-MinK reduced Kv2.1 current density (threefold and 40%, respectively) and slowed deactivation (60% and 80%, respectively). Compared to wild-type human MiRP1-Kv2.1 complexes, channels formed with M54T- or I57T-MiRP1 showed greatly slowed activation (tenfold and fivefold, respectively). The data broaden the potential roles of MinK and MiRP1 in cardiac physiology and support the possibility that inherited mutations in either subunit could contribute to cardiac arrhythmia by multiple mechanisms.

Figure 1. Immunoprecipitation of MinK-Kv2.1 and MiRP1-Kv2.1 complexes from rat heart

A. RT-PCR indicating expression of Kv2.1 mRNA in rat heart. Markers indicate migration of DNA ladder markers on agarose gel. mRNA was extracted from rat heart and then cDNA was produced by reverse transcription then amplified by Kv2.1-specific primers. Lanes: rHeart, cDNA amplified from rat heart mRNA; rKv2.1, positive control using Kv2.1 cDNA in a plasmid; No RT, as for rHeart lane but without reverse transcription (negative control). B. RT-PCR showing expression of MinK, MiRP1 and MiRP2 mRNA in rat heart. Markers indicate migration of DNA ladder markers on agarose gel. Lanes: rHeart, cDNA amplified from rat heart mRNA with primers as indicated; No RT, as for rHeart lanes but without reverse transcription (negative controls). C. Western blot analysis of MinK, MiRP1 and MiRP2 in CHO cells transfected with expression plasmids containing cDNA for MinK, MiRP1 or MiRP2, respectively. ‘−’ indicates non-transfected CHO cell lysate (negative control); ‘+’ indicates lysates from CHO cells transfected with MinK, MiRP1 or MiRP2, as indicated by the antibody used for immunoblotting (‘IB’, lower labels). Numbered labels to left of each blot indicate migration of molecular weight markers (in kD) on SDS-PAGE gel. D. Western blot analysis of native channel subunit expression in rat heart membrane fractions. Lanes: ‘−’ indicates non-transfected CHO cell lysate (negative control); ‘rHeart’ indicates membrane fraction from rat heart. Lower labels indicate antibody used for immunoblotting (‘IB’). Numbered labels to left of each blot indicate migration of molecular weight markers (in kD) on SDS-PAGE gel. E. Native co-immunoprecipitations of channel subunits from rat heart membrane fractions. Exemplar western blot using anti-Kv2.1 antibody for immunblotting (‘IB’) of Kv2.1 in Kv2.1-transfected CHO cell lysate, and in rat heart membrane fractions (‘rHeart IP’) immunoprecipitated by antibodies raised against Kv2.1, MinK or MiRP1 (n = 4 experiments). Kv2.1 was not detected in non-transfected CHO cell lysate or in rat heart membrane fractions immunoprecipitated by antibodies raised against the A1 adenosine receptor (A1-R) or MiRP2 (n = 2 experiments). Numbered labels to left of each blot indicate migration of molecular weight markers (in kD) on SDS-PAGE gel. F. Reciprocal native co-immunoprecipitations of channel subunits from rat heart membrane fractions. Western blots using anti-MinK (upper gel) or anti-MiRP1 (lower gel) antibodies for immunoblotting (‘IB’). MinK was present in rat heart membranes, MinK-transfected (‘+’) CHO cell lysate, and in rat heart membrane fractions (‘rHeart IP’) immunoprecipitated by antibodies raised against Kv2.1, MinK or ERG (n = 2 experiments). MiRP1 was present in rat heart membranes, MiRP1-transfected (‘+’) CHO cell lysate, and in rat heart membrane fractions (‘rHeart IP’) immunoprecipitated by antibodies raised against Kv2.1, MiRP1 or ERG (n = 2 experiments). Neither subunit was detected in non-transfected CHO cell lysate. Numbered labels to left of each blot indicate migration of molecular weight markers (in kD) on SDS-PAGE gel.

Figure 2. Modulation of Kv2.1 by rat MinK and MiRP1

A. Exemplar traces showing currents recorded from CHO cells transfected with Kv2.1 alone or co-transfected with rat MinK or MiRP1 as indicated. Insets: left, scale bars; right, voltage protocol. Vertical scale: 2nA, Kv2.1 and rMiRP1-Kv2.1; 0.5 nA, rMinK-Kv2.1. B. Mean peak current density from CHO cells transfected with Kv2.1 alone (filled squares, n = 61), or co-transfected with rMinK (open circles, n = 14) or rMiRP1 (triangles, n = 17). * statistical significance versus Kv2.1 alone, p < 0.05. C. Normalized exemplar traces showing expanded view of Kv2.1, rMinK-Kv2.1 and rMiRP1-Kv2.1 activation at 0 mV. D. Mean activation rates of Kv2.1, rMinK-Kv2.1 and rMiRP1-Kv2.1 channels at activation voltages between −20 mV and +60 mV, fitted with a single exponential function, expressed as τ_act. * statistical significance versus Kv2.1 alone, p < 0.05. Cells and symbols as in panel B. E. Normalized exemplar traces showing expanded view of Kv2.1, rMinK-Kv2.1 and rMiRP1-Kv2.1 deactivation at −40mV. F. Mean deactivation rates of Kv2.1, rMinK-Kv2.1 and rMiRP1-Kv2.1 currents at −40mV fitted with a double exponential function, showing slow (black) and fast (white) τ_deact components. * statistical significance versus Kv2.1 alone, p < 0.05. G. Mean fractional amplitude of the slow component of deactivation rates of Kv2.1, rMinK-Kv2.1 and rMiRP1-Kv2.1 currents at −40mV fitted with a double exponential function. * statistical significance versus Kv2.1 alone, p < 0.05.

Figure 3. Human MinK and MiRP1 slow Kv2.1 gating

A. Exemplar traces showing currents recorded from CHO cells transfected with Kv2.1 alone or co-transfected with human MinK or MiRP1 as indicated. Insets: left, scale bars; right, voltage protocol. B. Mean peak current density from CHO cells transfected with Kv2.1 alone (filled squares, n = 42), or co-transfected with hMinK (open circles, n = 24) or hMiRP1 (open triangles, n = 16). C. Mean normalized conductance-voltage relationships, cells and symbols as in panel B. Data were fit with a Boltzman function, G = G_max/[1 + exp(V–V_0.5/k)], giving values for midpoint voltage dependence of activation of: Kv2.1, 4.4 ± 0.7 mV; MinK-Kv2.1, 1.0 ± 1.0 mV; MiRP1-Kv2.1, −0.6 ± 0.8 mV; slopes were 11.3 ± 0.4 mV, 15.2 ± 0.9 mV, and 13.1 ± 0.5 mV, respectively. D. Normalized exemplar traces showing expanded view of Kv2.1, hMinK-Kv2.1 and hMiRP1-Kv2.1 activation at 0 mV. E. Mean activation rates of Kv2.1, hMinK-Kv2.1 and hMiRP1-Kv2.1 channels at activation voltages between −10 mV and +60 mV, fitted with a single exponential function, expressed as τ_act. F. Normalized exemplar traces showing expanded view of Kv2.1, hMinK-Kv2.1 and hMiRP1-Kv2.1 deactivation at −40mV. G. Mean deactivation rates of Kv2.1, hMinK-Kv2.1 and hMiRP1-Kv2.1 currents at −40mV fitted with a double exponential function, showing slow (black) and fast (white) τ_deact components. * statistical significance versus Kv2.1 alone, p < 0.05. H. Mean fractional amplitude of the slow component of deactivation rates of Kv2.1, hMinK-Kv2.1 and hMiRP1-Kv2.1 currents at −40mV fitted with a double exponential function. * statistical significance versus Kv2.1 alone, p < 0.05.

Figure 4. Effects of inherited human MinK mutations on MinK-Kv2.1 function

A. Exemplar traces showing currents recorded from CHO cells co-transfected with Kv2.1 + wild-type human MinK (‘+hMinK’), Kv2.1 + D76N-MinK (‘+D76N’) or Kv2.1 + S74L-MinK (‘+S74L’), as indicated. Insets: left, voltage protocol; right, scale bars. B. Mean peak current density from CHO cells transfected with Kv2.1 alone (filled squares) or with wild-type (open circles, n = 14), D76N (filled triangles, n = 9) or S74L (open triangles, n = 11) human MinK. * statistically significant difference (p < 0.001). C. Mean normalized conductance-voltage relationships, cells and symbols as in panel B. Fitting of the data with a Boltzman function, G = G_max/[1 + exp(V–V_0.5/k)], gave values for midpoint voltage dependence of activation of: Kv2.1, 4.4 ± 0.7 mV; wild-type human MinK-Kv2.1, 1.0 ± 1.0 mV; D76N-MinK-Kv2.1, 6.0 ± 1.6 mV; S74L-MinK-Kv2.1, 6.2 ± 0.7 mV; slopes were 11.3 ± 0.4, 15.2 ± 0.9, 18.0 ± 1.8, and 12.5 ± 0.5 mV, respectively. D. Mean activation rates, cells and symbols as in panel B. Activation rates at voltages between −10 mV and +60 mV, were fitted with a single exponential function, expressed as τ_act. E. Mean deactivation rates for cells as in panel B, subunits as indicated. Deactivation rates at −30 mV were fitted with a double exponential function, showing slow (black) and fast (white) τ_deact components. Asterisks denote statistically significant difference versus wild-type human MinK-MinK: *p < 0.05; **p < 0.01; *** p < 0.001. F. Mean fractional amplitude of the slow component of deactivation rates of Kv2.1, and Kv2.1 with wild-type or mutant hMinK, currents at −40mV fitted with a double exponential function. * statistical significance versus Kv2.1 + wild-type MinK, p < 0.05.

Figure 5. Effects of inherited human MiRP1 mutations on MiRP1-Kv2.1 function

A. Exemplar traces showing currents recorded from CHO cells co-transfected with Kv2.1 + wild-type human MiRP1 (‘+MiRP1’), Kv2.1 + I57T-MiRP1 (‘+I57T’) or Kv2.1 + M54T-MiRP1 (‘+M54T’), as indicated. Insets: left, voltage protocol; right, scale bars. B. Mean peak current density from CHO cells transfected with Kv2.1 alone (filled squares, n = 42) or with wild-type human MiRP1 (open triangles, n = 16), I57T-MiRP1 (open circles, n = 21) or M54T-MiRP1 (filled circles, n = 15). C. Mean normalized conductance-voltage relationships; cells and symbols as in panel B. Fitting of the data with a Boltzman function, G = G_max/[1 + exp(V–V_0.5/k)], gave values for midpoint voltage dependence of activation of: Kv2.1, 4.4 ± 0.7 mV; wild-type hMiRP1-Kv2.1, −0.6 ± 0.8 mV; M54T-MiRP1-Kv2.1, 10.4 ± 1.1 mV; I57T-MiRP1-Kv2.1, 1.1 ± 0.4 mV; slopes were 11.3 ±0.4, 13.1 ± 0.5, 13.4 ± 0.6, and 12.5 ± 0.3 mV, respectively. D. Normalized exemplar traces showing expanded view of Kv2.1 alone or with wild-type, M54T-or I57T-MiRP1 (as indicated) activation at 0 mV. E. Mean activation rates at activation voltages between −10 mV and +60 mV, fitted with a single exponential function, expressed as τ_act; cells and symbols as in panel B. F. Mean deactivation rates at −30 mV fitted with a double exponential function, showing slow (black) and fast (white) τ_deact components; cells as in panel B, subunits as indicated. * statistically significant difference versus Kv2.1 alone (p < 0.05). G. Mean fractional amplitude of the slow component of deactivation rates of Kv2.1, and Kv2.1 with wild-type or mutant hMiRP1, currents at −40mV fitted with a double exponential function. H. % inactivation at +40 mV over the initial 1 sec time period; cells as in panel B, subunits as indicated. * statistically significant difference versus wild-type hMiRP1-Kv2.1 (p < 0.05).