Gating of the HypoPP-1 mutations: I. Mutant-specific effects and cooperativity

Abstract

Hypokalemic periodic paralysis type 1 (HypoPP-1) is a hereditary muscular disorder caused by point mutations in the gene encoding the voltage-gated Ca(2+) channel alpha subunit (Ca(v)1.1). Despite extensive research, the results on HypoPP-1 mutations are minor and controversial, as it is difficult to analyse Ca(2+) channel activation macroscopically due to an existence of two open states. In this study, we heterologously expressed the wild-type and HypoPP-1 mutations introduced into the rabbit cardiac Ca(2+) channel (R650H, R1362H, R1362G) in HEK-293 cells. To examine the cooperative effects of the mutations on channel gating, we expressed two double mutants (R650H/R1362H, R650H/R1362G). We performed whole-cell patch-clamp and, to obtain more information, applied a global fitting procedure whereby several current traces elicited by different potentials were simultaneously fit to the kinetic model containing four closed, two open and two inactivated states. We found that all HypoPP-1 mutations have "loss-of-function" features: D4/S4 mutations shift the equilibrium to the closed states, which results in reduced open probability, shorter openings and, therefore, in smaller currents, and the D2/S4 mutant slows the activation. In addition, HypoPP-1 histidine mutants favored the second open state O(2) with a possibly lower channel selectivity. Cooperativity between the D2/S4 and D4/S4 HypoPP-1 mutations manifested in dominant effects of the D4/S4 mutations on kinetics of the double mutants, suggesting different roles of D2/S4 and D4/S4 voltage sensors in the gating of voltage-gated calcium channels.

Gating of the HypoPP-1 mutations: II. Effects of a calcium-channel agonist BayK 8644

“…L-type calcium channel mutations causing hypokalemic periodic paralysis type 1 (HypoPP-1) have pronounced "loss-of-function" features, as we demonstrated in the companion paper [11]. Here, we compared (±)BayK 8644 (BayK) effects on the wild-type (WT) calcium channel and on the HypoPP-1 mutant channels.…”

“…Two double mutations, R650H/R1362H (HH) and R650H/ R1362G (HG) were constructed by exchanging fragments from the three single mutations (encoding R650H, R1362H, and R1362G). Transfection was performed in the human embryonic kidney 293 (HEK-293) cell line using the standard calcium phosphate method, as described previously [11]. The rationale for using the cardiac isoform, the particular calcium-channel subunit composition, and the expression system is extensively discussed in the companion paper [11].…”

“…In the companion paper, we revealed that the HypoPP-1 histidine mutants shift the distribution of the open states towards the second open state [11]. The second open state in the L-type calcium channels was suggested to have a low selectivity for divalent cations, so that monovalent cations, such as Na + , K + , and Cs + , can extensively pass through the channel [9,11].…”

“…The second open state in the L-type calcium channels was suggested to have a low selectivity for divalent cations, so that monovalent cations, such as Na + , K + , and Cs + , can extensively pass through the channel [9,11]. Thus, the open-state distribution is a putative determinant of calcium-channel selectivity and is of a high physiological importance in HypoPP, because the membrane depolarization, the cause of paralytic attacks in HypoPP muscle, is the result of shifts in transmembrane gradients of electrolytes [16,19,20].…”

“…Thus, the open-state distribution is a putative determinant of calcium-channel selectivity and is of a high physiological importance in HypoPP, because the membrane depolarization, the cause of paralytic attacks in HypoPP muscle, is the result of shifts in transmembrane gradients of electrolytes [16,19,20]. As the fraction of the second open state in the calcium channels is small, the HypoPP-related changes of the open-state distribution are difficult to resolve [11]. To increase the O 2 fraction, we applied BayK.…”