1. Three mutations at the same site in the inactivation gate of the oc-subunit of the human muscle Nae channel, G1306E, G1306V and G1306A, cause three phenotypes of K+-aggravated myotonia: G1306E as the most severe and G1306A as the most benign form.2. Recombinant wildtype (WT) and mutant (G1306E, G1306V and G1306A) human Nae channels were expressed in human embryonic kidney cells (HEK293). G1306E and G1306V channels showed a distinct increase in the time constants of inactivation (Thl and rh2) and in the ratios of steady-state to peak currents (IS/Ipeak) (e.g. at 0 mV, G1306E vs. WT; Thl, showed only an increase in Thl (0 74 + 0 07 ms). For G1306E and G1306V channels, the steady-state inactivation curves, as well as the voltage dependence of the rate of recovery from inactivation, were shifted by +15 mV. For G1306A the h. curve was shifted by only +5 mV.3. G1306E and G1306V channels showed prolonged current rise times and later first openings suggesting slowing of activation. For G1306E channels only, the steady-state activation curve was shifted by -7 mV. For all mutants the deactivation time constants were increased. 4. We conclude that (i) the combination of alterations in inactivation and activation produces the slowing of the current decay, (ii) the slowed inactivation is most responsible for myotonia, and (iii) the shift of the steady-state activation curve, seen only with G1306E channels, may explain the severity of this phenotype. 5. The results suggest that two of the mutations in the Na+ channel inactivation gate also alter channel activation and deactivation.

1. Wild type (WT) and V1589M channels were expressed in human embryonic kidney (HEK293) cells for the study of the pathophysiology of the V1589M muscle Na+ channel mutation leading to K+-aggravated myotonia. 2. In comparison to WI, whole-cell recordings with V1589M channels showed an increased Na+ steady-state to peak current ratio (I/Ipeak) (3-15 + 0 70 vs. 0-87 + 0410%, at -15 mV) and a significantly faster recovery from inactivation. The recovery time constants, Trl and Tr2, were decreased from 1-28 + 0-12 to 0-92 + 0-08 ms and from 4-74 + 0 94 to 2-66 + 0-51 ms for the WT and mutant channels, respectively.3. Single-channel recordings with mutant channels showed higher probability of short isolated late openings (0 40 + 0 09 vs. 0-06 + 0-02, at -30 mV) and bursts of late openings (0-011 + 0 003 vs. 0 003 + 0 001, at -30 mV) compared to VVW.4. These results suggest that the mutation increases the probabilities for channel transitions from the inactivated to the closed and the opened states. 5. Increased extracellular concentrations of K+ had no effects on either V1589M or WI currents in HEK293 cells. The aggravation of myotonia seen in patients during increased serum K+ may arise from the associated membrane depolarization which favours the occurence of late openings in the mutant channel.