ClC-1 is the major muscle chloride channel responsible for the large resting anion conductance of adult skeletal muscle fibers. Dysfunction of human ClC-1 causes myotonia congenita, an inherited disease characterized by an over-excitability of muscle fibers resulting in muscle stiffness upon sudden forceful movement. We here investigate the functional consequences of two disease-causing mutations, C277Y and C277R, that were identified in patients with recessive generalized myotonia (Becker). We transiently expressed WT and mutant hClC-1 channels in tsA201 cells and studied currents through whole-cell patch clamp recordings. Cells expressing C277Y/R channels exhibited lower current amplitudes and altered voltage-dependent gating. WT currents rise instantaneously upon voltage steps followed by a pronounced deactivation at negative potentials and time independent current amplitudes at positive potentials. C277Y/R ClC-1 currents display bi-exponential activation upon hyperpolarization and slightly increasing currents upon depolarization. Non-stationary noise analysis revealed absolute open probabilities of C277Y below 5% at -80 mV. Single channel current amplitudes of C277Y (-0.135 ± 0.012 pA at -155 mV) were about one half of WT unitary currents. C277Y affected the selectivity filter of hClC-1 and changed permeability sequence to I = NO₃ > Br > Cl as compared to Cl > Br > NO₃ > I for WT channels. In contrast to WT channels, gating of C277Y/R ClC-1 is modified by intracellular [Cl^-]. Mimicking physiological conditions with low internal chloride (4 mM) dramatically reduced macroscopic C277Y/R conductances. ClC channels are dimers, and we constructed hetero-concatamers of WT and mutant C277Y/R channels to functionally study heterodimeric channels. Concatamer currents resembled a mixture of WT and mutant currents at neutral pH with a shifted activation curve to more positive potentials. With low external pH mutation C277Y altered gating of both protopores indicating an effect on the common gate that opens and closes both conductive pathways simultaneously. Our findings predict a significant reduction of muscle chloride conductance in affected muscle fibers. Moreover, they provide novel insights into molecular determinants of anion conduction by ClC-type chloride channels.